Thesis on

Cloning of erythropoietin gene in replicase based eukaryotic

vector and characterization of the recombinant plasmid

Submitted for the award of

DOCTOR OF PHILOSOPHY

Degree in

Biotechnology

By

Usha Tiwari

UNDER THE SUPERVISION OF

Prof. Anant Rai Prof. Kusum Agarwal IBIT, Bareilly Shobhit University, Meerut

Prof. Nishant Rai Graphic Era University, Dehradun

SHOBHIT INSTITUTE OF ENGINEERING & TECHNOLOGY

A DEEMED-TO-BE UNIVERSITY

MODIPURAM, MEERUT-250110 (INDIA)

2012

To my parents, my first teachers

Dr. Kusum Agarwal M.Sc., Ph.D.

Professor & Coordinator,

Department of Biotechnology,

Faculty of Biological Engineering,

Shobhit University, Meerut E-mail: kusum0211@yahoo.com

Certificate

This is to certify that the thesis entitled “Cloning of erythropoietin gene in

replicase based eukaryotic vector and characterization of the recombinant

plasmid ” which is being submitted by Ms. Usha Tiwari for the degree of Doctor of

Philosophy in Biotechnology to Faculty of Biological Engineering, Shobhit

University, Meerut, a deemed-to-be-University, established by GOI u/s 3 of UGC Act

1956, is a record of bonafide investigations and extensions of the problems carried

out by her under my supervision and guidance.

To the best of my knowledge, the thesis embodies the work of the candidate

herself and has not been submitted to any other University or Institution for the

award of any degree or diploma.

It is further certified that she worked with me for the required period in the

Department of Biotechnology, Faculty of Biological Engineering, Shobhit

University, Meerut.

Date: Prof. Kusum Agarwal

(Internal Supervisor)

Institute of Biotechnology & IT 197, Mudia Ahmadnagar, Bareilly-243122 (UP) Ph: 09219661948, 0581-2602907, Fax: 0581-2602035

www.ibit.org.in ibitbe@gmail.com

Prof. Anant Rai

Ph.D. Director

____________________________________________

Certificate This is to certify that the thesis entitled “Cloning of erythropoietin gene in

replicase based eukaryotic vector and characterization of the recombinant

plasmid ” which is being submitted by Ms Usha Tiwari for the degree of Doctor of

Philosophy in Biotechnology to Faculty of Biological Engineering, Shobhit

University, Meerut, a deemed-to-be-University, established by GOI u/s 3 of UGC Act

1956, is a record of bonafide investigations and extensions of the problems carried

out by her under my supervision and guidance.

To the best of my knowledge, the thesis embodies the work of the candidate

herself and has not been submitted to any other University or Institution for the

award of any degree or diploma.

It is further certified that she worked with me for the required period in the

Department of Biotechnology, Institute of Biotechnology and IT, Bareilly.

Date: Prof. Anant Rai (External Supervisor)

Prof. Nishant Rai Dept. of Biotechnology

Ph.D. Dehradun, UK, India

Associate Professor nishantrai1@gmail.com

Certificate

This is to certify that the thesis entitled “Cloning of erythropoietin gene in

replicase based eukaryotic vector and characterization of the recombinant

plasmid ” which is being submitted by Ms Usha Tiwari for the degree of Doctor of

Philosophy in Biotechnology to the Faculty of Biological Engineering, Shobhit

University, Meerut, a deemed-to-be-University, established by GOI u/s 3 of UGC Act

1956, is a record of bonafide investigations and extensions of the problems carried

out by her under my supervision and guidance.

To the best of my knowledge, the thesis embodies the work of the candidate

herself and has not been submitted to any other University or Institution for the

award of any degree or diploma.

It is further certified that she worked with me for the required period in the

Department of Biotechnology, Graphic Era University, Dehradun.

Date: (Prof. Nishant Rai)

Co-external Supervisor

Declaration

I, hereby, declare that the work presented in this thesis

entitled “Cloning of erythropoietin gene in replicase based

eukaryotic vector and characterization of the recombinant plasmid”

for the award of degree of Doctor of Philosophy, submitted to Faculty

of Biological Engineering, Shobhit University, Meerut, a deemed-to-be-

University, established by GOI u/s 3 of UGC Act 1956, is an authentic

record of my own research work carried out under the supervision of

Prof. Kusum Agarwal, Prof. Anant Rai and Prof. Nishant Rai.

I also declare that the work embodied in the present thesis

(i) is my original work and has not been copied from any

Journal/Thesis/Book, and

(ii) has not been submitted by me for any other degree or

diploma.

(Usha Tiwari)

Acknowledgements

Acknowledgement seems to be more difficult than the present

work. Many people were involved in one way or the other. Memory

and space both make it impossible to acknowledge the guidance

and assistance of multifarious nature received from teachers,

juniors and colleagues.

Words are inadequate to express my sincere and deepest

feelings of gratitude originated from the innermost core of my heart

for my Internal-Supervisor Prof. Kusum Agarwal Professor and

Coordinator, Department of Biotechnology, Faculty of Biological

Engineering, Shobhit University, Meerut, my External-Supervisor

Prof. Anant Rai Director, Institute of Biotechnology and IT, Bareilly

(Retd. Principal Scientist and Head, Division of Animal

Biotechnology, Indian Veterinary Research Institute, Izatnagar,

Bareilly), my Co-external Supervisor Prof. Nishant Rai Associate

Professor, Department of Biotechnology, Graphic Era University,

Dehradun for their unflinching interest, valuable advice, close

supervision, constructive criticism, healthy encouragement and

generosity throughout the course of this study.

I am thankful to the Honorable Chancellor Dr. Shobhit

Kumar, Honorable Pro Chancellor Kunwar Shekhar Vijendra,

Honorable Vice-Chancellor Prof. R. P. Agarwal of Shobhit University

for providing congenial environment for conducting research in the

University. I would always be thankful to my reverend teachers for

their affection, guidance, valuable inputs and blessings bestowed

upon me throughout the journey of my academic career.

I specially reflect my feelings and gratitude to Prof. D.V. Rai

Dean, Faculty of Biological Engineering, Prof. Ranjit Singh Former

Director, School of Pharmaceutical Sciences, Dr. Rekha Dixit, Dr.

Jayanad and Dr. Manish Kumar Gupta, Shobhit University, Meerut.

I am extremely thankful to Dr. D.V. Rai, Professor and

Head (Microbiology), and Mrs. Soni Gangwar, Assistant Professor

(Biotechnology), Institute of Biotechnology and IT, Bareilly for

providing assistance in the laboratory.

I express my gratitude to Prof. S.C. Agrawal, Dean,

Faculty of Humanities, Physical and Mathematical Sciences, Shobhit

University, Meerut, for his encouragement, constructive suggestions and

helpful attitude.

I thank my fellow lab mates Priyanka Pal, Rajveer

Maurya and Nitin Sharma for the stimulating discussions, I have

with them and for advising me from time to time for all those days we

worked together.

I extend my heartiest thanks to my parents Shri Upendra

nath Tiwari and Smt. Kanti Tiwari for sowing the seedling of

education, nurturing the plant of learning and being by my side

throughout my academic career.

(Usha Tiwari)

Research Papers Published/ Accepted/ Communicated in Refereed Journals/Proceedings

1. Tiwari, U., Agarwal, K., Rai, N., Rai, A. and Pal, P. (2011):

Cloning of human Erythropoietin gene in pSinCMV vector.

Biotechnology International, Vol.4, 31-35. (Online at www.bti.org.in)

2. Tiwari, U., Agarwal, K., Rai, N., Rai, A. and Pal, P. (2011):

Expression of recombinant plasmid containing human

erythropoietin gene in HeLa cell line using immunoperoxidase

test. Res J Pharm Sci and Biotech, Vol.1, 14-16. (Online at

www.rjpsb.info)

3. Tiwari, U., Agarwal, K., Rai, N., Rai, A. and Pal, P. (2010):

Isolation of plasmid DNA containing erythropoietin gene by TELT

method, accepted for publication in Transaction of physical and life

science. (accepted)

4. Tiwari, U., Agarwal, K., Rai, N., Rai, A. and Pal, P. (2011):

Expression of recombinant plasmid containing human

erythropoietin gene in HeLa cell line using SDS-PAGE and

western blotting, Communicated for publication in International

Journal of Pharmaceutical Applications. (submitted)

Participation in the Conferences/Workshops/Seminars

1. Attended National seminar-Cum Workshop on “Biomedical Research

and Clinical Application of Radioisotopes”, Shobhit University,

Meerut, March 31-01 April 2012.

2. Attended International Conference on “Indian Civilization through

the Millennia” held at Jamboo Dweep, Hastinapur, March 2-3, 2012. 3. Attended annual Conference on Vijnana Parishad of India and the

Global Society of Mathematical and Allied Sciences, held at School of

Basic and Applied Sciences, Shobhit University, Meerut, March 24-26,

2011. Presented paper entitled “Expression of recombinant plasmid

containing human erythropoietin gene in HeLa cells.”

4. Attended Conference on “Genes and Genomics: Qualitative and

Quantitative Approach” held at School of biotechnology, Shobhit

University, Meerut, September 11-12, 2010. Presented paper entitled

“Preparation of recombinant plasmid pSinCMV containing human

erythropoietin gene.”

5. Attended Workshop on “Nanomaterials: Recent Techniques and

Applications”, Shobhit University, Meerut, March 27, 2010.

Contents

List of Tables (i)

List of Figures (ii)

Abbreviations (iv)

Chapter 1: Introduction 1-10

Chapter 2: Review of Literature 11-54

2.1 Biology of erythropoietin 11

2.2 Recombinant erythropoietin 21

2.3 Biochemistry 29 2.4 Metabolism 31 2.5 Regulation 33 2.6 Cloning 38

2.7 Purification 42

2.8 Expression 44

Chapter 3: Materials and Methods 55- 73

3.1 Materials 55

3.1.1 Vector 55 3.1.2 Gene 55

3.1.3 Primers 56

3.1.4 Host Bacterial Strains 56 3.1.5 Cell Culture 56 3.1.6 Conjugates 56

3.1.7 Experimental Animals 56

3.1.8 Chemicals, Glass wares and Plastic wares 57

3.1.9 Solutions and Buffers 57

3.2 Methods 57 3.2.1 Revival of the E.coli culture containing 57

recombinant plasmid with human

erythropoietin gene

3.2.2 Isolation of plasmid DNA 57

3.2.3 Digestion of the recombinant plasmid with 58 RE (EcoRI) to release the epo.hu gene insert

3.2.4 Purification of the epo.hu gene insert 59 3.2.5 Preparation of the pSin Vector 59

3.2.6 Preparation of the epo.hu gene insert for 59 ligation

3.2.7 Digestion of the replicase based vector with 60 StuI enzyme

3.2.8 Dephosphorylation of vector DNA with 61

CIAP

3.2.9 Purification of replicase vector (pSin Vector) 62 3.2.10 Ligation of pSinVector and epo.hu gene 63 3.2.11 Transformation of E.coli DH5α 63

3.2.12 Screening of recombinant clones. 64 3.2.13 Selection of clone containing epo.hu gene in 64

right orientation 3.2.14 Expression study 67

3.2.15 Restriction enzyme analysis of the 72 recombinant plasmid, using different

restriction enzymes.

Chapter 4: Results 74-77

4.1 Cloning of epo.hu gene in pSin vector 74

4.2 Expression of the rplasmid in cell culture 76 4.3 Restriction enzyme analysis of the recombinant 76

plasmid, using different restriction enzymes

Chapter 5: Discussion 78-85

Chapter 6: Summary and Conclusion 86-88

Chapter 7: Future Scope 89

References 90-109

Appendix

List of Tables

Table No. Title Page No.

1 Primers with their sequences 56

2 Reaction mix for EcoRI digestion 58

3 Reaction mix for blunting 60

4 Reaction mix for StuI digestion 61

5 Reaction mix for Dephosphorylation 62

6 Ligation mixture 63

7 Reaction mix for kpnI digestion 65

8 Reaction mix for PCR 66

9 Component of resolving and stacking gel. 69

10 Reaction mix for different RE digestion 73

(i)

List of Figures Fig. No. Title Page No.

1 pSin Vector 55

2 Cloning strategy 57

3 SDS-PAGE System 68

4 Isolation of pTarget.epo.hu plasmid 74

5 pTarget.epo.hu plasmid digested with EcoRI 74 enzyme

6 Preparation of pSin Vector 74

7 Isolation of plasmid DNA (pSin.epo.hu) 75

8 RE Digestion of pSin.epo.hu with kpnI 75

9 PCR amplification of epo.hu gene 75

10 HeLa cells with pSin.epo.hu rplasmid showing 76 positive IPT test, 100X

11 Healthy Control HeLa cells showing no color 76 reaction, 100X

12 SDS-PAGE of HeLa cell extract for epo.hu 76 expression

13 Detection of epo.hu protein using Western 76 blotting

14 RE digestion analysis of the pSin.epo.hu rplasmid 77 with NheI

15 RE digestion analysis of the pSin.epo.hu rplasmid 77 with NotI

16 RE digestion analysis of the pSin.epo.hu rplasmid 77 with NruI

17 RE digestion analysis of the pSin.epo.hu rplasmid 77 with PmeI

18 RE digestion analysis of the pSin.epo.hu rplasmid 77 with PvuI

(ii)

19 RE digestion analysis of the pSin.epo.hu rplasmid 77

with SalI

(iii)

Abbreviations 0C Degree Celsius

% Percent

AA Amino Acid

Ab Antibody

BGH Bovine growth hormones

BSA Bovine serum albumin

CMV Cytomegalovirus

DMEM Dulbecco's Modified Eagle Medium

DNA Deoxyribonucleic acid

dNTP 2'- deoxy-nucleoside-5'-triphosphate

EDTA Ethylene Diamine Tetraacetic Acid

epo.hu Human erythropoietin

Fig. Figure

CIAP Calf Intestinal Phosphatase

IU International Unit

LB Luria Bertani

MW Molecular Weight

NaCl Sodium Chloride

FCS Fetal calf serum

AC Acridine Orange

No. Number

PBS Phosphate Buffered Saline

PCR Polymerase Chain Reaction

pH Log10 hydrogen ion concentration

RE Restriction endonuclease

RNA Ribonucleic acid

Rpm Revolution per minute (iv)

SDS Sodium Dodecyl Surface

TAE Tris-acetate-EDTA

TE Tris-EDTA

UV Ultraviolet

v/v Volume by volume

w/v Weight by volume

Units of Measurement

µg Microgram

µl Mirolittre

0C Degree Celsius

bp Base pair

g Gram

h Hours

Kb Kilo base

kDa Kilo Dalton

M Molar

mg Milligram

min Minute(s)

ml Milliliter

mM Millimolar

N Normal

Ng Nanogram

pmole Picomole

sec Second (s)

U Unit (s) (v)

Chapter-1

Introduction

Chapter-1

Introduction

The erythropoietin is a glycoprotein hormone and primarily

produced by the fetal liver and by the tubular cell fraction of the adult

kidney (Peschle et al., 1975; Schuster et al., 1987). Erythropoietin (epo)

was identified as hematopoietic cytokine that functions as the main

regulator of erythropoiesis. This function of epo was first formulated in

1906 by the French scientist Paul Carnot (Carnot et al., 1906). In 1948

the term 'erythropoietin' was introduced by Bonsdorff and Jalavisto. In

1977 the native human erythropoietin (epo.hu) was isolated for the first

time from human urine at the University of Chicago (Miyake et al.,

1977). The main roles of epo are inhibiting the apoptosis of erythroid

precursor cells and support their proliferation and differentiation into

normoblasts (Jelkmann, 1992).

The erythropoietin gene was successfully cloned in 1985 (Jacobs

et al., 1985). The human erythropoietin gene (epo.hu gene), a single-

copy gene located on chromosome 7 at position 99,929,820 bp to

99,932,720 bp from pter, consists of five exons and four introns. epo

gene is much conserved throughout the species. The human epo gene

shares 91% identity to monkey epo gene, 85% identity to cat and dog

epo gene, and 80% to 82% identity to pig, sheep, mouse, and rat epo

gene (Wen et al., 1993).

1

Human epo is a heavily glycosylated protein with a molecular

weight of about 34,000 Da. It consists of one 165 amino acid chain and

contains four oligosaccharide side chains: three N-linked and one O-

linked. The carbohydrate moiety represents approximately 40% of the

total molecular mass and is important for the stability and solubility of

the protein (Narhi et al., 1991), but not for receptor binding (Darling et

al., 2002). Thus, un-glycosylated erythropoietin has a very low in vivo

bioactivity due to rapid clearance from plasma by the liver (Tsuda et

al., 1990). epo was predicted by Wen in 1994 and confirmed by NMR

data in 1998 (Cheetham et al., 1998) to have a four-antiparallel

amphiphatic alpha-helical bundle structure (A, B, C and D), a structure

shared with other members of the cytokine family. The A and D helices

are linked by a disulfide bond between Cys7 and Cys161 and packed

against the helices B and C. Near the carboxy end of the AB loop is a

short alpha-helical segment (B‟) important for receptor binding

(Cheetham et al., 1998). The hydrophobic core of the protein is formed

by aromatic residues of the D-helix that are packed against

hydrophobic residues from the remaining helices. Species comparisons

of epo have shown that the core-forming amino acids are invariant.

Mutations in these domains lead to marked effects on protein folding.

Functionally important domains for (epo R) 2-binding have been

delineated in human epo by preparing amino acid replacement mutants

and testing them in three cell bioassay systems based on the human

UT7-epo leukemia cell line, the murine HCD57 erythroleukemia cell

line and murine erythroid spleen cells (Wen et al., 1994). Two distinct

patches were identified on the protein surface relevant for the

formation of a 2:1 homodimeric (epo receptor) 2: epo complex. A high-

2

affinity receptor binding site involves residues at the helix D:AB loop

interface and a low affinity receptor binding site comprises residues

Val11, Arg14, Tyr15, Ser100, Arg103, Ser104 and Leu108 (Wen et al.,

1994). epo expression in the fetal liver and adult kidney is induced

under hypoxic conditions via the hypoxia-inducible factor 1 (HIF-1)

(Semenza et al., 1992). HIF proteins are transcriptional regulators

targeting genes involved in angiogenesis, vasomotor control, energy

metabolism, apoptosis and erythropoiesis (Marti, 2004). HIF-1 is a

heterodimer composed of an α- and a β-subunit. The HIF-1β subunit is

a constitutive nuclear factor. HIF-1α is an oxygen-labile protein

containing two oxygen-dependent degradation domains (ODD) that is

rapidly degraded under normoxic conditions (Huang et al., 1998).

Regulators of this proteasome-mediated degradation are three prolyl-4-

hydroxylases (PHD 1-3) that require O2, iron and oxoglutarate as

cofactors. Under normoxic conditions the PHD proteins hydroxylase

key prolines in the HIF-1α subunit, thus leading to their ubiquitylation

and proteasomal degradation (Cai et al., 2003). Under hypoxic

conditions this hydroxylation cannot occur and HIF-1 accumulates in

the nucleus. HIF-1 binds to a highly conserved region 120 bp 3' to the

polyadenylation site of the epo gene, the hypoxically inducible

enhancer (Semenza et al., 1991). Depending on the severity of hypoxia,

epo mRNA levels can increase about 100-fold in vitro (Marti et al., 1996)

and about 1000-fold in vivo (Fandrey et al., 1993). Hypoxic conditions,

leading to transactivation of erythropoietin, can be mimicked by cobalt

chloride (CoCl2) and the iron chelator desferrioxamine (DSF). CoCl2 and

DSF treatment of murine neuronal and astroglial cultures induced epo

mRNA expression of about 4- to 5-folds (Bernaudin et al., 2000). Also

3

in vivo application of CoCl2 and DSF was found to stimulate epo gene

transcription in mice and rats (Bernaudin et al., 2000; Prass et al., 2002).

A possible mechanism for the actions of the transition metal Co2+ is the

substitution of the iron (Fe) atom in heme proteins, locking them

thereby in a„deoxy‟ state. In contrast, DSF binds intracellular iron and

thereby inhibits the Fe-catalyzed formation of reactive oxygen species.

First evidence for an erythropoietin binding receptor (epo R)

was published in 1987 from cross-linking experiments of iodinated epo

to immature murine erythroid cells (Sawyer et al., 1987). The binding

of epo to preformed (epo R) 2-dimers induces a conformational change

that brings constitutively associated Janus family tyrosine protein

kinases 2 (Jak2) in close proximity and stimulates their activation by

transphosphorylation (Witthuhn et al., 1993). In turn, Jak2

phosphorylates residues in the cytoplasmic domain of epo R, thereby

inducing several downstream signaling cascades. Depending on the cell

type, epo binding activates pathways involving the Signal Transducers

and Activators of Transcription (STATs), the Ras-mitogen-activated

protein kinase (MAPK) or the phosphatidylinositol 3-kinase (PI3K)

(Rossert et al., 2005). Several domains are essential for receptor

functioning: a WSXWS motif is necessary for proper protein folding

and a box 1 motif in the cytoplasmic part of the receptor is required for

Jak interaction and activation. Furthermore the protein contains a

cytoplasmic immune-receptor tyrosine-based inhibitor motif (ITIM)

that is involved in modulation of cellular responses by binding the

SH2-domains of several phosphatases. The existence of a fibronectin

type-III domain is shared with other cytokine receptor.

4

A few years ago, several splice variants of the epo R were

reported in human primary cancers and cancer cell lines. Splicing is the

crucial mechanism for messenger RNA (mRNA) maturation. This

process removes introns and joins exons in a primary transcript (pre-

mRNA). Introns usually contain a clear splicing signal, namely gu at

the 5‟ end of the splicing site, the splice donor, and ag at the 3‟ end, the

splice acceptor. These GU-AG dinucleotides flank more than 98% of

known intron sequences. The third important element of the splice site

is the branch site, which is located 20 - 50bp upstream of the acceptor

site. The branch site contains a CU (A/G) A(C/U) motif, whereas A is

conserved in all genes. Five snRNAs (U1, U2, U4, U5, and U6 snRNPs)

and their associated proteins form the spliceosome that catalyses a two-

step enzymatic reaction leading to removal of the intron and joining of

the two neighboring exons. Alternative splicing regulates differential

inclusion or exclusion of regions in the immature mRNA, invalidating

the old theory of „one-gene-one-protein‟. Four modes of alternative

splicing are known: alternative selection of promoters; alternative

polyadenylation; intron retaining and splicing out of exons The first

identified epo R splice variants, reported by Nakamura in 1992,

resulted from intron retaining leading to an early stop codon as in the

case of the soluble epo receptor (epo R-S), or to a prolonged amino acid

sequence as in the case of epo R-T. In the last years, several more splice

variants were revealed encoding soluble epo receptors or

membranebound epo receptors with intracellular carboxy-terminal

truncations (Arcasoy et al., 2003).

Erythropoietin (epo) was long thought to be exclusively

produced in kidney and fetal liver but recently brain and uterus have

5

been identified as additional production sites that regulate epo

expression in a tissue-specific manner (Chikuma et al., 2000). The

finding of a hematopoietic factor in the brain raised the question if

erythropoietin was merely a regulator of erythropoiesis. Furthermore,

the homodimeric erythropoietin receptor (epo R) 2 was not only found

on erythroid colony-forming units but also on neurons, astrocytes and

endothelial cells in the brain (Marti et al., 1996). It seems that a

paracrine epo/ (epoR) 2 systems exists in the central nervous system

independent of the endocrine system of adult erythropoiesis. In recent

years, epo was found to have multiple functions outside of the bone

marrow. Erythropoietin acts as an antiapoptotic and tissue-protective

cytokine in multiple in vitro and in vivo studies.

Two years later, the DNA sequence of this unidentified protein

could be identified by expression cloning from a pXM expression

library made from murine erythroleukemia cells (D'Andrea et al.,

1989). In 1990, the human erythropoietin receptor was finally cloned

from an erythroleukemia line (OCIM1) and from fetal liver (Jones et al.,

1990). On a genomic level the human epo R gene has a size of 6 kb,

contains 8 exons and is located on chromosome 19 (11,348,883 to

11,356,019 from pter). The corresponding glycoprotein has a size of

approximately 66 kDa and belongs to the type I cytokine receptor

family. In vitro it was shown that cortical neurons were protected by

epo against neurotoxic events such as NMDA (N-methyl-D-aspartate)

or nitric oxide (Digicaylioglu et al., 2001) and ischemic events such as

oxygen and glucose deprivation (Sinor et al., 2000). The oxygen and

glucose deprivation model (OGD-model) is an in vitro model of brain

ischemia. In medicine, ischemia is a restriction of blood supply,

6

resulting in damage or dysfunction of the affected tissue. The organs

most sensitive to inadequate blood supply are the heart, the kidneys

and the brain. The brain requires glucose and oxygen to maintain

neuronal metabolism and function. Hypoxia refers to inadequate

delivery of oxygen to the brain, and ischemia results from insufficient

cerebral blood flow. The consequences of cerebral ischemia depend on

the degree and the duration of reduced cerebral blood flow. Neurons

can tolerate ischemia for 30 - 60 minutes. If flow is not re-established to

the ischemic area, a cascade of metabolic processes ensues. The neurons

become depleted of ATP and switch to anaerobic glycolysis, a much

less efficient pathway. Lactate accumulates and the intracellular pH

decreases. Without an adequate supply of ATP, ion pumps in the

plasma membrane fail. The resulting influx of sodium, water and

calcium into the cell causes rapid swelling of neurons and glial cells.

Membrane depolarization also stimulates the massive release of the

amino acids glutamate and aspartate, both of which act as excitatory

neurotransmitters in the brain. Glutamate further activates sodium and

calcium channels in the neuronal cell membrane, namely the well-

characterized NMDA calcium channel. Excessive calcium influx causes

the disordered activation of a wide range of enzyme systems such as

proteases, lipases and nucleases. These enzymes and their metabolic

products, such as oxygen free radicals, damage cell membranes, genetic

material and structural proteins in the neurons, ultimately leading to

cell death (Dirnagl et al., 1999). In vivo models of ischemia, performed

in mouse, rat or gerbil, also revealed significant reduction in stroke

volumes after erythropoietin treatment (Bernaudin et al., 1999, Brines

et al., 2000; Morishita et al., 1997). This finding was independent from

7

the mode of application: intraventricularly in order to bypass the blood

brain barrier (BBB) or systemically. Although the BBB is considered

impermeable to large molecules, epo was demonstrated to cross the BBB

(Brines et al., 2000).

In addition to neuroprotective effects erythropoietin (epo) also

mediates robust cytoprotective effects in ischemic models of the retina

(Junk et al., 2002), the spinal cord (Celik et al., 2002), the heart

(Calvillo et al., 2003), the skin (Saray et al., 2003) and the intestine

(Akisu et al., 2001). Furthermore, epo promotes endothelial cell

proliferation and angiogenesis (Anagnostou et al., 1990; Anagnostou et al.,

1994) and has immunomodulatory effects on B-cells and T-cells (Imiela

et al., 1993; Katz et al., 2007).

Although endogenous brain epo is crucial for neuronal

survival in mild ischemia (Sakanaka et al., 1998), it is not sufficient to

significantly reduce brain injury after stroke. Therefore, exogenous

administration of epo has been considered. Promising results were

obtained in animal models, since treatment of mice with exogenous epo

reduced brain damage after hypoxia (Marti et al., 2004). In a pilot

double-blind clinical trial using recombinant erythropoietin (repo.hu),

Ehrenreich suggested that the protection may also apply to human

stroke patients (Ehrenreich et al., 2002). As erythropoietin is a general

cytoprotective factor, its clinical application is not restricted to ischemia

but might be considered for the treatment of chronic diseases associated

with neuronal or cellular degeneration such as Parkinson‟s or

Alzheimer‟s disease or for psychiatric disorders where

neurodegenerative processes are likely to contribute to the

pathophysiology of the disease. Actually, clinical trials for a psychiatric

8

disorder, namely schizophrenia, are currently underway (Ehrenreich et

al., 2007). One major drawback of erythropoietin in the treatment of

chronic diseases is its hematopoietic property, leading to undesired

rheological properties of the blood at chronic dosing. In the last year on

uncoupling the hematopoietic potential of epo from its neuro- and cyto-

protective characteristics. A first success was the invention of asialo-

erythropoietin (asialoepo) but it‟s very short plasma half-life made in

vivo applications inefficient (Erbayraktar et al., 2003). In 2004, a

carbamylated non-hematopoietic epo-derivative (Cepo) was published

by Leist et al., having preserved neuroprotective functions and a

normal plasma half-life. Leist showed, in a rat cerebral infarct model,

that Cepo had comparable tissue- protection capacities at equal doses as

reported for epo. Analogous to epo, Cepo was found to be

cardioprotective in a rat ischemia-reperfusion model (Fiordaliso et al.,

2005), kidney-protective in a rat ischemia-reperfusion model (Imamura

et al., 2007) and ameliorated disease and neuroinflammation in a rat

model of experimental autoimmune encephalomyelitis (Savino et al.,

2006).

The fundamental question is how neuroprotective actions can

be independent from erythropoietic actions in epo derivatives. The

nonerythropoietic characteristic of asialoepo, having only a short

plasma half-life, is based on the fact that formation of red blood cells

requires the continuous presence of epo, whereas a brief exposure is

sufficient for neuroprotection in vitro (Erbayraktar et al., 2003).

However, it is also conceivable that epo derivatives do not mediate

their neuroprotective activities via the classical erythropoietin receptor

(epoR) 2 as suggested for Cepo (Brines et al., 2004). Brines et al.

9

proposed a heterodimeric receptor complex as non-hematopoietic

receptor mediating epo protection, consisting of one epo R chain and

the beta common chain, the signaling subunit of many receptors such

as the granulocyte macrophage colony-stimulating factor (GM-CSF)

receptor, the interleukin-3 (IL-3) receptor and the IL-5 receptor.

Keeping in view the above facts the present study was envisaged with the

following objectives:

1. To clone the erythropoietin gene in replicase based eukaryotic

vector.

2. To study the expression of the recombinant plasmid in cell culture.

3. To analyze the recombinant plasmid using restriction enzymes.

10

Chapter-2

Review of Literature 2.1 Biology of erythropoietin

2.2 Recombinant erythropoietin

2.3 Biochemistry

2.4 Metabolism

2.5 Regulation

2.6 Cloning

2.7 Purification

2.8 Expression

Chapter-2

2. Review of Literature 2.1 Biology of erythropoietin

The erythropoietic response of snakes was examined after

injecting human urinary erythropoietin (epo), testosterone propionate

(TP), and l-thyroxine (T4), separately and in combinations, into starved

ophids. The effect of starvation was reflected by a decrease in the

number of erythrocytes, a fall in hemoglobin concentration, and a

decline in hematocrit. Statistically significant elevation of erythrocyte

number, hemoglobin concentration, and hematocrit was observed at 24

hr following the administration of epo + T4, and epo + TP + T4 into

starved ophids. The erythrocyte number was also increased by T4

treatment at 24 hr. Furthermore, while T4 and epo individually

increased the red blood cell number at 168 hr, T4, TP + T4, and epo +

TP + T4 elevated the hemoglobin concentration and epo + T4 and epo +

TP + T4 increased the hematocrit value. It is suggested that the

influence of any one of the hormones utilized in the previous study on

blood morphology of fasted snakes depends to a greater extent on the

presence or absence of the other hormone(s) (Pati and Thapliyal,

1984).

In 1977 small amounts of human erythropoietin from the

urine of patients with aplastic anemia were purified. Based on the

11

limited peptide sequence information obtained from this purified

material, the gene for human erythropoietin was then isolated and

cloned in 1983 (Lin et al.,1985) .

Another possibility for the anemia treatment is derived from a

cDNA encoding fusion protein of two complete human

erythropoietin domains linked by a 17-aminoacid flexible peptide. It

seems that a single subcutaneous dose of epo-epo fusion proteins

resulted in a significant increase in hematocrit within seven days,

whereas administration of an equivalent dose of conventional

recombinant epo did not produce any effect. Fusion proteins of epo with

hematopoietic growth factors have also been described (Weich et al., 1993;

Coscarella et al., 1997).

Erythropoietin (epo) controls the proliferation,

differentiation and survival of the erythroid progenitor cells. This

erythropoietin cytokine was cloned in 1985 and rapidly became used

for treatment of anemia of renal failure, opening the way to the first

clinical trials of a hematopoietic growth factor. The clonage of one

chain of the epo receptor followed in 1989, thereby opening the

research on intracellular signal transduction induced by epo. epo is

synthesized mainly by the kidney and the liver and sequences

required for tissue-specific expression have been localized in the epo

gene. A 3‟enhancer is responsible for hypoxia inducible epo gene

expression. HIF-1 a and b proteins bind to this enhancer. Gene

regulation by hypoxia is wide spread in many cells and involves

numerous genes in addition to the epo gene. The epo receptor belongs

to the cytokine receptor family and includes a p66 chain which is

dimerized upon epo activation; two accessory proteins defined by

12

cross-linking remain to be characterized. epo binding induces the

stimulation of Jak2 tyrosine kinase. Jak2 activation leads to the tyrosine

phosphorylation of several proteins including the epo receptor itself.

As a result, different intracellular pathways are activated: Ras/MAP

kinase, phosphatidylinositol 3-kinase and STAT transcription factors.

However, the exact mechanisms by which the proliferation and/or the

differentiation of erythroid cells are regulated after epo stimulation are

not known. Furthermore, target disruption of both epo and epo

receptor showed that epo was not involved in the commitment of the

erythroid lineage and seemed to act mainly as a survival factor

(Lacombe and Mayeux, 1998).

The epo-mimetics are small molecules capable of dimerizing

the epo receptor and act in the same way as epo. There are two groups

of epo-mimetics, the peptides and the nonpeptides. epo-mimetic

peptides were obtained from screening random peptide-phage

libraries in the search for an agonist peptide (Barbone et al., 1999;

Biazzo et al., 2000) .

The expiration of patents for epoetins, new versions of these

products and generics appeared in the market. Darbepoetin alpha was

created using site-directed mutagenesis to insert an additional two

additional N-linked glycosylation chains into the protein (at Asn-30 and

Asn-88). The strategy required the substitution of a total of five

aminoacids (Egrie et al., 2001).

Most of these molecules possess shorter in vivo half-lifes

than epo. However, Hematide, an epo-mimetic peptide attached to

13

polyethylene glycol, has a long circulating half-life and extended

duration of erythropoietic effect (Nissenson et al., 2002).

Human erythropoietin (epo.hu) is a glycoprotein produced

in response to the oxygen tension of the blood. It is mainly produced

by the peritubular fibroblast-like cells located in the cortex of the

kidney in adults and by hepatocytes during the fetal stage. epo

circulates to the bone morrow where it stimulates proliferation and

differentiation of the red blood cell progenitors, leading to more red

blood cells and increased oxygen-carrying capacity. epo is a

pleiotropic hormone. In addition to the kidney also liver, spleen, lung,

bone marrow and brain were shown to express epo mRNA (Jelkmann,

2004).

Global use of erythropoietin (epo) continues to increase as a

proven agent for the treatment of anemia. Yet, epo is no longer

believed to have exclusive biological activity in the hematopoietic

system and is now considered applicable for a variety of disorders

such as diabetes, Alzheimer‟s disease, and cardiovascular disease.

Treatment with epo is considered to be robust and can prevent

metabolic compromise, neuronal and vascular degeneration, and

inflammatory cell activation. On the converse side, observations that

epo administration is not without risk have fueled controversy. Here

they present recent advances that have elucidated a number of novel

cellular pathways governed by epo to open new therapeutic avenues

for this agent and avert its potential deleterious effects (Kenneth

Maiese et al., 2008).

14

Spinal cord injury (SCI) is a devastating condition for

individual patients and costly for health care systems requiring

significant long-term expenditures. Cytokine erythropoietin (epo) is a

glycoprotein mediating cytoprotection in a variety of tissues, including

spinal cord, through activation of multiple signaling pathways. It has

been reported that epo exerts its beneficial effects by apoptosis

blockage, reduction of inflammation, and restoration of vascular

integrity. Neuronal regeneration has been also suggested. In the past

review, the path physiology of SCI and the properties of endogenous

or exogenously administered epo are briefly described. Moreover, an

attempt to present the current traumatic, ischemic and inflammatory

animal models that mimic SCI is made. Currently, a clearly effective

pharmacological treatment is lacking. It is highlighted that

administration of epo or other recently generated epo analogues such

as asialo-epo and carbamylated- epo demonstrate exceptional

preclinical characteristics, rendering the evaluation of these tissue-

protective agents imperative in human clinical trials (Matis et al.,

2008).

Researchers have identified a gene called erythropoietin (epo)

that was linked to higher risk of severe retinopathy and nephropathy,

eye and kidney diseases that often affect diabetic patients. Diabetic

retinopathy (PDR) was the most common cause of legal blindness in

working-aged adults in the United States, accounting for 10% of new

cases of blindness. Diabetes was also the leading cause of kidney

disease, called end-stage renal disease (ESRD), in the U.S. and the

Western world. While researchers have known that these conditions in

15

diabetic patients can be hereditary, the actual genes involved have

been relatively unknown until now Researchers discovered the

involvement of the epo gene in a study of 1,618 people with diabetic

retinopathy and end-stage renal disease, and 954 diabetes patients

without any eye or kidney disease in three separate populations. Their

studied demonstrated that if a person has a copy of the mutant epo

gene, they have an increased risk of developing PDR and ESRD during

their lifetime. According to Dr. Dean Li from the Program in Human

Molecular Biology and Genetics at the University of Utah, while there

was no proven pharmacologic treatment for diabetic vascular eye

diseases, "inhibiting the growth of unwanted blood vessel growth

using antibodies directed against vascular endothelial growth factor

(anti-VEGF therapy) has been advocated. This genetic study suggests

that future therapeutic strategies need to consider blunting the effects

of erythropoietin in addition or as an alternative to an anti-VEGF

strategy (Featured Columns, 2008).

Abhary et al., (2010) determined whether sequence variation

in the erythropoietin gene (epo) was associated with the development

of diabetic retinopathy (DR).This was a multicenter study based on 518

subjects with long-standing diabetes mellitus (DM), 173 with type 1

DM (T1DM) and 345 with type 2 DM (T2DM). Study groups consisted

of 233 control subjects with no DR, 155 subjects with no proliferative

DR, 126 with proliferative DR, and 90 with clinically significant

macular edema. Subjects with end-stage renal disease were excluded.

DNA extracted from blood of each subject was genotyped for 3 epo

single-nucleotide polymorphisms (SNPs). All 3 SNPs in epo were

16

associated with overall DR status in the combined T1DM and T2DM

and T2DM alone groups (CC genotype of rs507392, P < .008; GG

genotype of rs1617640, P < .008; and CC genotype of rs551238, P <

.008) in the multivariate analysis. The GCC heliotype was also

associated with overall DR status in the combined DM and T2DM

alone groups (P = .008) by multivariate analysis. All SNPs and the

GCC heliotype were also associated with proliferative DR and

clinically significant macular edema in the combined DM and T2DM

alone groups. No associations were found with T1DM alone. Sequence

variation in epo is associated with the risk of DR independent of

duration of DM, degree of glycolic control, and

nephropathy. Identifying epo genetic markers for high risk of

developing DR could lead to the possibility of developing novel

treatments or preventive therapies.

Erythropoietin is a growth factor commonly used to manage

anemia in patients with chronic kidney disease. A significant clinical

challenge is relative resistance to erythropoietin, which leads to use of

successively higher erythropoietin doses, failure to achieve target

hemoglobin levels, and increased risk of adverse outcomes.

Erythropoietin acts through the erythropoietin receptor (epoR) present in

erythroblasts. Alternative mRNA splicing produces a soluble form of

epoR (sepoR) found in human blood, however its role in anemia is not

known (Khankin et al., 2010).

In newborn infants, the number of red blood cells in the

circulation decreases after birth. In infants born before term, this

decrease is exaggerated by frequent withdrawal of blood, which may

17

be necessary to monitor the infant‟s clinical condition. Therefore,

infants born before term are likely to require transfusions of red blood

cells. Low levels of erythropoietin (epo), a substance in the blood that

stimulates red blood cell production, in preterm infants provide a

rationale for the use of epo to prevent or treat anemia. epo can be

given “early” (before the infant reaches eight days of age) in order to

prevent or decrease the use of red blood cell transfusions. More than

2200 infants born before term have been enrolled in 27 studies that

used this approach. Early epo treatment reduces the number of red

blood cell transfusions and donor exposures following its use.

However, the overall benefit of epo may not be clinically important, as

many of these infants had been exposed to red blood cell transfusions

prior to entry into the trials. Treatment with early epo did not have

any important effects on mortality or common complications of

preterm birth with the exception that epo increased the risk for

retinopathy of prematurity, a serious complication that may cause

blindness in babies born before term. The addition of four new studies

enrolling 145 infants did not change the conclusions. Based on their

findings, epo is not recommended for routine use in preterm infants

(Ohlsson et al., 2010)

Over the past few decades, understanding of the physiologic

function of erythropoietin (epo) has evolved significantly. epo binds to

erythropoietin receptors (epoR), initiating signaling that stimulates

growth, inhibits apoptosis, and induces the differentiation of erythroid

progenitors to increase red blood cell mass. epo has additionally been

shown to exert tissue-protective effects on multiple tissues, suggesting

18

a pleiotropic mechanism of action. Erythropoiesis-stimulating agents

(ESA) are used clinically for treating cancer-related anemia

[chemotherapy-induced anemia (CIA)]. Clinical trials have reported

increased adverse events and/or reduced survival in ESA-treated

cancer patients receiving chemotherapy, potentially related to epo-

induced cancer progression. Signaling pathways downstream of

epo/epoR have been shown to influence numerous cellular functions

in both normal and tumor cells, including proliferation, apoptosis, and

drug resistance. Some studies have reported effects on proliferation,

reduced chemotherapy efficacy, reduction of apoptosis, and resistance

to selective therapies on cancer cell lines, whereas others have shown

null effects. In addition, newer targeted cancer therapies that are

directed toward specific signaling pathways may be antagonized by

ESAs. This molecular interplay between anticancer agents and

potential survival signals triggered by ESAs may have been

underestimated and may contribute toward decreased survival seen in

certain trials. As more targeted anticancer therapies become available,

these types of interactions may mitigate therapeutic efficacy by

allowing tumor cells to acquire drug resistance (Benjamin et. al.,

2011).

Periventricular leukomalacia (PVL) is the predominant

pathology in premature infants, characterized by prominent cerebral

white matter injury, and commonly caused by hypoxia-ischemia and

inflammation. Activated microglia trigger white matter damage and

play a major role in the development of PVL. Erythropoietin (epo) and

its derivative carbamylated erythropoietin (Cepo) have been shown to

19

be neuroprotective in several brain disease models. Here they

investigated whether epo and Cepo could provide protection in mouse

models of PVL induced by hypoxia-ischemia or hypoxia-ischemia-

inflammation. They administered epo or Cepo to mice with PVL, and

found that both epo and Cepo treatments decreased microglia

activation, oligodendrocyte damage and myelin depletion. They also

noted improved performance in neurological function assays.

Inhibited disease progression in PVL mice by epo or Cepo treatment

was associated with decreased poly-(ADP-ribose) polymerase-1

(PARP-1) activity. PARP-1 activity was increased dramatically in

activated microglia in untreated mice with PVL. Furthermore, they

demonstrated that the neuroprotective properties of epo and Cepo

were diminished after PARP-1 gene depletion. The therapeutic doses

of epo and Cepo used in that study did not interfere with normal

oligodendrocyte maturation and myelination. Together, their data

demonstrate that epo and Cepo are neuroprotective in cerebral white

matter injury via a novel microglial PARP-1 dependent mechanism,

and hold promise as a future treatment for PVL and other hypoxic-

ischemic/inflammatory white matter diseases (Liu et al., 2011).

20

2.2 Recombinant erythropoietin

Anemia due to impaired erythropoietin (epo) production is

associated with kidney failure. Recombinant proteins are commonly

administered to alleviate the symptoms of this dysfunction, whereas

gene therapy approaches envisaging the delivery of epo genes have

been tried in animal models in order to achieve stable and long-lasting

epo protein production. Naked DNA intramuscular injection is a safe

approach for gene delivery; however, transduction levels show high

inter-individual variability in rodents and very poor efficiency in non-

human primates. Transduction can be improved in several animal

models by application of electric pulses after DNA injection (Fattori et

al., 2005).

Recombinant human erythropoietin has been produced using

different mammalian cell lines as Chinese Hamster Ovary (CHO) cells,

Baby Hamster Kidney (BHK) cells or even human cells (HT1080). All

this recombinant epos have the same amino acid sequence but the

different preparations show differences in their degree of glycosilation

as well as in their glycan composition and/or structure due to

differences on the cell lines used to express the proteins and the

purification strategies used. Different formulations of recombinant epo

have been developed both in academia and by pharmaceutical

industries (Restelli et al., 2006; Montesino et al., 2008).

The terminal half-life of i.v. administered Darbepoetin-alpha

is three- to fourfold longer than that Epoetin-alpha and -beta (25.3 h vs

8.5 h), thus, affecting the biochemical and biological properties of

21

NESP Synthetic erythropoiesis protein is another erythropoietic

polymer. Using solid phase peptide synthesis and branched precision

polymer constructs, a 51 kDa protein-polymer construct has been

made containing a 166-amino-acids polypeptide chain (similar to the

sequence of epo) and two covalently attached polymer moieties. The

resulting polymer stimulates erythropoiesis through activation of the

erythropoietin receptor. It was reported that SEP had superior

duration of action in vivo and a longer circulation lifetime than epo

(Fishbane et al., 2007).

The administration of recombinant human erythropoietin

(repo.hu) and its analogues provides enormous benefit in the

prevention and reversal of anemia in chronic kidney disease (CKD),

malignancy and AIDS, and it supports autologous blood collection

(Jelkmann, 2007).

The growth factors erythropoietin and granulocyte-colony

stimulating factor have hematopoietic and non-hematopoietic

functions. Both are used clinically in their recombinant forms. Both also

have interesting tissue-protective effects in other organs, which are

unrelated to their hematopoietic functions. They have clinical

hematopoietic uses in neonatal populations and in experimental

nonhematopoietic research, and clinical potential as neuroprotective or

tissue-protective agents (Juul et al., 2007).

22

Recombinant human erythropoietin (repo.hu) has

revolutionized the treatment of anemia; recent clinical trials suggested

that repo.hu use may be associated with decreased survival in cancer

patients. Although the expression of erythropoietin (epo) receptor

(epoR) has been demonstrated in various human cancers, the effect of

exogenous epo on the growth and therapy resistance of epoR-bearing

tumor cells is unclear at present. In the previous study, they examined

the hypothesis that epoR may contribute to tumor growth

independent of epo in A2780 human ovarian carcinoma cells. A2780

human ovarian carcinoma cells showed high levels of epoR

expression, but lacked expression of epo mRNA and biologically

active epo protein under both normoxic and hypoxic conditions.

Exogenous epo did not stimulate epoR-mediated signaling,

proliferation, invasiveness, or resistance to cytotoxic drugs in A2780

cells. In contrast, specific inhibition of epoR expression using a short

hairpin RNA (shRNA) expression plasmid resulted in markedly

reduced proliferation and invasiveness in vitro. In addition, inhibition

of epoR expression led to abrogated in vivo ovarian cancer cell growth

in a tumor xenograft system and resulted in decreased epoR

signaling.Their findings suggest that epoR may be constitutively

active in some cancer cells in the absence of epo and provide the first

evidence for a potential role of an epo- independent, epoR-mediated

pathway in the growth of some human cancers (Paragh et al., 2008).

23

Evaluation of the pharmacokinetics (PKs) in a proper

physiological context is paramount to elucidate the factors that may

improve a drug‟s PK properties. Using modern system analysis-based

physiological modelling principles, that work applies a novel kinetic

analysis framework to a PK comparison of two erythropoietically

active drugs, C.E.R.A. (continuous erythropoietin receptor activator)

and recombinant human erythropoietin (epo), aimed at elucidating

the main factors responsible for the substantial PK differences seen.

The evaluation according to the new model is compared with a

compartmental model analysis. Sheep (n = 7 for epo; n = 8 for

C.E.R.A.) received intravenous bolus injections of epo and C.E.R.A.

Baseline and 20-30 blood samples per injection were assayed by

radioimmunoassay. Fundamental physiologically based PK building

block principles were introduced, proceeding to the construction of a

general PK model and several sub-models from which a final PK

model was selected based on information theoretical principles. The

compartmental comparison analysis uses a two-compartment model

with central Michaelis-Menten elimination. Several lines of evidence

support the hypothesis that the desirable slow elimination of C.E.R.A.

relative to epo is mainly caused by a smaller recirculation extraction

fraction, which appears more influential on the elimination kinetics

than the mean circulation transit time. The compartmental analysis

demonstrates large differences in several PK parameters that

contribute to C.E.R.A.‟s slower elimination, consistent with the

recirculation model analysis. It is hypothesized that C.E.R.A.‟s smaller

recirculatory extraction fraction is due to a reduced receptor-mediated

elimination, consistent with in-vitro measurements where C.E.R.A.

24

shows epo-receptor binding with a lower association constant and a

larger dissociation constant (Pedersen et al., 2008).

Anemia is the most common complication of inflammatory

bowel disease (IBD). Control and inadequate treatment leads to a

worse quality of life and increased morbidity and hospitalization.

Blood loss, and to a lesser extent, malabsorption of iron are the main

causes of iron deficiency in IBD. There is also a variable component of

anemia related to chronic inflammation. The anemia of chronic renal

failure has been treated for many years with recombinant human

erythropoietin (repo.hu), which significantly improves quality of life

and survival. Subsequently, repo.hu has been used progressively in

other conditions that occur with anemia of chronic processes such as

cancer, rheumatoid arthritis or IBD, and anemia associated with the

treatment of hepatitis C virus. Erythropoietic agents complete the

range of available therapeutic options for treatment of anemia

associated with IBD, which begins by treating the basis of the

inflammatory disease, along with intravenous iron therapy as first

choice. In cases of resistance to treatment with iron, combined therapy

with erythropoietic agents aims to achieve near-normal levels of

hemoglobin/hematocrit (11-12 g/dL). New formulations of

intravenous iron (iron carboxymaltose) and the new generation of

erythropoietic agents (darbepoetin and continuous erythropoietin

receptor activator) will allow better dosing with the same efficacy and

safety ( López et al., 2009).

Multiple complications can ensue in the cardiovascular,

renal, and nervous systems during diabetes mellitus (DM). Given that

25

endothelial cells (ECs) are susceptible targets to elevated serum D-

glucose, identification of novel cellular mechanisms that can protect

ECs may foster the development of unique strategies for the

prevention and treatment of DM complications. Erythropoietin (epo)

represents one of these novel strategies but the dependence of epo

upon Wnt1 and its downstream signaling in a clinically relevant

model of DM with elevated D-glucose has not been elucidated. Here

they show that epo can not only maintain the integrity of EC

membranes, but also prevent apoptotic nuclear DNA degradation and

the externalization of membrane phosphatidylserine (PS) residues

during elevated D-glucose over a 48-hours period. epo modulates the

expression of Wnt1 and utilizes Wnt1 to confer EC protection during

elevated D-glucose exposure, since application of a Wnt1 neutralizing

antibody, treatment with the Wnt1 antagonist DKK-1, or gene

silencing of Wnt1 with Wnt1 siRNA transfection abrogates the

protective capability of epo. epo through a novel Wnt1 dependent

mechanism controls the post-translational phosphorylation of the

“pro-apoptotic” fork head member FoxO3a and blocks the trafficking

of FoxO3a to the cell nucleus to prevent apoptotic demise. epo also

employs the activation of protein kinase B (Akt1) to foster

phosphorylation of GSK-3β that appears required for epo vascular

protection. Through this inhibition of GSK-3β, epo maintains β-

catenin activity, allows the translocation of β-catenin from the EC

cytoplasm to the nucleus through a Wnt1 pathway, and requires β-

catenin for protection against elevated D-glucose since gene silencing

of β-catenin eliminates the ability of epo as well as Wnt1 to increase

EC survival. Subsequently, they show that epo requires modulation of

26

both Wnt1 and FoxO3a to oversee mitochondrial membrane

depolarization, cytochrome c release, and caspase activation during

elevated D-glucose (Chong et al., 2011).

Sullivan et al., (2011) developed a novel mutant form of

epo that is neuroprotective but no longer erythropoietic by altering a

single amino acid (arginine to glutamate at position 76, R76E). They

hypothesized that a single intramuscular injection of recombinant

adeno-associated virus carrying epoR76E (rAAV2/5.CMV.EpoR76E)

would protect retinal ganglion cells in a mouse model of glaucoma

without inducing polycythemia. This systemic treatment not only

protected the retinal ganglion cell somata located within the retina; it

also preserved axonal projections within the optic nerve, while

maintaining the hematocrit within normal limits. The rescued retinal

ganglion cells retained their visual function demonstrated by flash -

visual evoked potentials. To their knowledge, that was the first

demonstration of a therapy that protected neurons from death and

prevented loss of visual function from the slow neurodegenerative

effects of glaucoma. Because of its broad range of cellular targets, epo-

R76E is likely to be successful in treating other neurodegenerative

diseases as well.

Productivity and sialylation are two important factors for the

production of recombinant glycoproteins in mammalian cell culture.

In previous study, they found that silkworm hemolymph increased

the sialylation of recombinant secreted human placental alkaline

phosphatase in the insect cells, promoted the transfer of sialic acids

onto the glycoprotein oligosaccharides in an in vitro asialofetuin

27

sialylation system, and enhanced recombinant protein production in

the Chinese hamster ovary (CHO) cells. These beneficial effects were

mainly due to the 30K proteins, which consist of five isoforms.

Among the 30K proteins, 30Kc19 was determined to be the major

component. In that study, the 30Kc19 gene was introduced into a

CHO cell line producing recombinant human erythropoietin, and its

effects on productivity and sialylation were investigated. The

transient expression of 30Kc19 significantly improved the production

and sialylation of epo. A stable cell line containing 30Kc19 was also

established to investigate the effect of 30Kc19 gene expression. The

stable expression of 30Kc19 increased the production and sialylation

by 102.6% and 87.1%, respectively. The enhanced productivity from

30Kc19 expression is believed to occur because the 30Kc19 protein

suppresses the loss of mitochondrial membrane potential and

consequently improves the generation of intracellular ATP. In

addition, the positive effect of 30Kc19 expression on sialylation is

believed to be due to its ability to maintain sialyltransferase activity.

In conclusion, 30Kc19 expression is a novel approach to improve the

production and sialylation of recombinant glycoproteins in CHO cells

(Wang et al., 2011).

Recombinant human erythropoietin (repo.hu) is

arguably the most successful therapeutic application of recombinant

DNA technology till date. It was isolated in 1977 and the gene

decoded in 1985. Since then, it has found varied applications,

especially in stimulating erythropoiesis in anemia due to chronic

conditions like renal failure, myelodysplasia, infections like HIV, in

28

prematurity, and in reducing peri-operative blood transfusions. The

discovery of erythropoietin receptor (epo-R) and its presence in

nonerythroid cells has led to several areas of research. Various types of

repo.hu are commercially available today with different dosage

schedules and modes of delivery. Their efficacy in stimulating

erythropoiesis is dose dependent and differs according to the patient's

disease and nutritional status. epo should be used carefully according to

guidelines as unsolicited use can result in serious adverse effects. Because

of its capacity to improve oxygenation, it has been abused by athletes

participating in endurance sports and detecting this has proved to be a

challenge (John et al., 2012).

2.3 Biochemistry

The human epo gene is located on the long arm of

chromosome 7 (q11-q22). It contains five exons, which encode a 193

amino acid pro-hormone including a 27 aa signaling peptide, and four

introns. The 166-amino acid protein has a molecular weight of 19,398

Da. The resulting glycoproitein hormone has a molecular mass of 34

kDa. The peptide core of mature epo consists of a single 165 aa

polypeptide chain (the signaling peptide is cleaved prior to secretion

and the circulating human epo lacks the carboxy-terminal arginine). It

has two disulfide bonds (Cys-7 - Cys-161 and Cys-29 - Cys-33) and

four glycosylation sites that provide three N-linked (Asn-24, 38, 83)

and one O-linked (Ser-126) oligosaccharide chains. The resulting

carbohydrate content accounts for roughly 40 % of the total molecular

mass of the erythropoietin glycoprotein. The N-glycosilation is

essential for the in vivo biological activity of epo, especially, the

29

terminal sialic acid residues of these glycans .When these residues are

removed from epo (e.g. with sialidase), the resulting molecules have an

increased activity in vitro, but less activity in vivo, presumably due to

removal from circulating by the asialo-glycoprotein receptor in the liver.

When these residues are removed from epo (e.g.with sialidase), the

resulting molecules have an increased activity in vitro but less activity

in vivo, presumably due to removal from circulating by the

asialo-glycoprotein receptor in liver ( Briggs et al., 1974;Dordal et. al.,

1985; Weikert et. al., 1999; Toledo et. al., 2005).

Banks et al., (2009) described that the first kinetic folding

studies of erythropoietin, a glycosylated four-helical bundle cytokine

responsible for the regulation of red blood cell production. Kinetic

responses for folding and unfolding reactions initiated by manual

mixing were monitored by far-ultraviolet circular dichroism and

fluorescence spectroscopy, and folding reactions initiated by stopped-

flow mixing were monitored by fluorescence. The urea concentration

dependence of the observed kinetics were best described by a three-

state model with a transiently populated intermediate species that is

on-pathway and obligatory. This folding scheme was further

supported by the excellent agreement between the free energy of

unfolding and m-value calculated from the microscopic rate constants

derived from this model and these parameters determined from

separate equilibrium unfolding experiments. Compared to the

kinetics of other members of the four-helical bundle cytokine family,

erythropoietin folding and unfolding reactions were slower and less

susceptible to aggregation. They tentatively attribute these slower

30

rates and protection from association events to the large amount of

carbohydrate attached to erythropoietin at four sites.

2.4 Metabolism

epo is distributed largely intravascular and it is cleared from

circulation with a fairly short half-life. However, the mechanisms

responsible for clearance of epo from the circulation are still under

investigation. Different studies suggested that to a minor degree, epo

may be cleared by the kidneys following glomerular filtration (by the

galactose receptor), once it is desialylated by action of tissue and blood

sialidases in the liver (Jelkmann et al., 2002). However, there is

evidence to assume that epo is mainly removed from circulation by

uptake into erythrocytic and other cells possessing the epo receptor.

The glycoprotein hormone erythropoietin (epo) is an essential viability

and growth factor for the erythrocytic progenitors. epo is mainly

produced in the kidneys. epo gene expression is induced by hypoxia-

inducible transcription factors (HIF). The principal representative of

the HIF-family (HIF-1, -2 and -3) is HIF-1, which is composed of an O2-

labile alpha-subunit and a constant nuclear beta-subunit. In normoxia,

the alpha-subunit of HIF is inactivated following prolyl- and

asparaginyl-hydroxylation by means of alpha-oxoglutarate and Fe(2+)-

dependent HIF specific dioxygenases. While HIF-1 and HIF-2 activate

the epo gene, HIF-3, GATA-2 and NFkappaB are likely inhibitors of

epo gene transcription. epo signaling involves tyrosine

phosphorylation of the homodimeric epo receptor and subsequent

activation of intracellular antiapoptotic proteins, kinases and

transcription factors. Lack of epo leads to anemia. Treatment with

31

recombinant human epo (repo.hu) is efficient and safe in improving the

management of the anemia associated with chronic renal failure. repo.hu

analogues with prolonged survival in circulation have been developed

(Jelkmann, 2004).

Hematopoietic stem cells (HSC), which are responsible for

maintaining continuous pool of blood cells, are being used for bone

marrow transplantation (BMT). However, the programmed cell death/

apoptosis pose a serious problem for their optimum proliferation and

differentiation after radio- and/ or chemotherapy. The role of Bcl-2 (B

Cell Lymphoma) protein, a Bcl-2 family member, is well established in

suppressing apoptosis of HSC on irradiation and serum withdrawal.

The anti-apoptotic activity of Bcl-2 is regulated by inter- and intra-

family homo-/ heterodimerization. Here they were proposing that the

potential of Bcl-2 and its survival enhancing mutants, such as D34A

and S70E, may be harnessed (gene therapy) to suppress the radiation

and growth factor withdrawal induced apoptosis provided the

neoplastic outcomes of these genes are regulated. The suggested

hypothetical model is likely to be helpful in treating blood borne

disorders and radiation injury through BMT (Gangenahalli et al.,

2006).

Differentiating erythroid cells execute a unique gene

expression program that insures synthesis of the appropriate proteome

at each stage of maturation. Standard expression microarrays provide

important insight into erythroid gene expression but cannot detect

qualitative changes in transcript structure, mediated by RNA

32

processing, that alter structure and function of encoded proteins. They

analyzed stage-specific changes in the late erythroid transcriptome via

use of high-resolution microarrays that detect altered expression of

individual exons. Ten differentiations- associated changes in

erythroblast splicing patterns were identified, including the previously

known activation of protein 4.1R exon 16 splicing. Six new alternatives

splicing switches involving enhanced inclusion of internal cassette

exons were discovered, as well as 3 changes in use of alternative first

exons. All of these erythroid stage-specific splicing events represent

activated inclusion of authentic annotated exons, suggesting they

represent an active regulatory process rather than a general loss of

splicing fidelity. The observation that 3 of the regulated transcripts

encode RNA binding proteins (SNRP70, HNRPLL, MBNL2) may

indicate significant changes in the RNA processing machinery of late

erythroblasts. Together, these results support the existence of a

regulated alternative pre-mRNA splicing program that is critical for late

erythroid differentiation (Yamamoto et al., 2008).

2.5 Regulation

They examined regulation of the human erythropoietin

(epo) gene through the GATA sequence in the epo promoter, and

demonstrated that Hep3B and HepG2 cells express human GATA-2

(hGATA-2) mRNA and protein. Nuclear extracts of QT6 cells

transfected with hGATA-1, -2 or -3 transcription factors revealed

specific binding to the GATA element in the human epo gene

promoter by gel mobility shift assay. Transient transfection of Hep3B

cells with hGATA-1, -2 or -3 demonstrated that each of these

33

transcription factors significantly decreased the level of expression of epo

mRNA as assessed by a competitive polymerase chain reaction.

Furthermore, transient transfection of Hep3B cells with hGATA-1, -2 and

-3 and an epo reporter gene construct showed significant inhibition

of the epo promoter. They concluded that the hGATA-1, -2 and -3

transcription factors specifically bind to the GATA element in the human

epo gene promoter and negatively regulate epo gene expression.

(Imagawa et al., 1996)

Tissue hypoxia is the main stimulus of epo production and

secretion. epo is not only produced when oxygen capacity of the blood

decreases (hypoxia), but also when arterial pO2 decreases or when the

oxygen affinity of the blood increases. In most tissues, including

kidney, liver, uterus and other organs like brain, the epo gene

expression is induced by hypoxia-inducible transcription factors

(HIFs). The principal representative of the HIF-family is HIF-1, a

heterodimeric protein composed of an alpha subunit (HIF-1alpha, 120

kDa) and a beta subunit (HIF-1Beta, 91-94 kDa) that is activated by a

variety of stressors, including hypoxia (Maiese et al., 2004). However

there are other transcription factors which can modulate epo gene

transcription. epos-receptor binding induces a conformational change

and a tighter connection of the two receptor molecules (Maiese et

al.,2005). As a result, two Janus kinase 2 (JAK2) tyrosine kinase, which

are in contact with the cytoplasmic region of the epo receptor, are

activated. Then, several tyrosine residues of the epo receptor are

phosphorylated and exhibit docking sites for signalling proteins

containing SRC homology 2 (SH2) domains. As a result, several signal

transduction pathways are channelled, including phosphatidyl-inositol

34

3-kinasa (PI-3K/Akt), JAK2, STAT 5, MAP kinases and protein kinase

C. However, the specific roles of the various enzymes and

transcriptional cofactors are only beginning to be understood. The

effect of epo is terminated by the action of the hemopoietic cell

phosphatase (HCP) which catalyses JAK2 de-phosphorylation.

Erythropoietin (epo) was the primary regulator of

erythropoiesis, controlling the proliferation, maturation, and survival

of erythroid progenitor cells. The functions of epo were mediated

through its specific receptor (epoR) expressed mainly on the surface of

erythroid progenitor cells, and the expression of both responds to

hypoxia. The subterranean mole rat (Spalax) was a unique model

system to study the molecular mechanisms for adaptation to hypoxia.

Here, they cloned two forms of Spalax epoR: a completed epoR cDNA

as well as a novel truncated bone marrow specific epoR form. In the

full-length Spalax epoR (sepoR), two out of the eight conserved

tyrosine- phosphorylation sites were substituted (Y481F and Y499G),

suggesting that Spalax Epo signaling pathways may be modulated.

The level of the sepoR mRNA in the spleen and in bone marrow was

relatively low and similar in Spalax newborns and adults, with no

significant response to hypoxia. The truncated sepoR was not detected

in the spleen and comprised only approximately 1% of the sepoR

expressed in the bone marrow. In Rattus, the truncated epoR form was

approximately 15% of the total expressed receptor. The level of Rattus

epoR in newborn spleens was three- to fourfold higher than in Spalax

newborns and decreased toward adulthood. Severe hypoxia induces a

significant increase in adult Rattus epoR. There data provided further

insight into the adaptive mechanisms of Spalax to the extreme

35

conditions of hypoxia in its subterranean environment (Savino et al.,

2005).

Baek et al., (2011) allowed that Hypoxia inducible factor

(HIF-1α) is a master regulator of tissue adaptive responses to hypoxia

whose stability is controlled by an iron containing prolyl hydroxylase

domain (PHD) protein. A catalytic redox cycle in the PHD's iron center

that results in the formation of a ferryl (Fe(+4)) intermediate has been

reported to be responsible for the hydroxylation and subsequent

degradation of HIF-1α under normoxia. They showed that induction of

HIF-1α in rat kidneys can be achieved by iron reduction by the

hydroxypyridin-4 one (CP94), an iron chelator administered

intraperitoneally in rats. The extent of HIF protein stabilization as well

as the expression of HIF target genes, including erythropoietin (epo),

in kidney tissues was comparable to those induced by known

inhibitors of the PHD enzyme, such as desferrioxamine (DFO) and

cobalt chloride (CoCl(2)). In human kidney cells and in vitro PHD

activity assay, they observed that the HIF-1α protein can be stabilized

by addition of CP94. This appears to inactivate PHD; and thus

prevents the hydroxylation of HIF-1α. In conclusion, they identified

the inhibition of iron-binding pocket of PHD as an underlying

mechanism of HIF induction in vivo and in vitro by a bidentate

hydroxypyridinone.

The novel multifunctional brain permeable iron, chelator M30

[5-(N-methyl-N-propargyaminomethyl)-8-hydroxyquinoline] was

shown to possess neuroprotective activities in vitro and in vivo,

against several insults applicable to various neurodegenerative

36

diseases, such as Alzheimer's disease, Parkinson's disease, and

amyotrophic lateral sclerosis. In the previous study, they

demonstrated that systemic chronic administration of M30 resulted in

up-regulation of hypoxia-inducible factor (HIF)-1α protein levels in

various brain regions (e.g. cortex, striatum, and hippocampus) and

spinal cord of adult mice. Real-time RT-PCR revealed that M30

differentially induced HIF-1α-dependent target genes, including

vascular endothelial growth factor (VEGF), erythropoietin (epo),

enolase-1, transferrin receptor (TfR), heme oxygenase-1 (HO-1),

inducible nitric oxide synthase (iNOS), and glucose transporter

(GLUT)-1. In addition, mRNA expression levels of the growth factors,

brain-derived neurotrophic factor (BDNF) and glial cell-derived

neurotrophic factor (GDNF) and three antioxidant enzymes (catalase,

superoxide dismutase (SOD)-1, and glutathione peroxidase (GPx))

were up-regulated by M30 treatment in a brain-region-dependent

manner. Signal transduction immunoblotting studies revealed that

M30 induced a differential enhanced phosphorylation of protein

kinase C (PKC), mitogen-activated protein kinase (MAPK)/ERK kinase

(MEK), protein kinase B (PKB/Akt), and glycogen synthase kinase-3β

(GSK-3β). Together, these results suggest that the multifunctional iron

chelator M30 can up-regulate a number of neuroprotective-adaptive

mechanisms and pro-survival signaling pathways in the brain that

might function as important therapeutic targets for the drug in the

context of neurodegenerative disease therapy (Kupershmidt et al.,

2011).

37

Polycythaemia has been reported rarely as a familial

condition. There is evidence to suggest transmission as a Mendelian

dominant trait, but recessive inheritance has also been described. They

present here a case of benign familial polycythaemia in a 25-year-old

male with similar presentation in his family members. Their patient

presented with reddish discolouration of the eyes, early satiety, and

weight loss and itching at intervals, for four years. An additional

examination revealed red beefy tongue and Grade III clubbing. The

importance of presenting this case lies in the fact that the prognosis

appears to be good in these patients, but regular observation is

necessary as Kiladjian and colleagues have mentioned that there is a

risk of leukaemia, thrombosis and myelofibrosis in these patients later

on, as the idiopathic erythrocytosis group contains a certain number of

polycythaemia patients (Maitra and Bhowmik, 2012).

2.5 Cloning

The human erythropoietin gene has been isolated from a

genomric phage library by using mixed 20-mer and 17-mer

oligonucleotide probes. The entire coding region of the gene is

contained in a 5.4-kilobase HindIII-BamHIl fragment. The gene

contains four intervening sequences (1562 basepairs) and five exons

(582 base pairs). It encodes a 27-amino acid signal peptide and a 166-

amino acid mature protein with a calculated Mr of 18,399. The

erythropoietin gene, when introduced into Chinese hamster ovary

cells, produces erythropoietin that is biologically active in vitro and in

vivo (Lin et. al., 1985).

A 600 bp synthetic erythropoietin (epo) gene encoding all 166

amino acids of the epo protein and 27 amino acids of the signal peptide

38

has been constructed. The whole gene was divided into three large

fragments consisting of a total of 32 oligonucleotides. These

oligonucleotides were synthesized by the solid-phase

phosphoramidite method and ligated into three large fragments. These

latter three were separately cloned into vector M13mp19 and

then transformed into E. coli JM 103. Positive clones were screened with

32P-labeled probes. The sequences of the fragments were confirmed by

DNA sequencing, and the sequence of the whole synthetic epo gene was

confirmed by enzymatic digestion and sequencing. The results

indicated that the nucleotide sequence of the synthetic epo gene is

identical to that of the original (Cai, 1992).

A gene encoding the extracellular domain of the human

erythropoietin receptor (epo-R) was constructed using

oligonucleotides, with a view to maintaining preferred codon usage for

the Streptomycetes. The gene was sub cloned into a multicopy

Streptomyces-Escherichia coli shuttle vector, pCAN46 (derived from

pIJ680), containing a strong constitutive promoter from the S. fradiae

aph gene, a signal peptide coding region derived from the protease B

gene of S. griseus, and a transcription terminator sequence also

derived from the S. fradiae aph gene. Extracellular expression of

authentic epo-R by S. lividans was demonstrated using SDS-PAGE and

Western blot analysis, followed by direct amino terminal sequencing of

the purified product. Specific binding of S. lividans-expressed epo-R to

recombinant human glycosylated epo was demonstrated using

BIAcore (surface plasmon resonance) analysis and native gel shift

assays. (Binnie et al., 1997)

39

Oxygen is essential for life, and the body has developed an

exquisite method to collect oxygen in the lungs and transport it to the

tissues. Hb contained within red blood cells (RBCs), is the key oxygen-

carrying component in blood, and levels of RBCs are tightly controlled

according to demand for oxygen. The availability of oxygen plays a

critical role in athletic performance, and agents that enhance oxygen

delivery to tissues increase aerobic power. A breakthrough in

understanding how RBC formation is controlled included the

discovery of erythropoietin (epo) and cloning of the epo gene. Cloning

of the epo gene was followed by commercial development of

recombinant human epo (repo.hu). Legitimate use of this and other

agents that affect oxygen delivery is important in the treatment of

anemia (low Hb levels) in patients with chronic kidney disease or in

cancer patients with chemotherapy-induced anemia. However,

competitive sports were affected by illicit use of repo.hu to enhance

performance. Testing methods for these agents resulted in a cat-and-

mouse game, with testing labs attempting to detect the use of a drug or

blood product to improve athletic performance (doping) and certain

athletes developing methods to use the agents without being detected

(Elliott, 2008) .

Garg et al., (2010) suggested that the cardioprotective

potential of epo-induced preconditioning in isolated rat heart was due to

interplay of the JAK-2, PI-3K and PKC pathways. Inhibition of any one of

the three pathways was sufficient to block the cardioprotective effect of

epo-induced preconditioning in isolated rat heart.

40

Zhou et al., (2010) hypothesized that there should be several

loci in the genome of Chinese hamster ovary (CHO) cells that allow

not only high-level, but also stable gene expression. Based on this

hypothesis, they constructed a plasmid pMCEscan, which introduced a

site-specific recombinase-recognition sequence, FRT, for gene targeting

into those sites. Another targeting vector, pcDNA5/FRT, has an FRT

sequence fused to a promoter less hygromycin-resistance gene that can

be expressed only when correct gene targeting occurs. Using the

pMCEscan, which is a novel and stringent selection system used to

create a few high protein-producing clones, they constructed

engineered CHO strains that can be used for high-level production of

foreign proteins by gene targeting. they selected 28 CHO strains that

expressed a high-level of reporter genes and carried one copy of the

pMCEscan in their chromosomes, and they treated these strains with

methotrexate (MTX) to evaluate dihydrofolate reductase (DHFR)-

mediated gene amplification. Nine clones showed high-level tissue

plasminogen activator (tPA) production without amplification. They

then targeted other genes (tPA, secreted alkaline phosphatase (SEAP),

erythropoietin (epo)) to test the basal expression ability of nine CHO

strains. CHO strains 8-1 and 8-11 consistently expressed high basal

levels of the recombinant genes. Using this cell-vector system, they

obtained the tPA stable high producers by gene targeting and gene

amplification. This system allows for rapid generation of recombinant

proteins without cloning and greatly simplifies selection of cell lines

for the production of potential therapeutic proteins.

41

Erythropoietin (epo) acts on erythroblasts in the bone

marrow (BM) to stimulate the formation of red blood cells. In that

study, they wanted to determine whether BM-derived mesenchymal

stromal cells (MSCs) can be used as cellular vehicles to deliver epo in

mice without the use of viral vectors. After isolation and

characterization of murine MSCs (mMSCs), different transient

transfection procedures were compared for their efficacy of gene

transfer of the pEGFP-N2 plasmid. Nucleofection outperformed

magnetofection and lipofection. Stably transfected mMSCs were

generated by selection with G418-disulfate and single-cell-colony-

forming unit (sc-CFU) assays without changes in their proliferation

capacity and osteogenic/adipogenic differentiation potential. Next,

murine epo was stably introduced into mMSCs by nucleofection of a

plasmid encoding the epo and egfp genes. Intraperitoneal

transplantation of epo-expressing mMSCs increased serum epo levels,

hematocrit and hemoglobin of C57BL/6 mice within 1 week. The

hematocrit remained elevated for 5 weeks, but production of

antibodies against both transgenes was detected in the hosts and

serum epo levels normalized (Scheibe et al., 2011).

2.7 Purification

The isolation and purification of naturally occurring epo is a

difficult task given the large amount of starting material needed and

the optimization of the assay required. It was clear from early studies

that epo was not stored in great quantities in any organ of the body, so

there were no clusters of epo-producing cells that could be isolated

readily from which substantial amounts of hormone could be purified

42

(Lewis et al., 1965). Potentially sources of naturally occurring epo

included the urine or plasma of anemic large animals, including

humans and various organs such as the kidney, and cell lines derived

from tumors such as renal tumors that spontaneously produced epo

(Cotes et al., 1966; Miyake, 1977).

Recombinant human erythropoietin (repo.hu) was purified

from the conditioned media of Chinese hamster ovary cells with a

transfected human erythropoietin gene. They investigated the effects

of the repo.hu in rats with renal anemia induced by partial

nephrectomy. Five-sixth nephrectomy resulted in renal failure with

anemia. Twenty-five days after the operation plasma urea nitrogen

was increased about 2.5 times, and the red blood cell count,

hematocrit, and hemoglobin concentration fell to 85% of normal. The

reticulocyte count and plasma erythropoietin level did not change such

as they do in patients with anemia due to chronic renal failure. Both

total red blood cell volume and the plasma iron turnover rate were

depressed in five-sixth nephrectomized rats compared with normal

rats. The five-sixth nephrectomized rats were injected with repo.hu (60

IU/kg) intravenously every second day for a total of six injections.

After three injections of repo.hu, circulation volume of total red blood

cells was increased from 9.9 ml to 14.6 ml, and the plasma iron

turnover rate was increased from 1.03 mg/kg/day to 2.12 mg/kg/day,

and the reticulocyte count was also increased. After six injections, a

marked increase of the red blood cell count, hematocrit, and

hemoglobin concentration were observed. Plasma urea nitrogen and

the creatinine levels as indications for renal function did not change

43

after repo.hu administration in both normal and five-sixth

nephrectomized rats. In conclusion repo.hu has a potent erythropoietic

action and it is possible to cure the anemia caused by renal failure

(Kawamura et al., 1990).

Simple two step chromatographic purification process was

developed with relatively high yield and purity of epo using blue

sepharose affinity chromatography combined with Q-sepharose ion

exchange chromatography. A single protein zone with molecular mass of

32-38kDa was appeared in SDS-PAGE analysis of the purified repo.hu

(Surabattula et al., 2011).

Expression

The glycoprotein hormone erythropoietin plays a major role

in regulating erythropoiesis and deficiencies of erythropoietin result in

anemia. Detailed studies of the hormone and attempts at replacement

therapy have been difficult due to the scarcity of purified material.

They used a cloned human erythropoietin gene to develop stably

transfected mammalian cell lines that secrete large amounts of the

hormone with potent biological activity. These cell lines were

produced by cotransfection of mammalian cells with a plasmid

containing a selectable marker and plasmid constructions containing a

doned human erythropoietin gene inserted next to a strong promoter.

The protein secreted by these cells stimulated the proliferation and

differentiation of erythroid progenitor cells and, with increased

selection, several of these cell lines secrete up to 80 mg of the protein

per liter of supernatant. Hybridization analysis of DNA from human

44

chromosomes isolated by high resolution dual laser sorting provides

evidence that the gene for human erythropoietin is located on human

chromosome 7 (Powell et al., 1986).

Royer et al., (2004) reported that stable expression of cloned

genes in mammalian cells has been achieved in the past by retroviral

transduction using bicistronic retroviral vectors. In these vectors, the

use of an Internal Ribosome Entry Site (IRES) allows simultaneous

expression of a protein of interest and a fluorescence marker.

However, traditional cDNA cloning in these vectors is often difficult.

They reported the construction of a high-throughput retroviral vector

using the Invitrogen "Gateway" Cloning system. The Gateway

recombination sequences (attR) flanking the ccdB and chloramphenicol

resistance genes were incorporated at the 5' of the IRES of pMX-IRES-

GFP, -CD2, or -CD4 vectors. Through recombination, these vectors can

acquire cDNAs coding for genes of interest, which will result in

simultaneous expression of the recombined gene and the marker

protein. They constructed Gateway bicistronic vectors coding for the

erythropoietin receptor (epoR) and GFP, CD4, or CD2. epo-dependent

proliferation assays and analysis of Jak2-dependent epoR cell-surface

expression showed that these vectors were able to function

indistinguishable from the original pMX-epoR-IRES-GFP. The

expression levels of the genes cloned upstream the IRES were

proportional to the levels of expression of GFP, which was cloned

downstream of the IRES. They used the same approach and generated

Ba/F3 cells that overexpress STAT5a, STAT5b, or a constitutively

active form of STAT5. Overexpression of STAT5 lead to a significant

45

effect on the intrinsic adherence to plastic of these cells, but did not

change their proliferative responses to cytokines.

Related protein (Sar1p) plays an essential role in protein

transport from the endoplasmic reticulum to the Golgi apparatus.

Here, authors reported the molecular analysis of Sara2 in erythroid cell

culture. A 1250 bp long cDNA, encoding a 198 amino-acid protein very

similar to Sar1 proteins from other organisms, was obtained.

Furthermore, they also reported a functional study of Sara2 with Real-

time quantitative PCR analysis, demonstrating that expression of Sara2

mRNA increased during the initial stages of erythroid differentiation

with epo and that a two-fold increase in expression occurs following

the addition of hydroxyurea (HU). In K562 cells, Sara2 mRNA was

observed to have a constant expression and the addition of HU

upregulated proliferation and differentiation and could be valuable for

understanding the vesicular transport system during erythropoiesis.

These results suggested that Sara2 is an important gene in

erythropoiesis processes (Jardim et al., 2005).

Random integration linking genomic amplification is widely

used to generate desired cell lines for stable and high-level expressing

recombinant proteins. But this technique is laborious, and the

expression level is unpredictable due to position effects. Uroplakin II

(UPII) gene expression is highly tissue and cell specific, with mRNA

present in the suprabasal cell layers of the bladder and urethra.

Previous reports described the mouse UPII (mUPII) promoter as

primarily urothelium selective. However, ectopic expression of a

transgene under the 3.6 kb mUPII promoter was also detected in brain,

46

kidney, and testis in some transgenic mouse lines. An 8.8 kb pig UPII

(pUPII) promoter region was cloned and investigated which cells

within the bladder and urethra express a transgene consisting of the

pUPII promoter fused to human erythropoietin (hu.epo) or a luciferase

gene. pUPII-luciferase expression vectors with various deletions of the

promoter region were introduced into mouse fibroblast (NIH3T3),

Chinese hamster ovary (CHO), and human bladder transitional

carcinoma (RT4). A 2.1 kb pUPII promoter fragment displayed high

levels of luciferase activity in transiently transfected RT4 cells, whereas

the 8.8 kb pUPII promoter region displayed only low levels of activity.

The pUPII-hu.epo expression vector was injected into the pronucleus of

zygotes to make transgenic mice. To elucidate the in vivo molecular

mechanisms controlling the tissue- and cell-specific expression of the

pUPII promoter gene, transgenic mice containing 2.1 and 8.8 kb pUPII

promoter fragments linked to the genomic hu.epo gene were generated.

An erythropoietin (epo) assay showed that all nine transgenic lines

carrying the 8.8 kb construct expressed recombinant human

erythropoietin (rhu.epo) only in their urethra and bladder, whereas two

transgenic lines carrying the 2.1 kb pUPII promoter displayed hu.epo

expression in several organs including bladder, kidney, spleen, heart,

and brain. These studies demonstrate that the 2.1 kb promoter contains

the DNA elements necessary for high levels of expression, but lacks

critical sequences necessary for tissue-specific expression. They

compared binding sites in the 2.1 and 8.8 kb promoter sequences and

found five peroxisome proliferator responsive elements (PPREs) in the

8.8 kb promoter. The data demonstrated that proliferator-activated

receptor (PPAR)-gamma activator treatment in RT4 cells induced the

47

elevated expression of hu.epo mRNA under the control of the 8.8 kb

pUPII promoter, but not the 2.1 kb promoter. Collectively, the data

suggested that all the major trans-regulatory elements required for

bladder- and urethra-specific transcription are located in the 8.8 kb

upstream region and that it may enhance tissue-specific protein

production and be of interest to clinicians who are searching for

therapeutic modalities with high efficacy and low toxicity (Kwon et al.,

2006).

The mechanisms controlling the expression of the gene

encoding for the hormone erythropoietin (epo) were exemplary for

oxygen-regulated gene expression. In humans and other mammals,

hypoxia modulated epo levels by increasing expression of the epo gene.

An association between polycythaemia and people living at high

altitudes was first reported more than 100 years ago. Since the

identification of epo as the humoral regulator of red blood cell

production and the cloning of the epo gene, considerable progress has

been made in understanding the regulation of epo gene expression.

This has finally led to the identification of a widespread cellular

oxygen-sensing mechanism. Central to this mechanism was the

transcription factor complex hypoxia-inducible factor (HIF)-1. The

abundance and activity of HIF-1, a heterodimer of an alpha- and beta-

subunit, was predominantly regulated by oxygen-dependent post-

translational hydroxylation of the alpha-subunit. Non-heme ferrous

iron containing hydroxylases use dioxygen and 2-oxoglutarate to

specifically target proline and an asparagine residue in HIF-1alpha. As

such, the three prolyl hydroxylases (prolyl hydroxylase domain-

48

containing protein (PHD) 1, PHD2 and PHD3) and the asparagyl

hydroxylase (factor inhibiting HIF (FIH)-1) act as cellular oxygen

sensors. In addition to erythropoiesis, HIF-1 regulated a broad range of

physiologically relevant genes involved in angiogenesis, apoptosis,

vasomotor control and energy metabolism. Therefore, the HIF system was

implicated in the pathophysiology of many human diseases. In addition

to the tight regulation by oxygen tension, temporal and tissuespecific

signals limit expression of the epo gene primarily to the fetal liver and the

adult kidney (Stockmann et al., 2006).

Authors previously found increased expression of

erythropoietin receptor (epo-R) in peripheral dog lung during postnatal

and postpneumonectomy (PNX) lung growth. To studied the upstream

regulation of epo-R, They analyzed the expression of hypoxia-inducible

factors (HIF)-1alpha, -2alpha, and -3alpha during postnatal lung

growth in immature and mature (2.5 and 12 mo old, respectively) dogs

and during compensatory lung growth 3 wk and 10 mo after right PNX.

Relative to their respective controls, HIF-1alpha transcript was 52-95%

higher in immature lungs and 284% higher in the remaining lung 3 wk

post-PNX. HIF-2alpha transcript did not change during maturation but

was 42% lower 3 wk post-PNX. HIF-3alpha transcript was 53-65%

lower in both the immature lung and 3 wk post-PNX. Changes were no

longer detectable 10 mo post-PNX. No change in HIF transcripts was

observed in kidney and liver post-PNX. Consistent with the mRNA

changes, HIF-1alpha protein was 120 and 196% higher in growing lungs

and 3 wk post-PNX relative to their respective controls. Over

expression of HIF-1alpha in cultured HEK-293 cells increased

49

endogenous expression of epo-R protein. These results demonstrate

regulated expression of the HIF system and parallel changes in HIF-

1alpha and epo-R expression during two types of lung growth. Because

the normal growing lung was not hypoxic, the HIF system likely

responds to other signals encountered during sustained lung strain

(Zhang et al., 2006).

After a century of research and medical use, erythropoietin

(epo) has more therapeutic approaches than ever in history. After

cloning its gene in 1984, epo obtained FDA license for clinical use in

1989. epo and its analogues were mainly used for treatment of the

anemia of chronic renal failure and malignancies. Regarding research of

the past 15 years, tremendous efforts were made for improvement of

bioactivity, half-life and alternative application. Today, there were

human cell-lined derived epo, SEP, Cepo, CERA and drugs which were

linked to different pathways of signaling. Due to the fact that these

substances were not detectable with standardized methods of detection,

it must be assumed that the abuse in sport is still possible. Moreover it

was found out that the epo receptor and epo synthesis were also

expressed by non-hematopoietic tissues, e. g. heart myocytes, ovarian

and glial cells. On these tissues epo was linked to promote cell

proliferation and differentiation, angiogenesis or inhibition of

apoptosis. This detection offered approaches in treatment for apoplexia

and cardiac infarction and even in preventive treatment of

cardiovascular diseases which led to an interest of manifold subject

categories (Schöffel et al., 2008).

Over expression of the transcription factor Spi-1/PU.1 by

50

transgenesis in mice induces a maturation arrest at the

proerythroblastic stage of differentiation. They have previously isolated

a panel of spi-1 transgenic erythroleukemic cell lines that proliferated in

the presence of either erythropoietin (epo) or stem cell factor (SCF).

Using these cell lines, they observed that epoR stimulation by epo

down- regulated expression of the SCF receptor Kit and induced

expression of the Src kinase Lyn. Furthermore, enforced expression of

Lyn in the cell lines increased cell proliferation in response to epo, but

reduced cell growth in response to SCF in accordance with Lyn ability

to down-regulate Kit expression. Together, the data suggest that epo-

R/Lyn signaling pathway is essential for extinction of SCF signaling

leading the proerythroblast to strict epo dependency. These results

highlight a new role for Lyn as an effector of epoR in controlling Kit

expression. They suggest that Lyn may play a central role in during

erythroid differentiation at the switch between proliferation and

maturation (Kosmider et al., 2009).

Ramadori et al., (2009) reported that an acute-phase response

is the systemic reaction of an organism to insult (e.g. infection, trauma

and burning). It represents the „first line‟ of defense of the body to

tissue-damaging attacks. In the previous work, they used a rat model of

intra-muscular turpentine oil (TO) injection to analyze erythropoietin

(epo) gene expression changes in the liver, one of the main target

organs of acute-phase cytokines. epo began to increase in the serum of

TO-treated animals 6 h after injection and reached a maximum at 24 h

(125±20 pg/ml). The detection of total RNA by polymerase chain

reaction analysis showed that the levels of epo gene expression in the

51

liver were considerably increased between 2 and 12 h by up to 20-fold

at the peak after TO administration, followed by a gradual decrease

over the next 48 h, although the values remained significantly higher

compared with the control group. In the kidney, after a sudden slight

increase, the values declined progressively to 3.5-fold decrease at 12 h

after the injection. In the liver, a parallel up regulation of the hypoxia-

inducible factor-1 (HIF-1) α gene was observed (up to 4.7-fold increase),

while HIF-2 α gene expression remained unaltered. On the other hand,

the protein of both genes became detectable after the injection and

increased progressively over 24 h, with a subsequent decline. These

results suggest that epo may be added to the increasing group of

positive acute-phase proteins and the liver might represent the major

source of the hormone under these conditions in the rat.

Lent viral gene transfer can provide long-term expression of

therapeutic genes such as erythropoietin. Because over expression of

erythropoietin can be toxic, regulated expression is needed.

Doxycycline inducible vectors can regulate expression of therapeutic

transgenes efficiently. However, because they express an immunogenic

transactivator (rtTA), their utility for gene therapy is limited. In

addition to immunogenic proteins that are expressed from inducible

vectors, injection of the vector itself is likely to elicit an immune

response because viral capsid proteins will induce “danger signals”

that trigger an innate response and inflammatory cells (David et at.,

2010).

Hypoxia inducible factor-1 (HIF-1) has been considered as a

critical transcriptional factor in response to hypoxia. It can increase P-

52

glycoprotein (P-Gp) thus generating the resistant effect to

chemotherapy. At present, the mechanism regulating HIF-1 alpha is

still not fully clear in hypoxic tumor cells. Intracellular redox status is

closely correlated with hypoxic micro-environment, so they investigate

whether alterations in the cellular redox status lead to the changes of

HIF-1 alpha expression. HepG2 cells were exposed to Buthionine

sulphoximine (BSO) for 12h prior to hypoxia treatment. The level of

HIF-1 alpha expression was measured by western blot and

immunocytochemistry assays. Reduce glutathione (GSH)

concentrations in hypoxic cells were determined using glutathione

reductase/5, 5‟-dithiobis-(2-nitrob- enzoic acid) (DTNB) recycling

assay. To further confirm the effect of intracellular redox status on HIF-

1 alpha expression, N-acetylcysteine (NAC) was added to culture cells

for 8h before the hypoxia treatment. The levels of multidrug resistance

gene-1 (MDR-1) and erythropoietin (epo) mRNA targeted by HIF-1

alpha in hypoxic cells were further determined with RT-PCR, and then

the expression of P-Gp protein was observed by western blotting. The

results showed that BSO pretreatment down-regulated HIF-1 alpha and

the effect was concentration-dependent, on the other hand, the

increases of intracellular GSH contents by NAC could partly elevate the

levels of HIF-1 alpha expression. The levels of P-Gp (MDR-1) and epo

were concomitant with the trend of HIF-1 alpha expression. Therefore,

the data indicate that the changes of redox status in hypoxic cells may

regulate HIF-1 alpha expression and provide valuable information on

tumor chemotherapy (Jin et al., 2011).

53

Expression of epo rapidly increased with increasing MTX

concentration up to 1000 nM, further increased to 2000 nM does not

affect the expression. After the MTX selection, cells grown in the

presence of MTX were more stable and retained similar amounts of epo

expression & gene copies until 50 doublings. Whereas, cells grown in

the absence of MTX were unstable and retained only 50% of initial epo

expression & gene copies at the 50th doubling. Scale-up from culture

flask to wave bioreactor increases epo yield, the novel wave bioreactor

with a working volume of 1 liter could produce more than 0.56 g of epo

in 3 weeks, with a volumetric productivity of 24 mg/l/day and specific

productivity of 5.93 µg/106cells/day (Surabattula et al., 2011).

54

Chapter-3

Materials and Methods 3.1 Materials

3.1.1 Vector

3.1.2 Gene

3.1.3 Primers

3.1.4 Host Bacterial Strains

3.1.5 Cell Culture

3.1.6 Conjugates

3.1.7 Experimental Animals

3.1.8 Chemicals, Glass wares and Plastic wares

3.1.9 Solutions and Buffers

3.2 Methods

3.2.1 Revival of the E.coli culture containing recombinant plasmid with human erythropoietin gene

3.2.2 Isolation of plasmid DNA

3.2.3 Digestion of the recombinant plasmid with RE EcoRI to release the epo.hu gene insert

3.2.4 Purification of the epo.hu gene insert

3.2.5 Preparation of the pSin Vector

3.2.6 Preparation of the epo.hu gene insert for ligation

3.2.7 Digestion of the replicase based vector with StuI

enzyme

3.2.8 Dephosphorylation of vector DNA with CIAP

3.2.9 Purification of replicase vector (pSin Vector)

3.2.10 Ligation of pSin Vector and epo.hu gene.

3.2.11 Transformation of E.coli DH5α

3.2.12 Screening of recombinant clones

3.2.13 Selection of the clone containing epo.hu gene in right orientation

3.2.14 Expression study.

3.2.15 Restriction enzyme analysis of the recombinant plasmid, using different restriction enzymes.

Chapter-3

3. Materials and methods

3.1 Materials

3.1.1 Vector

The pSin vector (Fig. 1) is derived from an alpha virus (Sindbis

virus). The subgenomic promoter of alpha virus is very strong so that it

makes large number of target mRNA from the sequence downstream to

it. In comparative studies of conventional (nonreplicating) plasmid

DNA vectors and alphavirus DNA-based replicon vectors, the latter

generally produces larger quantity of DNA concentrations than does

conventional vectors.

3.1.2 Gene

The epo gene of human used in this study was cloned in pTarget

vector in Biotechnology laboratory of IBIT, Bareilly.

3.1.3 Primers

Primers used in the study were synthesized from Chromous

biotech. (India).

55

BssHII SphI StuI ApaI

BGH Poly A

pSin Vector

10.8 kb

Replicasegene

Fig. 1: pSin Vector

SI.No Primers Primer sequence

1. epo.hu gene (Forward primer) 5’ATTGACGGCGTAGTACAC 3’

2. BGH (Reverse primer) 5’TAGAAGGCACAGTCGAGG 3’

Table: 1 Primers with their sequences

3.1.4. Host Bacterial Strains

Escherichia coli (E. coli) DH5α (Proteges, Madison) host strain was

used for transformation with recombinant plasmids.

3.1.5. Cell Culture

HeLa cell line was obtained from National Centre for Cell Science

(NCCS), Pune. This cell line was used in the study for gene expression of

recombinant plasmid and was maintained in DMEM (Gibco, NY)

supplemented with 50 µg/ml gentamycin (Amresco, USA) and 10% fetal

calf serum (Hyclone, USA).

3.1.6. Conjugates

Rabbit anti-mouse HRP conjugated antibody was obtained from

Bangalore Genei, Bangalore, (India).

3.1. 7 Experimental Animals

Swiss albino mice, 4 weeks old were procured and kept in animal

room for raising hyper immune sera.

56

3.1.8. Chemicals, glasswares and plastic wares

All chemicals used in the study were either from Fermentas

(UK), or Promega, Bangalore Genei (Bangalore, India), Medox, Qiagen

(Germany). Glasswares and Plastic wares were from Borosil, Axygen

and Greiner.

3.1.9. Solution sand buffers

The composition of chemical solutions and buffers used in the

study is given in appendix.

3.2 Methods

The cloning strategy is shown in Fig. 2

3.2.1 Revival of the E.coli culture containing recombinant

plasmid (pTarget.epo.hu) with epo.hu gene

The E.coli having recombinant pTarget vector

(pTarget.epo.hu) was revived in LB broth overnight at 370C following the

protocol of (Sambrook and Russell, 2001). The E.coli containing

rplasmid was incubated in 5ml LB broth containing 100 µg/ml.

3.2.2 Isolation of plasmid DNA (pTarget.epo.hu)

Plasmid DNA was isolated following TELT method (He et al.,

1990). Grown 1.5 ml E.coli culture with plasmid in LB medium

containing 100µg/ml ampicillin for 16 h. Pelleted cells for 30 sec. in

57

pTarget.

pSin epo.hu

Vector

↓

↓ Digestion with EcoRI for Release of epo.hu gene

Digestion with StuI enzyme

↓ ↓

Dephosphorylation

Blunting

Ligation

rpSin.epo.hu

rplasmid

↓

Transformation of E.coli DH5α

↓

Selection of rplasmid containing gene in right orientation

Fig. 2: Cloning strategy

micro centrifuge and resuspended in 100 µl TELT solutions and added

an equal volume of 1:1phenol/chloroform. Vortexed vigorously for 15

sec. and spinned 1 min in microcentrifuge at 22ºC. Collected the upper

phase of nucleic acid and mixed with 2 vol of 100% ethanol. After 2

min, spinned 10 min in microcentrifuge at 4ºC. Washed pellet with 1

ml of 70% ethanol, allowed to dry and resuspended in 30 µl TE buffer.

Plasmid DNA was checked on 1% agarose gel electrophoresis.

3.2.3 Digestion of the recombinant plasmid with restriction

enzyme (EcoRI) to release the epo.hu gene insert

After checking the integrity and presence of plasmid in

eluted buffer, the plasmid was digested with EcoRI Enzyme (12 U/µl) as

per the guidelines provided by the supplier. The reaction mixture was

prepared as mentioned below in table 2.

Component Volume

rplasmid pTarget.epo.hu (200 µg/ml) 20.0 µl

EcoRI Enzyme (12U/µl) 3.0 µl

Enzyme buffer (10X) 5.0 µl

Nuclease free water 22.0 µl

Total 50.0 µl

Table 2: Reaction mix for EcoRI digestion

The reaction mixture was incubated at 37°C 12 overnight. The

digestion was checked on 1.5% agarose gel and the released insert was

58

extracted from agarose gel.

3.2.4 Purification of the epo.hu gene insert

The epo.hu gene insert was cut out and purified using

phenol: chloroform method (Sambrook and Russell, 2001) briefly:

Added equal volume of phenol: chloroform: isoamyl alcohol and

mixed vigorously. Centrifuged at 12000g for 15 min. to separated the

aqueous and phenol phases. Collected the upper aqueous phase

containing the plasmid DNA into another microfuge tube. Precipitated

the plasmid DNA by adding 0.8 volume of isopropanol. Centrifuged at

12000g for 10 min to get the precipitate DNA into pellet form.

Discarded the supernatant and saved the pellet. To wash the pallet

added 1ml of 70% ethanol to microfuge tube containing pellet gently

and wait for 5 min. then centrifuged at 12000g for 10 min. discarded

the supernatant and saved the pellet air-dried the pellet and dissolved

in 30µl TE buffer.

3.2.5 Preparation of the pSin vector

Growth of rE.coli cells containing pSin vector was done in LB

broth, as well on LB agar plates.

3.2.6 Preparation of the epo.hu gene insert for ligation

Because of limited choice of restriction sites in replicase

based mammalian expression vector, blunt end cloning was thought to

be a desirable approach. Although comparatively difficult, it had the

dual advantage of getting the cloned insert in right orientation with a

probability of 50%. So for blunting of staggered ends generated by

59

EcoRI enzyme T4 DNA polymerase (Fermentase) was used as follows in

table 3:

Component Volume

epo.hu gene Insert 20.0 µl

T4 DNA polymerase buffer 10X 5.0 µl

T4 DNA polymerase (5 U/µl) 2.0 µl

dNTPS mix.(10mM each) Fermentas 1.0 µl

Nuclease free water 22.0 µl

Total volume 50.0 µl

Table 3: Reaction mix for blunting

The reaction mixture was incubated at 37˚C for 10 minutes. The

reaction was stopped by heating at 75°C for 10 minutes and then

purified by phenol: chloroform method. It was now ready for use in

ligation.

3.2.7 Digestion of the replicase vector with restriction enzyme

(StuI) for ligation

StuI was chosen as preferred site to create blunt end. A

reaction mixture was prepared with Stu1 enzyme as per the following

protocol to digest the pSin vector plasmid.

60

Component Volume

pSinVectorDNA(200µg/µl) 20.0 µl

StuIenzyme(10 U/µl) 3.0 µl

NEBuffer(10X) 5.0 µl

Nuclease free water 22.0 µl

Total 50 .0µl

Table 4: Reaction mix for StuI digestion

The reaction mixture was incubated at 37°C overnight. The linearised

plasmid was checked and quantitated on 1% agarose gel

electrophoresis by method described earlier. The linearized plasmid was

then gel extracted and quantified.

3.2.8 Dephosphorylation of vector DNA with CIAP

The calf intestinal alkaline phosphatase (CIAP) was used to

remove 5` phosphate group from both the ends of linearized plasmid. The

following reaction mixture was prepared in 50 µl volume.

Component Volume

Linearized vector pSin (200 µg/µl) 11.0 µl

CIAP enzyme (1 U/µl) 1.0 µl

Buffer (10X) 5.0 µl

Nuclease free water 33.0 µl

61

Total 50.0 µl

Table 5: Reaction mix for dephosphorylation

The pSin Vector was dephosphorylated using CIAP to prevent self

ligation. The protocol provided with fermentas CIAP was followed

with slight modification. Briefly, the reaction mixture was incubated at

37°C for 30 minutes. But to be on safer side 1 µl of CIAP was added

again and reincubated at 37°C for 30 minutes. Enzyme was then

inactivated by heating the reaction mixture at 75°C for 10 minutes.

3.2.9 Purification of replicase vector (pSin Vector)

The above preparation on was purified using phenol:

chloroform method (Sambrook and Russell, 2011) briefly: Added

equal volume of phenol: chloroform: isoamyl alcohol and mixed

vigorously. Centrifuged at 12000g for 15 min to separated the aqueous

and phenol phases. Collected the upper aqueous phase containing the

plasmid DNA into another microfuge tube. Precipitated the plasmid

DNA by adding 0.8 (80 ml) volume of isopropanol. Centrifuged at

12000g for 10 min to get the precipitated DNA into pellet form.

Discarded the supernatant and save the pellet. To washed the pallet

add 1ml of 70% ethanol to microfuge tube containing pellet gently and

wait for 5 min than centrifuged at 12000g for 10 min. Discarded the

supernatant and save the pellet air-dry the pellet and dissolved in 30 µl

TE buffer .

62

3.2.10 Ligation of pSin Vector and epo.hu gene

A 10µl reaction mixture was standardized with 30% PEG

8000 for blunt end ligation. The components were mixed in the

following concentrations.

Component Volume

T4 DNA Ligase (Promaga) (1-3U/ µl) 2.0 µl

pSin Vector (200 µg/ml) 0.5 µl

epo.hu gene(150 µg/ml) 4.0 µl

Ligation buffer (10X) 1.0µl

30% PEG8000 (Amresco) 1.5 µl

Nuclease free water 1.0 µl

Total 10 .0 µl

Table 6: Ligation mixture

The time temperature relationship was maintained as described in

datasheet of T4 DNA Ligase provided by Fermentas. The reaction

mixture was incubated for overnight at 16°C.

3.2.11 Transformation of E.coli DH5α

The single step method of competent cell preparation and

transformation was used (Chung et al., 1989). A fresh overnight

culture of bacteria was diluted into prewarmed LB Broth and the cells

were incubated at 37˚C in shaking incubator. The cells were pelleted

63

by centrifugation at 1000xg for 10 min at 4˚C, supernatant removed

and resuspended at 1/10th of original in ice cold 1X TSS. The cell

suspension was mixed gently. For transformation, a 0.1 ml aliquot of

cells was pipetted into a cold polypropylene tube containing 1 µl of

plasmid DNA and the cell/ DNA suspension was mixed gently. The

cell/ DNA mixture was incubated for 5 minute at 4˚C. A 0.9 ml aliquot

of TSS plus 20 mM glucose was added and the cells were incubated at

37˚C in shaking incubator at 200 rpm for 1 h. The above transformed

cells were plated on LB agar plates containing appropriate antibiotic

(ampicillin 50 µg/ ml) and the plates were incubated at 37˚C for 16

h/overnight.

3.2.12 Screening of recombinant clones

A large number of colonies were seen but 25 were picked

from the overnight grown transformants. The individual colonies were

inoculated in fresh ampicillin (50µg/ml) containing LB broth and

allowed to grow for 16 hours. Plasmid was isolated from these colonies by

TELT method.

3.2.13 Selection of the clone containing human erythropoietin

gene in right orientation.

It was done using following three methods.

i. Restriction enzyme analysis

The clone selection by Restriction enzyme analysis of isolated

pSin.epo.hu in map draw of DNASTAR to find restriction enzyme was

done. It was found that in RE digestion analysis, the restriction enzyme

kpnI has three sites in the recombinant plasmid DNA, one in the insert

64

and the other two in the vector. The digestion with kpnI enzyme

released three fragment of size 506, 2300 and 8560 bp when in wrong

orientation; and fragment size 152, 3359, 7856 bp when in right

orientation. The presence of epo.hu gene inert in right orientation in

the recombinant plasmids was confirmed by Restriction enzyme

analysis using kpnI restriction enzyme. The digestion mixture was

prepared by mixing the components in amounts as mentioned below.

Component Volume

Plasmid DNA (pSin.epo.hu) (200µg/ml) 4.0 µl

kpnI restriction enzyme (8-12 U/µl) 1.0 µl

NE Buffer (10X) 2.0 µl

Nuclease free water 8.0 µl

Total 15.0 µl

Table 7: Reaction mix for kpnI digestion

The reaction mixture was vortexes and spun and incubated in a water bath

at 37°C overnight. The digested mixture was then electrophoresis in 1%

agarose (Low EEO, Banglore Genei). The released fragments after

digestion were compared 1kb DNA ladder.

ii. Polymerase chain reaction

The presence of epo.hu gene inert in right orientation in the

recombinant plasmids was confirmed by PCR using epo.hu gene

specific forward primer and BGH as reverse primer. The PCR reaction

mixture (50µl) contained of recombinant plasmid 50 pmole each of

65

gene specific forward primer and BGH as reverse primer and 3 units of

Taq DNA polymerase in 1X PCR buffer. The reaction was carried as

follows.

Component Volume

Auautoclaved distilled water 33.0 µl

rplasmid(pSin.epo.hu) (200µg/ml) 5.0 µl

Forwardprimerof epo.hu (50 pmole/µl) 2.0 µl

ReverseBGH (50 pmole/µl) 2.0 µl

dNTPs(10mM) 2.0 µl

Buffer(10X) 5.0 µl

TaqDNApolymerase (3 Units/µl) 1.0 µl

Total 50.0 µl

Table 8: Reaction mix for PCR

The epo.hu gene was amplified following initial denaturation at 940C

for 2 minutes and 40 cycles of denaturation at 940C for 35 seconds,

annealing at 500C for 35 seconds and amplification at 720C for 50

seconds. After amplification an aliquot of 10 µl was subjected to

agarose gel electrophoresis along with 100 bp DNA molecular weight

markers through 1.5% agarose gel at 60 volts for appropriate time for the

analysis of PCR product.

iii. DNA sequencing

Once the recombinant plasmids were identified as containing

66

epo.hu gene in correct orientation, a part of the same plasmid lot was sent

for sequencing to Chromous Biotech Ltd. The sample was

sequenced with BGH reverse primer. The obtained sequence was

analyzed by BLAST (NCBI) in the light of its similarly with existing

sequence for Human erythropoietin gene and the sequence from which

primer was designed.

3.2.14 Expression study

i. Preparation of antibody against recombinant plasmid in mice

Primary polyclonal antibody against the human erythropoietin

gene was raised in mouse by hyper immunization of six mice with

pSin.epo.hu plasmid. 50 µg of rplasmid DNA was injected

intramuscularly in lateral region of thigh muscles of each mouse and

repeated every week for four weeks consecutively. One week after last

injection bled through inner canthus of eyes with a capillary and

serum was prepared.

ii. Detection of expressed protein in cell culture using

immunoperoxidase technique (Gerna et al., 1976).

Cell culture was trypsinised using TVS. 100 µl of cell culture

suspension was plated in 96 well microtitre plate. Prepared the

calcium phosphate-DNA co-precipitate as follows: Combined 50 µl of

2.5 M CaCl2 with 10 µg of rplasmid and 40 µl distilled water in a sterile

microfuge tube. Immediately transferred the calcium phosphate-DNA

suspension into the above 96 well microtitre plate containing the cell

suspension. Transfected cells were incubated at 370C in a humidified

67

chamber with an atmosphere of 5% CO2 for 72 hours. After 72 hours of

incubation, the cells were assayed for expression of transfected gene.

Cells were washed with PBS twice and fixed with 100 µl of chilled

acetone. After washing the fixed cells with PBS, cells were treated with

2% H2O2 in PBS for 10 minutes and again washed with PBS twice for 5

minutes each. Cells were incubated with mouse anti epo.hu hyper

immune sera for 1 hour at 37 ºC and washed thrice with PBS, 5

minutes each. Rabbit anti mouse globulin HRP conjugate antibody was

added to wells and incubated for 1 hour at ºC. The cells were washed

with PBS thrice and incubated with Nadi reagent containing freshly

added H2O2 for 5 minutes at room temperature. After the development

of color, cells were washed with PBS, dried in air and observed under

microscope and photographed.

iii. Detection of expressed protein using SDS-PAGE and Western

blotting

Sodium dedecyl sulfate-polyacrylamide gel electrophoresis (Fig. 3)

HeLa cells were transfected in 96 well plates with pSin.epo.hu

rplasmid and pSin vector. After 48 hours of transfection cells were

processed following the method (Rodriguez and Tail, 1983).

Gel preparation: The gels consist of a lower resolving gel and an

upper stocking gel that concentrate the sample before its entry into the

resolving gel. The buffer system was the discontinuous system of

laemmli. Stored all solution in brown bottle at 4 °C and SDS solution at

room temperature. The volumes below were for slab gels of 15 ml vol with

a 6 ml stacking gel. Volumes are gives in ml.

68

Fig. 3: SDS-PAGE

Reagents

Lower gel buffer

Upper gel buffer

Acrylamide stock

Distilled water

10 % SDS

10%ammonium

persulphate

TEMED

Resolving gel (10%) Stacking gel (4%)

3.75 -----

----- 1.25

5.0 0.80

6.05 3.87

0.15 0.06

0.05 0.02

0.07 0.004

Table 9: Component of resolving and stacking gel

The gel plates were assembled and sealed against leakage. The

resolving gel was poured and covered. After polymerization, the

butanol was removed by flushing briefly with water, then the stacking

gel poured and a comb inserted to form sample wells. Following

polymerization of the stacking gel, the comb was removed and the

wells rinsed to remove unpolymerized acryl amide. The gel was then

assembled in the running apparatus and running buffer added. epo.hu

Protein samples (25 µl) were prepared by adding an equal volume i.e.

25 µl of 2X SDS sample buffer and heating at 100°C for 5 min to ensure

denaturation of the samples. Gels were typically run at 40 mA for the

marker dye reaches the end of the gel. The gel apparatus was

dissembled and the gel removed. The gel was immediately dried on

Whatman No.1 filter paper and stained for direct visualizing of

69

polypeptide bands. Placed gel in a staining container with lid. Added

50 ml staining solution. Covered and placed at room temperature for

15 min. Poured off solution, replaced with 50ml staining solution +

5ml stain. Covered and placed at room temperature for overnight. The

gel was turned blue at that time. Poured off the staining solution,

added 100ml destaining solution covered and returned to room

temprature for 15min. Gentle mixing improved the destaining process.

Poured off the destaining solution and replaced. Continued to repeat

this step many times. Completed destaining to produce a clear gel with

blue polypeptide bands depends on gel thickness and required 24 h.

Western blot analysis

After completion of Sodium dedecyl sulfate-polyacrylamide gel

electrophoresis, the epo.hu protein bands from unstained gel were

blotted on to nitrocellulose membrane using SNAP i.d. system. This

Millipore‟s patent pending SNAP i.d. Protein Detection System

provides a fast and convenient method for the detection of immune

reactive proteins on western blots and the Millipore‟s protocol was

followed.

System Set-up

Placed the SNAP i.d. system on a level bench top and attached the

vacuum tubing to the back of the system by pushing the coupling

epo.hu insert on the end of the tubing into the quick disconnect fitting

at the back of the system base. Connected the other end of the tubing

to a vacuum source, used a one-liter vacuum flask as a trap and a

Millex-FA50 filter to protect the vacuum source from contamination.

70

Rolled gently with blot roller to remove any air bubbles trapped

between the blot holder and the blot. Wet the inner white face of the

blot holder with Milli-Q water, until it turns gray. Removed excess

liquid using the blot roller, was preventing movement of the blot

during assembly. Placed the pre-wet blot membrane in the center of

the blot holder and the protein side down Open the blot holder lid.

Blot Assembly

Before starting the blot assembly procedure, prepared the antibody,

blocking, and wash buffers solutions. Placed the spacer on top of the blot,

making sure it completely covers all edges. Rolled blot again to ensure

completed contact of blot spacer with blot membrane, Closed the blot

holder lid Squeezed firmly at the base of the tab area to secure lid.

Opened lid of system by squeezing latch between thumb and

forefingers and lifting upwards Placed blot holder in system chamber

with the well side up, aligning the blot holder tabs with notches of

chamber. Repeated the assembly procedure for all blots being

processed Closed and latch the system lid.

Immunodetection Protocol

Added the appropriate volume of blocking solution to each well being

used after the well(s) had emptied completely (20 seconds), turn

vacuum off using the vacuum control knob(s). Antibody collection

trays were sold separately. Added the appropriate volume (100 µl) of

primary antibody (mouse anti-epo Ab) to each well being used

incubated the primary antibody (mouse anti-epo Ab) for 10 minutes at

room temperature, with the vacuum off. Turned the vacuum on and

71

wait 20 seconds to make sure that antibody solution had been

completely emptied from the blot holder the solution was absorbed

into the blot holder and the surface appeared dry. With vacuum

running continuously, washed the blot with 10 ml of wash buffer

(PBS), three sequential washes were required for optimal performance.

Each washed had been taken 20 seconds to complete and the blot

holder was empty, turn vacuum off. With the vacuum off, applied the

appropriate volume (100µl) of secondary antibody (rabbit anti-mouse

HRP conjugate) evenly across the entire surface of the blot holder

Incubated the secondary antibody (rabbit anti-mouse HRP conjugate)

for 10 minutes at room temperature with vacuum off. Again, the

antibody solution was absorbed into the blot holder and the surfaces

appear dry. Turned on the vacuum and waited 20 seconds to make

sure that antibody solution had been completely emptied from the blot

holder. With vacuum running continuously, washed the blot with 10

ml of wash buffer, three sequential washes were required for optimal

performance Removed blot holder from the system, placed it on the

bench with the well-side down, and opened the lid. With forceps,

removed and discarded the spacer. Removed blot and incubated with

the appropriate detection reagent such as Immobilon® Western HRP

Substrate, visualize. Discarded the single-use blot holder.

3.2.15 Restriction enzyme analysis of the recombinant plasmid,

using different restriction enzymes

Restriction enzyme analysis of the recombinant plasmid, using Pvul,

NheI, PmeI, NruI, SalI and NotI was done.

72

Component Volum in µl

pSin.epo.hu 4.0 4.0 4.0 4.0 4.0 4.0

plasmid DNA

NE Buffer 1.0 1.0 1.0 1.0 1.0 1.0

(10X) Buffer 3 Buffer 4 Buffer 4 Buffer 3 Buffer 3 Buffer 3

Restriction 1.0 1.0 1.0 1.0 1.0 1.0

Enzyme PvuI NheI PmeI NruI SalI NotI

Nuclease free 3.5 3.5 3.5 4.0 3.5 3.5

water

BSA 0.5 0.5 0.5 0.5 0.5

Total 10.0 10.0 10.0 10.0 10.0 10.0

Table 10: Reaction mix for RE analysis

The reaction mixtures were vortexed and spun and incubated in a

water bath at 37°C overnight. The digested mixtures were then

electrophoresis in 1% agarose (Low EEO, Banglore Genei). The

released fragments after digestion were compared to 1kb DNA ladder.

73

Chapter-4

Results 4.1 Cloning of epo.hu gene in pSin vector

4.2 Expression of the recombinant plasmid in cell culture

4.3 Restriction enzyme analysis of the recombinant plasmid, using different restriction enzymes.

Chapter-4

Results 4.1 Cloning of epo.hu gene in pSin Vector

The epo.hu gene was already cloned in pTarget cloning vector

was transformed with E.coli DH5α cells and plasmid DNA

(pTarget.epo.hu) was isolated (Fig. 4). The entire sequence of insert

(epo.hu gene) and vector pTarget was analyzed in mapdraw of

DNASTAR laser gene program to find appropriate site for restriction

enzyme which cut in vector flanking the insert. The E.coRI enzyme

located in MCS of the vector (pTarget vector) was found suitable to

release the epo.hu gene insert without having any cutting site in the

epo.hu gene insert digestion with this E.coRI enzyme released a

product of approximately 588 base pairs including some sequences

from the vector‟s MCS and recombinant vector backbone of 5670 bp

(Fig. 5).

Plasmid Vector (pSin vector) was isolated and was

linearized by digestion with StuI enzyme (Fig. 6).

Blunt end ligation was done in the presence of PEG 8000

and ligated pSin vector was transformed in E.coli (DH5α) cells. A

large number of colonies were observed 25 and were picked from the

overnight grown transformants. The individual colonies were

74

M 1

6.2 kb

5.0 kb

4.0 kb 3.0 b

1.0kb

Fig.4 : Isolation of pTarget.epo.hu plasmid Lane

M : 1 kb DNA Marker

Lane 1 : pTarget.epo.hu plasmid DNA

0.5 kb

2.0 kb

3.0 kb

4.0 kb

5.0 kb5670 bp

588 bp

Fig. 5 pTarget.epo.hu digested with EcoRIenzymeLane M : Marker 1kb ladderLane 1 : pTarget.epo.hu digested with EcoRI releasing a gene insert 588 bp and vector 5670 bp

M 1

M 1

10.8kb

10 kb

4 kb

3 kb

2 kb

1 kb

Fig.6: Preparation of pSin Vector

Lane M : 1 kb DNA Marker

Lane 1 : pSin Vector digested with StuI enzyme

inoculated in fresh ampicillin (50µg/ml) containing LB broth and

allowed to grow for 18 hours. Plasmid DNA (pSin.epo.hu) was

isolated from these colonies by TELT methods (Fig. 7).

Selection of the clone containing human erythropoietin gene

in right orientation was done using restriction enzyme analysis with

kpnI restriction enzyme, Polymerase chain reaction and DNA

sequencing.

The RE digestion results confirmed that clone contained

insert in right orientation (Fig. 8) fragments sizes were in agreement with

the prediction from map draw analysis which confirmed that the

epo.hu gene was in right orientation.

Finally, the clone was selected again by PCR amplification of

isolated plasmids with epo.hu gene specific primers and BGH as

reverse primers. It resulted in amplification of epo.hu gene giving a

product of 588 bp on 1% agarose gel electrophoresis perfectly

matching with the fragment size amplified with gene specific

primers on positive recombinant clone pSin vector (Fig. 9).

The rplasmid clone with insert in right orientation

(pSin.epo.hu) was sequenced with BGH reverse primer. The obtained

sequence was analyzed by BLAST (NCBI) in the light of its similarly with

existing sequence for human erythropoietin gene and the sequence

from which primer was designed, which confirmed that gene was in

right orientation.

75

1.0 kb

2.0 kb

4.0 kb

6.0 kb

8.0 kb10.0 kb

M 1

Fig.7 : Isolation of plasmid DNA (pSin.epo.hu)

Lane M : Marker 1kb ladder

Lane 1 : Isolation of pSin.epo.hu plasmid DNA

11.38 kb

Fig.8: RE digestion of pSin.epo.hu with KpnI .

Lane M : 1kb DNA Marker

Lane 1 &3, undigested rplasmid, Lane 2, rplasmid cut with KpnI producing fragments of 3.359, 7.856 kb

1 2 M 3

7.856Kb

3.359Kb

588bp

M 1

Fig.9: PCR amplification of epo.hu gene.

Lane M: 100 bp DNA ladder;

Lane1: epo.hu PCR product of 588 bp.

200 bp

500 bp

300 bp

400 bp

4.2 Expression of the rplasmid in cell culture

The expression of rplasmid (pSin.epo.hu) was checked by

immunoperoxidase test (IPT) in HeLa cell line and cells were found

to express the epo.hu protein by development of purple color

precipitate. Intense purple coloration of cells was observed in which

HeLa cells were transfected with pSin.epo.hu (Fig. 10). The vector

alone and untransfected healthy cells (Fig. 11) failed to show any

coloration indicating that the epo.hu gene was expressed in cells due

to the presence of rpSisn.epo.hu plasmid. In this highly expression

technique 90% cells were found to express epo.hu protein.

The SDS-PAGE (Sodium dedecyl sulfate-polyacrylamide gel

electrophoresis) analysis of expressed epo.hu protein indicated a

specific isolated thick band (Blue color) of 34 kDa (Fig. 12). The

vector transfected cells and mock transfected cells failed to show

thick band.

The western blot analysis of expressed protein indicated a

specific isolated band seen in nitrocellulose membrane of 34kDa size

(Fig. 13). The vector transfected cells and mock transfected cells fail to

show thick bands.

4.3 Recombinant plasmid digested with different Restriction

enzyme

The entire sequence of epo.hu gene insert and vector pSin was

analyzed in mapdraw of DNASTAR laser gene program to find

different sites for restriction enzymes and were again checked for the

presence of restriction sites for odifferent restriction enzymes PvuI,

76

Fig.10: HeLa cells with pSin.epo.hu. rplasmid showing positive IPT test, 100X

Fig.11: Healthy Control HeLa cells showing no color reaction, 100X

Fig.12: SDS-PAGE of HeLa cell extract for epo expression.

Lane M, Protein marker

Lane 1- transfected cells showing expressed protein band.

Lane 2-mock transfected Cell extract showing no expression.

M 1 2

20kDa

30 kDa

35 kDa

43 kDa

68 kDa

34 kDa

34 kDa

1 2 3 4 M

68 kDa

30 kDa

20 kDa

Fig.13: Detection of epo protein using Western blotting Lane

: M 1 kDa protein marker

Lane : 1 Mock transfected

Lane : 2 Vector control

Lane : 3 & 4 epo.hu protein

NheI, PmeI, NruI, SalI and NotI which could release the product of

desired length. The enzymes PvuI, SalI and NotI had one site each.

Digestion with PvuI and NotI enzymes released a product size

approximately between the ranges of 8000 to 10000 bp while digestion

with enzyme SalI released a product size approximately between the

ranges of 4000 to 6000 bp. The enzyme NheI was chosen which had

five sites. Digestion with this enzyme released products size near

about 2000 to above 10000 bp. The restriction enzymes PmeI and NruI

were chosen which had two sites for each enzyme. Digestion with

PmeI released a product size approximately between the ranges of

8000 to above 10000 bp while digestion with enzyme NruI released a

product size between the ranges of 2000 to above 1000 bp (Fig. 14, 15,

16, 17, 18 and 19).

77

1.0 kb

2.0 kb

4.0 kb

6.0 kb

8.0 kb

10.0 kb

M 1

Fig.14: RE Digestion of pSin.epo.hu rplasmid with NheI Enzyme

Lane M : Marker 1kb ladder

Lane 1 : rplasmid digested with NheI Enzyme

1.0 kb

2.0 kb

4.0 kb

6.0 kb

8.0 kb

10.0 kb

M 1

Fig.15: RE Digestion of pSin.epo.hu rplasmid with NotI Enzyme

Lane M : Marker 1kb ladder

Lane 1 : rplasmid digested with NotI Enzyme

1.0 kb

2.0 kb

4.0 kb

6.0 kb

8.0 kb

10.0 kb

M 1

Fig.16: RE Digestion of pSin.epo.hu rplasmid with NruI Enzyme

Lane M : Marker 1kb ladder

Lane 1 : rplasmid digested with NruI Enzyme

1.0 kb

2.0 kb

4.0 kb

6.0 kb

8.0 kb

10.0 kb

M 1

Fig.17: RE Digestion of pSin.epo.hu rplasmid with PmeI Enzyme

Lane M : Marker 1kb ladder

Lane 1 : rplasmid digested with PmeI Enzyme

1.0 kb

2.0 kb

4.0 kb

6.0 kb

8.0 kb

10.0 kb

M 1

Fig.18: RE Digestion of pSin.epo.hu rplasmid with PvuI Enzyme

Lane M : Marker 1kb ladder

Lane 1 : rplasmid digested with PvuI Enzyme

1.0 kb

2.0 kb

4.0 kb

6.0 kb

8.0 kb

10.0 kb

M 1

Fig.19: RE Digestion of pSin.epo.hu rplasmid with SalI Enzyme

Lane M : Marker 1kb ladder

Lane 1 : rplasmid digested with SalI Enzyme

Chapter-5

Discussion

Chapter-5

Discussion

Erythropoietin (epo) is a 34 kDa glycoprotein produced

mainly by kidney paratubular cells in response to reduced oxygen

delivery. epo stimulates erythroid progenitor cell proliferation,

differentiation and maturation. It also inhibits cell apoptosis, which

results in increased erythrocyte formation. The recombinant human

erythropoietin (repo.hu) is widely used to compensate for the

reduced production of endogenous epo in renal failure and to correct

the associated anemia. Administration of repo.hu alleviates the

necessity for blood transfusion and greatly improves the quality of

life for patients. Clinical studies have shown that repo.hu can also be

effective in the treatment of other chronic anemia. Injections of high

doses of repo.hu have been shown to increase blood hemoglobin

levels and occasionally to alleviate the need for transfusion.

In the present study human erythropoietin gene was

successfully cloned in pSin Vector. This recombinant plasmid was

designated as pSin.epo.hu. This cloning vector is derived from an

alpha virus (Sindbis virus) and sub genomic promoter of alpha virus is

very strong so that it makes large number of mRNA from the

sequence downstream to it, hence it is a good vector.

78

The cloning resulted in the appearance of many colonies of

which colonies were found to be carrying the epo.hu gene insert. The

presence of epo.hu gene insert and its orientation was checked by

digestion with Restriction enzymes and PCR amplification with

epo.hu gene specific primers. On MapDraw (DNASTAR) the

recombinant pSin vector was predicted to be of 11.38 kb (10.8 bp

vector+0.588 bp epo.hu gene insert). The recombinant plasmid clone

(pSin.epo.hu) was sequenced with BGH reverse primer. The sequence

was matched with available sequences by BLAST in NCBI gene

databank and was found to be homologous with epo.hu gene. In the

past whole gene of epo.hu was divided into three large fragments

consisting of a total of 32 oligonucleotides. These oligonucleotides

were synthesized by the solid-phase phosphor-amidite method and

ligated into three large fragments. The human erythropoietin gene

has been isolated from a genomric phage library by using mixed 20-

mer and 17-mer oligonucleotide probes (Lin et al., 1985). The gene

for murine erythropoietin (epo) was isolated from a mouse genomic

library with a human epo cDNA probe. (Shoemaker and Mitsock,

1986). These latter three were separately cloned into vector M13mp19

and then transformed into E. coli JM 103 (Cai, 1992). Erythropoietin

(epo) genomic gene was cloned and its expression vector pOP13/epo

was constructed. CHO-K12 cell was transfected by this vector using

lipofectin method. A stable expression cell strain C10 cell with the epo

production at 160IU/d in 106 cells were obtained at 400 µg/mL G418.

Based on the C10 cell, another vector pHY/dhfr (dihydrofolate

reductase) that carries a dhfr gene and a selecting marker of

hygromycin B resistant gene was transferred to this cell. Several cell

79

clones were obtained at 200 µg/mL hygromycin B. These cell clones

that can express both epo gene and exogenous dhfr gene were

selected under the progressively increased concentration to 1 µmol

methotrexate (MTX). Some high epo expression cell clones were

obtained, the highest expression was 2400 IU/d in 106 cells, 15 times

higher than that without MTX pressure. Then, a method of epo high

expression by using un-dhfr negative cell was primarily established.

(Cui et al., 1996). The M13mp19 is derivatives of the single-stranded,

male-specific filamentous DNA bacteriophage M13 and it has no

CMV pramoter. epo.hu was amplified from epo cDNA by PCR

methods. The PCR product was further cloned into pUC18 plasmid at

Sma I site, and then precisely engineered into a intermediate vector

pSK43SB which were digested with Hind III, Mung bean nuclease,

and Sal I. Then digest pSK43SB-epo plasmid with EcoR I and Cla I, the

EC fragment with an alpha-factor leading sequence, epo gene and

CYC1 terminate were produced. It was then cloned into a typical high

efficiency episomal expression vector YEpHC8. Human epo protein

with highly mannose glycosylated was identified by Western blot

methods in both secreted and in cells proteins (Zhang et al., 1997). A

gene encoding for mature epo.hu was amplified from human kidney

total RNA by RT-PCR using specific primers. The product was cloned

into the pCR2.1 TOPO cloning vector (invitrogen) and the nucleotide

sequence containing 498 bp encoding for mature epo.hu with 166

amino acids was determined for by ABI PRISM 3100 Avant Genetic

Analyzer (TC Y hoc Viet, 2006) .The human erythropoietin gene

epo.hu was also cloned in pTarget vector (Gangwar et al., 2009). epo

gene cloning was done by RT-PCR from the total RNA of human fetal

80

liver and expressed it in E. coli after insertion of the gene in the

expression vector pBV220. The cloned gene was confirmed by

sequence analysis and gene product was confirmed by both Western

blot and its first 11 amino acid residues sequence of the N-terminal. In

vitro bioassay showed that the purified gene product can repress the

growth of TF-cells in the presence of epo (Zhang et al., 2000).

In comparative studies of conventional (nonreplicating)

plasmid DNA vectors and alpha virus DNA-based replicon vectors

the latter generally produces larger quantity of DNA concentrations

than does conventional vectors. The constructed recombinant

(pSin.epo.hu) has replicase gene which produces large amount of

erythropoietin protein and thus only small amount of DNA will be

needed to be injected for therapeutic agent. The DNA can be stored

and transported at room temperature and thus suitable for use in

Indian conditions where refrigeration increases the cost of drugs.

epo gene were selected under the progressively increased

concentration to 1 µmol methotrexate (MTX) (Cui et al., 1996) . epo-

cDNA construct pcDNA3.1-epo containing only mature epo protein

sequence (495 bp) was provided by CEMB (Centre of Excellence in

Molecular Biology, University of the Punjab, Lahore). For the

construction of epo secretary plasmid, they used this plasmid as a

starting material. PCR-based addition of secretary signal was

performed for extra-cellular protein secretion using the six over

lapping primers at 5' end of epo-cDNA construct. Final PCR product

was a fragment of 588 bp, having 495 bp of mature epo protein

sequence, 81 bp of secretary signal peptide and 12 bp of EcoR1 and

81

BamH1 restriction sites at 5' and 3' end respectively. PCR-product was

digested with EcoR1 and BamH1 (Fermentas, USA) and used for

ligation into plasmid pcDNA3.1/zeo the resulting plasmid was

named as pcDNA3.1-SS-epo In another approach six nucleotides of

Kozak sequence (GCCACC), was also added before the start codon of

epo-cDNA construct by using the primer K as forward and primer R

as reverse primers . The size of this fragment was 594 bp including

the Xho1 and BamH1 restriction sites at 5' and 3' end respectively.

PCR-product was digested with Xho1 and BamH1 and used for

ligation into plasmid pcDNA3.1/zeo the resulting plasmid was

named as pcDNA3.1-K-SS-epo. Following transformation and

subsequent plasmid purification, of randomly selected clones,

restriction digestion and sequencing analysis was performed using

Big Dye chain termination reaction on an ABI 3100 DNA sequence

(Applied BioSystem) to verify the cloning of epo-cDNA constructs in

expression vector (Kausar et al., 2011).

The expression of recombinant plasmid pSin.epo.hu was studied

in HeLa cell culture using immunoperxidase test (IPT), SDSPAGE and

western blotting. The expression of rplasmid was detected by IPT in HeLa

cell line and 90% of the cells were found to express the epo.hu protein by

development of purple color precipitate. The SDSPASE (Sodium dedecyl

sulfate-polyacrylamide gel electrophoresis) analysis of expressed

protein indicated a specific thick band (Blue color) seen in sodium

dedecyl sulfate-polyacrylamide gel and the western blot analysis of

expressed protein indicated a specific band (0range color) seen in

nitrocellulose membrane.

82

The erythropoietin gene, when introduced into Chinese hamster

ovary cells, produces erythropoietin that is biologically active in vitro

and in vivo (Lin et al., 1985). Transformation of COS-1 cells with a

mammalian cell expression vector containing the murine epo coding

region resulted in secretion of murine epo with biological activity on

both murine and human erythroid progenitor cells. The transcription

start site for the murine epo gene in kidneys was determined. This

permitted tentative identification of the transcription control region.

The region included 140 base pairs upstream of the cap site which

was over 90% conserved between the murine and human genes.

Surprisingly, the first intron and much of the 5'- and 3'-untranslated

sequences were also substantially conserved between the genes of the

two species (Shoemaker and Mitsock, 1986). Adaptive responses to

hypoxia occur in many biological systems. A well-characterized

example is the hypoxic induction of the synthesis of erythropoietin, a

hormone which regulates erythropoiesis and hence blood oxygen

content. The restricted expression of the erythropoietin gene in

subsets of cells within kidney and liver has suggested that this

specific oxygen-sensing mechanism is restricted to specialized cells in

those organs. Using transient transfection of reporter genes coupled

to a transcriptional enhancer lying 3' to the erythropoietin gene, they

show that an oxygen-sensing system similar, or identical, to that

controlling erythropoietin expression is widespread in mammalian

cells. The extensive distribution of this sensing mechanism contrasts

with the restricted expression of erythropoietin, suggesting that it

mediates other adaptive responses to hypoxia (Maxwell et al., 1992).

CHO-K12 cell was transfected by pOP13/epo vector using lipofectin

83

method. A stable expression cell strain C10 cell with the epo

production at 160IU/d in 106 cells were obtained at 400 µg/mL G418.

Based on the C10 cell, another vector pHY/dhfr (dihydrofolate

reductase) that carries a dhfr gene and a selecting marker of

hygromycin B resistant gene was transferred to this cell. Several cell

clones were obtained at 200 µg/mL hygromycin B. These cell clones

that can express both epo gene and exogenous dhfr gene (Cui et al.,

1996). Expressed epo.hu in E.coli cells, the epo.hu gene was sub

cloned into pET21d(+) vector (Novagen) by ligase reaction and was

transformed into BL21 (DE3) cells. The expression of the epo.hu was

analyzed by SDS-PAGE and confirmed by Western Blotting using anti

epo.hu antibody (TC Y hoc Viet, 2006). The Pichia pastoris expression

system was used to produce recombinant human erythropoietin, a

protein synthesized by the adult kidney and responsible for the

regulation of red blood cell production. The entire recombinant

human erythropoietin (repo.hu) gene was constructed using the

Splicing by Overlap Extension by PCR (SOE-PCR) technique, cloned and

expressed through the secretory pathway of the Pichia expression system.

Recombinant erythropoietin was successfully expressed in P. pastoris.

The estimated molecular mass of the expressed protein ranged

from 32 kDa to 75 kDa, with the variation in size being attributed to

the presence of repo.hu glycosylation analogs. A crude functional

analysis of the soluble proteins showed that all of the forms were active in

vivo (Maleki et al., 2010).

The IPT technique also known as immune enzyme technique

(IET) is used for the detection of several viruses and highly sensitive

84

technique. It is replaces the immune fluorescence technique. The

stained studies can be observed in ordinary microscope and stained

slides can be preserved for longer period with at any deterioration, it

can be used for smears, culture monolayer, as well as thin section.

This method is easy to detected expressed protein in HeLa cells or cell

culture. Viral proteins and higher molecular weight proteins are

separated by SDS-PAGE using a pure gene preparation. In western

blot analysis SNAP i.d. Protein Detection System was used. It was

Millipore‟s patent pending SNAP i.d. Protein Detection System

provides a fast and convenient method for the detection of immune

reactive proteins on western blots. With this unique vacuum-driven

system, the amount of time required for immune detection is greatly

reduced. What previously took 4 to 24 hours by traditional western

blotting methods now takes only 30 minutes to complete without any

loss of signal intensity or reduction in blot quality. All

immunodetection steps after protein transfer to a membrane (i.e.,

blocking, washing, and primary and secondary antibody incubations)

are performed utilizing the SNAP i.d. Protein Detection System.

Thus in the present work human epo gene was successfully

cloned in pSin Vector and it was found to produce high level of

expression in HeLa cells.

85

Chapter-6

Summary and Conclusion

Chapter-6

Summary and Conclusion

Erythropoietin (epo) is a glycoprotein hormone responsible

for the regulation of red blood cell production. This hormone triggers

the proliferation, differentiation and maturation of bone marrow

erythroid precursors into functional erythrocytes when blood oxygen

availability is decreased, such as during hypoxia. epo binds to and

activates the receptor on erythroid progenitor cells. The treatment of

anemic patients with epo significantly reduces their dependence on

blood transfusions and minimizes potential side effects such as iron

overload, infections and adverse reactions to leukocyte antigens. In

addition to its role in hematopoeisis, epo is neuroprotective in the

nervous system and can also protect other organs (Genc et. al., 2004).

The erythropoietin (epo.hu) gene already cloned in

pTarget vector designated as pTarget.epo.hu was used to obtain

epo.hu gene. The entire sequence of insert (epo.hu gene) and vector

pTarget was analyzed in map draw of DNASTAR laser gene program

to find appropriate site for restriction enzyme which cut in vector

flanking the insert. The E.coRI enzyme located in MCS of the vector

(pTarget vector) was found suitable to release the epo.hu gene insert

without having any cutting site in the epo.hu gene insert digestion

with this E.coRI enzyme released a product of approximately 588

86

base pairs including some sequences from the vector‟s MCS and

recombinant vector backbone of 5670 bp. The pSin Vector was isolated

by TELT method and it was linearized by digestion with StuI

restriction enzyme. Blunt end ligation was done in the presence of

PEG 8000 and ligated pSin vector was transformed in E.coli (DH5α)

cells. A large number of colonies (25) were picked from the overnight

grown transformants. The individual colonies were inoculated in

fresh ampicillin (50µg/ml) containing LB broth and allowed to grow

for 18 hours. Plasmid (pSin.epo.hu) was isolated from these colonies

by TELT methods.

Selection of the clone containing human erythropoietin

gene in right orientation was done using restriction enzyme analysis

with kpnI restriction enzyme, Polymerase chain reaction and DNA

sequencing. The clone was selected by Restriction enzyme analysis of

isolated plasmid (pSin.epo.hu) check for the presence of epo.hu gene

insert was analyzed in map draw to find kpnI restriction enzyme

which could release the product of desired length and was selected

again by PCR amplification of isolated plasmids with epo.hu gene

specific primers and BGH as reverse primers. Finally the rplasmid

clone with insert in right orientation (pSin.epo.hu) was sequenced

with BGH reverse primer. The obtained sequence was analyzed by

BLAST (NCBI) in the light of its similarly with existing sequence for

human erythropoietin gene and the sequence from which primer was

designed and it conformed the gene in right orientation.

The expression of rplasmid (pSin.epo.hu) was checked

by immunoperoxidase test (IPT) in HeLa cell line and cells were

87

found to express the epo.hu protein by development of purple color

precipitate. The expression of rplasmid was again checked by using

SDS-PAGE and western blot analysis. The SDS-PASE and western

blot (Sodium dedecyl sulfate-polyacrylamide gel electrophoresis)

analysis of expressed epo.hu protein indicated a specific isolated thick

band in gel as well as nitrocellulose membrane. The entire sequence of

pSin.epo.hu DNA was analyzed in mapdraw of DNASTAR lasergene

program to find different sites for restriction enzymes and were again

checked for the presence of restriction sites. Different restriction

enzymes PvuI, NheI, PmeI, NruI, SalI and NotI could release the

product of expected size. The enzymes PvuI, SalI and NotI were chosen

which had one sites for each enzyme. Digestion with PvuI and NotI

enzymes released a product size approximately between the ranges of

8000 to 10000 bp while digestion with enzyme SalI released a product

size approximately between the ranges of 4000 to 6000 bp. The

enzyme NheI was chosen which had five sites. Digestion with this

enzyme released products size near about 2000 to above 10000 bp. The

restriction enzymes PmeI and NruI were chosen which had two sites for

each enzyme. Digestion with PmeI released a product size

approximately between the ranges of 8000 to above 10000 bp while

digestion with enzyme NruI released a product size between the

ranges of 2000 to above 1000 bp.

88

Chapter-7

Future Scope

Chapter-7

Future Scope

Anemia is commonly prevalent in Indian

population. The condition of having less than the normal number of red

blood cells or less than the normal quantity of hemoglobin in the blood

are known as anemia. Erythropoietin induces erythropoiesis / formation

of erythrocyte cells in human blood.

A recombinant plasmid DNA (pSin.epo.hu) has been

developed which contains human erythropoietin gene. This plasmid

DNA if injected will produce human erythropoietin protein which in

turn will enhance the red blood cell formation. Thus, it can be

successfully used as therapy in cases of anemia in human. It is in

liquid form ready for direct injection and no diluent is needed. Hence

evaluation of the recombinant plasmid may be studied for clinical

trails after obtaining relevant permissions.

89

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Schweitzer, A.C., Wickrema, A. and .Conboy, J.G. (2009): Alternative

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109

Appendix

Appendix

A. PLASMID DNA ISOLATION BY TELT METHOD

TELT Solution

Tris Cl 50 mM

Na2 EDTA 62.5 mM

LiCl 2.5 M

Triton X-100 4% v/v

Phenol/ chloroform 1:1 v/v

Adjust the pH to 8.0 with 5NaOH.

B.REAGENTS USED IN AGAROSE GEL ELECTROPHORESIS

1. Tris-acetate-EDTA (TAE) buffer 50X

Tris base 242 g

Glacial acetic acid 57.1 ml

0.5 M EDTA (pH 8.0) 100 ml

Distilled water was added to make final volume up to 100 ml. A

working solution of 1X was used.

2. Ethidium bromide stock solution (10 mg/ ml)

Ethidium bromide 100 mg

Distilled water 10.0 ml

The solution was mixed and stored at 40C. A concentration of 0.5 µg/ ml

was used in preparing agarose gel.

3. Loading Dye (6X)

Bromophenol blue

Xulene cyanol FF

Sucrose

0.25 % (w/v)

0.25% (w/v)

40% (w/v)

C. REAGENTS USED FOR BACTERIOLOGICAL PROCEDURES

1. LB (Luria-Bertani) broth

Bacto tryptone 10 g

Bacto yeast extract 5 g

NaCl 10 g

Distilled water 950 ml

Adjust the pH to 7.0 with 5N NaOH.

Volume was made up to 1L and sterilized by autoclaving at 15 psi (1.05 Kg/

cm2) for 20 mins and the solution was stored at 40C.

2. LB agar

1.5% Agar in LB medium.

Sterilized by autoclaving at 15 psi (1.05 Kg/ cm2) for 15 mins and stored

at 40C.

3. SOB Medium

Bacto tryptone 20 g

Bacto Yeast extract 5 g NaCl 0.5 g

Deionized water 950 ml

Dissolve by shaking. Add 10 ml of a 250 mM solution of KCl. Adjust the pH

to 7.0 with 5N NaOH and make volume up to 100 0ml. Sterilize by

autoclaving. Just before use, add 5 ml of a sterile 2M MgCl2.

4. SOC Medium

To SOB medium, 2mM glucose (filter sterilized) was added.

5. 1XTSS (Transformation and storage solution)

LB broth 95 ml

PEG 8000 (w/v) 10 g

MgSO4 (50 mM final Conc.) 1.23g

DMSO 5.0 ml

LB broth was filtered through 0.22µ filter and added after autoclaving of

other ingradients at 1210C, 15 psi for 10 min., DMSO

6. Ampicillin (50 mg/ ml)

Ampicillin 250 mg

Distilled water 5 ml

Sterilized by filtration through 0.22 I filter and stored at - 200C.

D. REAGENTS USED IN CELL CULTURE

1. PBS

NaCl 8.00 g

KCl 11.95 g

Na2HPO4 (anhydrous) 1.44 g

KH2PO4 0.24 g

Distilled water was added to make the final volume upto 1 litre. The pH

was adjusted to 7.4 with HCl. The resulting solution was autoclaved and

stored.

2. DMEM (Gibco)

DMEM powder 13.4 g

With high glucose

With L-glutamine

With pyridoxine hydrochloride

Without sodium bicarbonate

Without sodium pyruvate

Distilled water (up to) 1000 ml

Dissolved in 950 ml of distilled water and mixed 3.7 gm of NaHCO3 per

liter of medium. Distilled was added to make final volume up to 1000

ml. The pH was adjusted to 0.2-0.3 below desired working pH. Sterilized

by filtration.

3. Growth medium

DMEM 90 ml

FCS 10 ml

4. Trypsin Versene Solution (0.17%)

NaCl 10.0 g

KCl 0.250 g

Na2HPO4 1.9 g

KH2PO4 0.250 g

Trypsin 1.7 g

Versene 1.4 g

0.4%Phenol red 1.0 µl

Distilled water to make up to 1000 ml

E. REAGENTS USED IN IMMUNOPEROXIDASE TEST

Nadi reagent

Alpha naphthol 15 mg

Phenylenediamine 22 mg

PBS 20 ml

F. SOLUTIONS USED FOR SDS-PAGE & WESTERN BLOTTING

1. Lower gel buffer (pH 8.8)

Tris 18.15g

SDS 0.40 g

Distilled water was added to make the final volume to 100ml.

2. Upper gel buffer (pH 6.8)

Tris 5.98 g

Distilled water was added to make the final volume to 100ml.

3.Resolving gel 10%

Lower gel buffer 3.75 ml

Acrylamide stock 5.00ml

D/W 6.05ml

10% SDS 0.15ml

10% Ammonium persulfate 0.05ml

TEMED 0.007ml

4.Stacking gel 4%

Upper gel buffer 1.25ml

Acrylamide stock 0.80ml

D/W 3.87ml

10% SDS 0.06ml

10% Ammonium persulfate 0.02ml

TEMED 0.007ml

5. Tris Glycine electrophroesis buffer (5X)

Tris base 15.1 g

Glycine (pH 8.3) 93.8g

6. Sample buffer (2X)

Stacking gel buffer 1.7 ml

10 % SDS 4.5 ml

Glycerol 1.0 ml

Bromophenol blue 2.0 ml

Distilled water 2.3 ml

7. Ammonium persulphate (10%)

APS 1.0 g

Distilled water 10.0 ml

9. Acrylamide (30%)

Acrylamide 29.2 g

N-N-methylene-bis- acrylamide 0.80 g

Dissolved in 60 ml distilled water by heating at 37ºC and adjust

volume to 100 ml. Filtered and stored in amber colored bottle at 4 ºC.

10. Gel staining solution

Coomassie brilliant blue 0.25 g

Methanol: deionized water (1:1) 0.80 g

Glacial acetic acid 10 .00 ml

The solution was filtered through Whatman filter paper No. 1

11. Gel destaining desolution

Methanol: deionized water (1:1) 90 .00 ml

Glacial acetic acid 10 00 ml

12. Transfer buffer

Tris base 2.90 g

Glycine 11.95 g

SDS-PAGE 0.185 g

Methanol 100 ml

Distilled water was added to make the final volume up to 50 ml.

13. Blocking buffer

PBS 1000ml

Tween-20 0.5 %

Skimmed milk powder 5%

14. Dilution buffer

PBS 1000ml

Tween-20 0.5 %

Skimmed milk powder 2.5%

31

Biotechnology International 4(2): 31-35, April-June 2011

ISSN 0974-1453 Online www.bti.org.in

Cloning of human erythropoietin gene in pSinCMV vector

Usha Tiwari *, Kusum Agarwal *, Nishant Rai #, Anant Rai +, Priyanka Pal*

*School of Biotechnology, Shobhit University, Modipuram, Meerut-250110, U.P.

# Department of Biotechnology, Graphic Era University, Clement Town, -Dehradun,

Uttarakhand, + Institute of Biotechnology and IT, Mudia Ahmed nagar, Pilibhit Road,

Bareilly243122, U.P. *Author for Correspondence, ushatiwari@rocketmail.com

Summary: The erythropoietin (epo.hu) gene already cloned in pTarget vector was released by

digesting with EcoRI and cloned in StuI site of pSinCMV vector. The clone containing gene in

right orientation was confirmed using RE digestion, PCR and sequencing and designated as

pSinCMV.epo.hu.

Keywords: Erythropoietin gene, pSinCMV vector, replicase vector, gene cloning

Introduction

Erythropoietin is a glycoprotein hormone that controls erythropoiesis or red blood cell

production. It is a cytokine for erythrocyte (red blood cell) precursors in the bone marrow also

called hematopoietin or hemopoietin, it is produced by the capillary endothelial cells in

the kidney and liver, it is the hormone that regulates red blood cell production. It also has other

known biological functions. For example, erythropoietin plays an important role in the brain's

response to neuronal injury. Erythropoietin is also involved in the wound healing process (Lin et.

al.,1985). Erythropoietin expression increases in five-sixths nephrectomized rats, after muscle-

targeted gene transfer by in vivo electroporation, using plasmid DNA expressing rat epo

(pCAGGS-epo) (Ataka et.al.,2003). Myelodysplastic syndrome (MDS) may be induced by certain

mutagenic environmental or chemotherapeutic toxins; however, the role of susceptibility genes

remains unclear. The G/G genotype of the single-nucleotide polymorphism (SNP) rs1617640 in

the erythropoietin (epo) promoter has been shown to be associated with decreased epo expression

(Ma et. al., 2010). Keeping the above facts in view, the present work was undertaken to clone the

human erythropoietin gene in replicase based eukaryotic pSinCMV vector.

Materials and Methods

Vectors: pTarget.epo.hu. recombinant plasmidand pSinCMV vector were available in

Biotechnology Laboratory of IBIT, Bareilly.

E.coli culture: Escherichia coli DH5 host strain for transformation of recombinant plasmids was

available in the lab.

Isolation of plasmid DNA: Revival of the E.coli culture containing pTarget vector with

32

erythropoietin gene was done in LB broth and plasmid DNA was isolated following TELT

method (He et. al., 1990). Grown 1.5 ml E.coli culture with plasmid in LB medium containing

100µg/ml ampicillin for 16 h. Pelleted cells for 30 sec in microcentrifuge and resuspended in

100 µl TELT solution and added an equal volume of 1:1phenol/chloroform. Vortexed

vigorously for 15 sec and spinned 1 min in microcentrifuge at 220C . Collected the upper

phase of nucleic acid and mixed with 2 vol of 100% ethanol. After 2 min, spinned 10 min in

microcentrifuge at 40C. Washed pellet with 1 ml of 70% ethanol, allowed to dry and

resuspended in 30 µl TE buffer. Plasmid DNA was checked on 1% agarose gel

electrophoresis.

RE digestion and DNA extraction from agarose gel: RE digestion of pTarget.epo.hu with EcoRI

was done. The gel extraction of DNA fragment was done using MinElute gel extraction kit (Qiagen,

Germany) following manufacturers instruction.

Blunting of EcoRI generated human erythropoietin gene staggered ends: For

blunting of staggered ends generated by EcoRI enzyme, T4 DNA polymerase was used.

Reaction mixture consisted of epo gene insert 20 µl, T4 DNA polymerase buffer 10X 5

µl, T4 DNA polymerase 2 µl, dNTP mix. (10mM each) 1 µl, nuclease free water 22 µl,

making total volume 50 µl. The reaction mixture was incubated at 37˚C for 10 min.

Purification of blunted insert: The blunted epo gene insert was purified using phenol

chloroform, following the protocol of Sambrook and Russell (2001).The presence of purified

blunted DNA was checked by running 1 µl of DNA on 1% agarose gel.

Preparation of pSinCMV vector: The pSinCMV vector DNA was digested with

StuI to create blunt end using 50 µl reaction mixture. The linearised plasmid was checked on

1% agarose gel electrophoresis. It was than dephosphorylated using Calf Intestinal Alkaline

Phosphatase (CIAP) to prevent self-ligation. Purification of dephosphorylated linearized

vector was done using phenol chloroform as per method of Sambrook and Russell (2001).

Blunt end ligation of pSinCMV vector and epo gene: The reaction mixture consisted

of 30% PEG 8000 for blunt end ligation. The reaction mix consisted of: T4 DNA Ligase

(Fermentas) 2.0 µl, pSinCMV vector 0.5 µl, epo gene 4.0 µl , ligation buffer (10x) 1.0µl, 30%

PEG8000 (Amresco) 1.5 µl nuclease free water 1.0 µl , making total volume to10 µl in a

microfuge tube. The reaction was incubated overnight at 15˚C.

Transformation of E.coli (DH5 ) cells with the ligated product: The single step method of

competent cell preparation and transformation was used (Chung et. al., 1989).

Screening of recombinant clones: Few colonies were picked from the overnight

grown transformants. The individual colonies were inoculated in fresh ampicillin

(50µg /ml) containing LB broth and allowed to grow for 18 to 24 hours. Plasmid was

isolated from these colonies by TELT method. Plasmid was checked on 1% agarose gel

electrophoresis.

33

Digestion of plasmid with kpnI enzyme to check the presence and right orientation

of insert: Isolated plasmid was checked for the presence of insert and its right orientation

by digestion with enzyme kpnI .The digestion mixture (15 µl) contained: Plasmid 4.0 µl,

kpnI 1.0µl, buffer (10X) 1.5 µl, nuclease free water 8.5 µl. The reaction mixture was

vortexed and spun and kept in incubator at 37˚C overnight. The digested mixture was then

electrophoresis in 1.5% agarose. The released fragments after digestion were compared

against 1 kb marker.

Colony PCR: The presence of gene insert in right orientation in the recombinant plasmid

was confirmed by PCR using gene specific forward primer and BGH as reverse primer.

The PCR reaction mixture (50 µl) contained 100 ng of recombinant plasmid, 50 pmol

each of gene specific forward primer and BGH as reverse primer and 3 units of Taq DNA

polymerase in 1x PCR buffer. The reaction was carried as follows: distilled water 33 µl,

rplasmid 5 µl, Forward primer of epo gene 2 µl, BGH Reverse primer 2 µl, dNTPs 2 µl,

buffer (10X) 5 µl , Taq DNA polymerase 1 µl. The epo gene was amplified following

initial denaturation at 94˚C for 5 min and 30 cycles of denaturation at 94˚C for 30

seconds, annealing at 50˚C for 50 seconds, amplification at 72˚C for 7 min and final

amplification at 72˚C for 10 min. After amplification, an aliquot of 10 µl was subjected to 1%

agarose gel electrophoresis along with 100 bp DNA molecular weight marker.

Sequencing: The recombinant plasmid selected after the above two methods was

sequenced using BGH primer.

Results and Discussion

The epo gene already cloned in pTarget vector was released by digesting with EcoRI

enzyme and the plasmid DNA was gel eluted and purified. It was successfully cloned in

pSinCMV vector using blunt ligation method. Vector sequence along with the cloned

insert was analysed in Mapdraw using DNASTAR software to find the restriction

enzymes, which could release the product so as to identify the plasmid containing gene in

right orientation. The enzyme kpnI was chosen which released three fragments viz.

0.152kb, 3.359 kb and 7.856 kb if the gene was in right orientation. The fragment sizes

obtained were in agreement with the prediction from Mapdraw analysis, which confirmed

that the gene was in right orientation. PCR with epo gene specific forward primer and

BGH as reverse primer further confirmed that clone was in right orientation. Sequencing

of the rplasmid further confirmed that the gene was in right orientation. This recombinant

plasmid was designated as pSinCMV.epo.hu. pSin is derived from an alpha virus (Sindbis

virus). The sub genomic promoter of alpha virus is very strong so that it makes large

number of target mRNA from the sequence downstream to it. Erythropoietin (epo)

genomic gene was also cloned and its expression vector pOP13/epo was constructed (Cui

et.al., 1998). In another study, A 600 bp synthetic erythropoietin gene encoding all 166

amino acids of the epo protein and 27 amino acids of the signal peptide had been

constructed. The results indicated that the nucleotide sequence of the synthetic epo gene

was identical to that of the original (Yi et. al., 1992). In comparative studies of

conventional (nonreplicating) plasmid DNA vectors and alpha virus DNA-based replicon

34

vectors the latter generally produces larger quantity of DNA concentrations than does

conventional vectors.

1 2 M 3

M 1

7.856 kb

3.359 kb

588bp

Fig.1. RE digestion of Fig.2. Lanes M: 1 kb pSinCMV.epo.hu with KpnI . ladder; 1: Epo 588 bp PCR Lane M, 1kb DNA ladder; 1 &3, product

undigested rplasmid; 2, rplasmid

cut with KpnI producing

fragments of 3.359, 7.856 kb

References

Ataka K, Maruyama H, Neichi T, Miyazaki J, Gejyo F, (2003). Effects of

erythropoietin- gene electrotransfer in rats with adenine-induced renal failure. Am J

Nephrol, 23: 315-323.

Chung CT, Niemela SL, Miller RH, (1989) . One step preparation of

competent Escherichia coli: Transformation and storage of bacterial cells in the same

solution. Proc Natl Acad Sci USA, 86: 2172-2175.

Cui Z, Liu P, Qi S, Shen H,(1998 ).High expression of epo gene using

unChinese Science Bulletin. 43: DOI: 10.1007/BF03186992.

Elliott S, (2008) .Erythropoietin- stimulating agents and other method to enhance

oxygen transport. British J Pharmacology . 154: 529-41.

35

Gerna G, Mccloud CJ, Chambers R (1976). Immunoperoxidase technique for

detection of antibodies to human cytomegalovirus. J Clinical

Microbiology. 3: 364-372.

He Wilde, Kaderbhai MA (1990). A simple single step procedure

for small scale preparation of Escherichia Coli plasmid. Nucleic Acids Res, 18:

1660.

Imagawa S, Yamamoto M, Miura Y, (1996). GATA Transcription

Factors Negatively Regulate Erythropoietin Gene Expression. Acta

Haematological, 95: 3-4.

Lin FK, Suggs S, Lin CH, Browne JK, Smalling R, Egrie JC, Chen

K. K, Fox G. M, Martin F, Stabinsky Z, (1985). Cloning and

expression of the erythropoietin gene. Proc Natl Acad Sci USA, 82: 7580-

7584.

López RM, Aladrén BS, García FG, (2009).Use of agents stimulating

erythropoiesis in digestive diseases .World J Gastroenterol, 15: 4675-4685.

Mastrogiannaki M, Matak P, Keith BM, Simon MC, Sophie

V, Eyssonnaux C, (2009). HIF , but not HIF-1 , promotes iron absorption

in mice. J Clin Invest, 119: 1159-1166.

Ma W, Kantarjian H , Zhang K , Zhangm X , Wang X , ChenC , Donahue A C, Zhang

Z , Yeh C , O'Brien S , Manero G , Caporaso N, Landgren O,

Albitar M (2010).Significant association between polymorphism of the

erythropoietin gene promoter and myelodysplastic syndrome. BMC Medical

Genetics.11:163doi:10.1186/1471-2350-11-163.

Markusic DM, Waart DR, Seppen J (2010). Separating lentiviral

vector injection and induction of gene expression in time, does not prevent an

immune response in rats . PLoS One. 5: e9974.

Sambrook J, Russell DW (2001). Molecular Cloning. A Laboratory

Manual,3rd ed.,Cold Spring Harbour Laboratory Press, cold Spring Harbour,

NewYork.

Schoffel N, BorqerJ.Z, Quarcoo D, Scutaru C, Groneberq DA (2008).

Erythropoietin- state of science. Sportverletz Sportschaden, 224:201-206.

Yi Z, Ke X, Yuan X, Bao X (1992) Synthesis and cloning of the whole

human erythropoietin (epo) gene. Cai Y,14:173-8.

Res J Pharm Sci Biotech 2011;1(1):14-16

RESEARCH ARTICLE

RESEARCH JOURNAL OF PHARMACEUTICAL SCIENCE AND BIOTECHNOLOGY Available online at: www.rjpsb.info

EXPRESSION OF RECOMBINANT PLASMID CONTAINING HUMAN ERYTHROPOIETIN

GENE IN HELA CELLS LINE USING IMMUNOPEROXIDASE TEST

*Usha Tiwari1, Kusum Agarwal1, Nishant Rai2, Anant Rai3, Priyanka Pal1

1School of Biotechnology, Shobhit University, Modipuram, Meerut, India, 2Department of Biotechnology, Graphic Era University,

Clement Town, Dehradun, Uttarakhand, India. 3Institute of Biotechnology and IT, 197, Mudia Ahmadnagar, Bareilly, India.

ABSTRACT

The erythropoietin (epo.hu) gene cloned in pSinCMV vector was analysed for its expression by immunoperoxidase test

in HeLa cells. The cells were found to express the protein by development of purple colour precipitate. Intense purple

coloration of cells was observed in which HeLa cells were transfected with rpSinCMV.epo.hu. The vector alone and

untransfected healthy cells failed to show any colouration indicating that the epo gene was expressed in cells due to the

presence of rpSinCMV.epo.hu. Plasmid

Keywords: HeLa cells, Erythropoietin gene, pSinCMV vector, replicase vector, gene expression, immunoperoxidase test.

Abbreviations: LB- Luria Bertani, dNTPS- dinucleotide triphosphates, ʅ ů- microlitre, PEG- Polyethylene glucose

INTRODUCTION

Erythropoietin (epo) is the glycoprotein

hormone which regulates mammalian erythrocyte

production and, as a result, tissue oxygen delivery.

epo RNA levels increase several hundredfold in ro-

dent liver and kidney in response to hypoxia or

anemia[1]. A hormone produced by the kidney that

promotes the formation of red blood cells in the

bone marrow. epo is a glycoprotein (a protein with

a sugar attached to it). Human epo has a molecular

weight of 34,000[2]. Keeping the above facts in view,

the present work was been undertaken to expres-

sion of recombinant plasmid containing human

erythropoietin gene in HeLa cell line using IPT Test.

MATERIALS AND METHODS

Cell line

Corresponding Author: Usha Tiwari

School of Biotechnology, Shobhit University, Modipuram,

Meerut, India

E mail: ushatiwari@rocketmail.com

HeLa cells were available in Biotechnology

lab of IBIT, Bareilly.

Plasmid DNA

pSinCMV.epo.hu. recombinant plasmid was

constructed and available in Biotechnology lab of

IBIT, Bareilly[3].

Preparation of antibody against recombinant

plasmid in mice

Primary polyclonal antibody against the hu-

man erythropoietin gene was raised in mouse by

hyper immunization with pSinCMV.epo.hu recom-

binant plasmid.

Detection of expressed protein in cell culture using

immunoperoxidase Technique[4].

Transfection of HeLa cells

Calcium-Phosphate-Mediated Transfection

of eukaryotic cells with plasmid DNA: Harvested ex-

ponentially growing cells trypsinization and pre- Tiwari U, et al.: Expression of recombinant plasmid containing human erythropoietin Gene in Hela cells line using Immunoperoxidase test

Res J Pharm Sci Biotech 2011;1(1):14-16

pared cell suspension growth medium. Prepared

the calcium-phosphate-DNA corecipitate as follows:

combined 100 ʅ ů ŽĨ Ϯ ͘ϱ D Ă ůϮ with 20 ʅ ů ŽĨ

ƉůĂs-

ŵŝĚ E ŝŶ Ă ƐƚĞƌŝůĞ ŵŝĐƌŽĨƵƐĞ ƚƵďĞ ͘ ĚĚĞĚ ϴϬ

ʅ ů

D/W keep at RT for 1min. immediately transferd the

calcium phosphate- E ƐƵƐƉĞŶƐŝŽŶ ƵƐŝŶŐ ϮϬ

ʅ ů

suspension for each well. Added ϭϬϬ ʅ ů ŽĨ ĐĞůů ĐƵl-

ture suspension in each well gently to mix the me-

dium, which was became yellow orange and turbid.

Carry out that step as quickly as possible because

the efficiency of transfection declined rapidly once

the DNA precipitated was formed. Kept controled

wells without transfection. Incubated at 37°C in a

humidified incubator with an atmosphere of 7%

CO2 for 72 hours.Examination for gene expression

by IPT.

Immunoperoxidase test (IPT)

/Ŷ ϱϲ ǁĞůů plates HeLa cells with epo protein

and vector each in duplicate along with healthy

controls were incubated for 48 hours at 37°C. Cells

were washed with 1X PBS twice and fired with

chilled acetone. After washing the fixed cells with

PBS, cells were treated with 2% H2O2 in PBS for 10

ŵŝŶƵƚĞƐ ĂŶĚ ĂŐĂŝŶ ǁĂƐŚĞĚ ǁŝƚŚ W ^ ƚǁŝĐĞ ĨŽƌ ϱ

minutes each. Cells were incubated with ϭϬʅ ů

mouse anti epo protein serums for 2 hours and

ǁĂƐŚĞĚ ƚŚƌŝĐĞ ǁŝƚŚ W ^͕ ϱ ŵŝŶƵƚĞƐ ĞĂĐŚ ͘ ϭϬʅ ů

ZĂb-

bit anti mouse HRP conjugate antibody was added

to wells and incubated for 1 hour. The cells were

again washed with PBS thrice and incubated in CO2

incubator. After the development of colour, cells

were washed with PBS, dried in air and observed

and photographed.

RESULT AND DISCUSSION

Cells showing expression of epo protein

while control cells did not show any colour devel-

opment. The immunoperoxidase technique (IPT),

also Known as immunoenzyme technique (IET), is

used for the detection of several viruses and highly

sensitive technique. It is replaces the immunofluo-

rescence technique. The stained slides can be ob-

served in ordinary microscope and stained slides

can be preserved for a longer period with deteriora-

tion. It can be used for smears, culture monolayer,

as well as thin sections. This method is easy to de-

tect expressed protein in cell culture.

CONCLUSION

From the study we conclude that the im-

munoperoxidase test is easy to carry out, it does

not require any special technical equipment and

moreover, all reagents are easy to obtain. It has the

distinct advantage in that an ultraviolet microscope

is not required and the preparation lasts indefi-

nitely.

Res J Pharm Sci Biotech 2011;1(1):14-16

REFERENCES

1. L Gerna Semenza, LW Guang. A nuclear factor induced by hypoxia via de novo protein syn-

thesis binds to the human erythropoietin gene enhancer at a site required for transcrip- tional activation. Molecular and Cellular Biol- ogy 1992;12 ͗ϱϰϰϳ-ϰϱ.

2. CX Zhang, YL Jiang, C Hu, XF Wu. High expres- sion of human EPO gene in the larvae and pu-

pae of the silkworm, Bombyx mori Sheng Wu Gong Cheng Xue Baoϭ͘ϲ 2000:ϰϲ-ϱϬ.

3. P Pal, K Agarwal, A Rai, U Tiwari. Cloning of chicken anemia virus VP3 gene in pSinCMV

vector. Biotechnology International 2011;4:ϭϲ-21.

4. J Sambrook, DW Russell. Molecular cloning. A Laboratory Manual, 3rd ed, Cold Spring Harbor Laboratory Press, Spring Harbor, New York, 2001.

Acknowledgements I express my sincere thanks to director

of Institute of Biotechnology and IT Bareilly, In- dia and vice chancellor of School of Biotechnol- ogy, Shobhit University, Modipuram, Meerut, India, whose gave me an opportunity to work in the institute and university for completing my research work.

Tiwari U, et al.: Expression of recombinant plasmid containing human erythropoietin Gene in Hela cells line using Immunoperoxidase test

Isolation of recombinant plasmid DNA containing

Erythropoietin gene by TELT Method.

Usha Tiwari *, Priyanka Pal *, Kusum Agarwal *, Anant Rai **, Nishant

Rai**, Swati Saxena**, Raj Veer Maurya** and Nitin Sharma **

* School of Biotechnology, Shobhit University, Meerut.

** Institute of Biotechnology and Information Technology, Bareilly.

ABSTRACT

Erythropoietin is the only haematopoietic growth factor that behaves

like a hormone. Produced in the kidneys and the liver, erythropoietin

interacts with erythroid progenitor cells in the bone marrow. Erythropoietin

(Epo) is a glycoprotein hormone that promotes the production of red blood

cells. The E.coli culture containing recombinant plasmid with human

Erythropoietin gene was received plasmid DNA and was isolated by TELT

Method.

KEYWORDS

Erythropoietin gene, Cloning vector.

INTRODUCTION

Erythropoietin (Fig) is the principal hormone involved in the regulation and

maintenance of the physiological level of circulating erythrocyte mass. The

hormone is produced primarily by the kidney in the adult and by the liver during fetal

life and is maintained in the circulation.

1

Fig: ERYTHROPOIETIN (Epo)

Production of Epo is stimulated under conditions of hypoxia Lin [1].

Erythropoietin (Epo) was the primary regulator of erythropoiesis, controlling the

proliferation, maturation, and survival of erythroid progenitor cells. The functions of Epo were

mediated through its specific receptor (EpoR) expressed mainly on the surface of erythroid

progenitor cells, and the expression of both responds to hypoxia.

Researchers have identified a gene called erythropoietin (Epo) that was linked to

higher risk of severe retinopathy and nephropathy, eye and kidney diseases that

often affect diabetic patients Spivak [2].

MATERIALS AND METHODS

MATERIALS

Host Bacterial strains

Escherichia coli (E.coli) DH5α (Protégés, Madison) host strain was used for isolation of

recombinant plasmid.

METHODS

Revival of the E.coli culture.

Preparation of competent cell and Transformation by TSS Method

Chung [3].

Took DH5α bacterial culture. Add equal volume of ice cold 2XTSS and the cell

suspension was mixed gently. Add 2µl of plasmid DNA and competent cell 100µl

and mixed gently. Incubated 60 minute at 4ºC.0.9 ml aliquot of SOC was added.

Incubated at 37ºC with shaking for 1hrs to allow expression of the antibiotic-

Resistance gene. Transformants were selected by standard methods.

Growth of rE.coli cells.

Preparation of LB broth: 2.5gm LB broth in d/w, sterilized by autoclaving at 15 lbs psi

(1210C ) for 15 min. Preparation of LB Agar: 4.0gm LB Agar powder in 100ml d/w

sterilized by autoclaving as above. LB agar plate: LB agar plate prepared by streaking.

LB agar cooled to 420C and added ampicillin to provided concentration of 100µg/ml.Add

given rE.coli culture by streaking method.

Observation.

White colonies grow on Petri plate. For preparing E.coli culture in LB broth

containing 100µ g/ml ampicillin, inoculated 200µl culture in 100µl broth and kept in

incubator at room temperature(370C) for 24 hours.

Isolation of plasmid DNA.

Isolation of plasmid DNA miniprep by TENS method.

Took 1.5 ml freshly bacterial culture in 1.5 ml tube. Spin for 30 sec at 10000xg. Removed

all supernatant leaving around 50µl LB. (it is better drain over tissue paper to remove

excess LB after discarding supernatant). Resuspend the cells uniformly by vortexing or

tapping and add 300µ of TENS and vortex for 10 seconds to mix properly. (It is better to

add TENS in one tube at a time mix thereafter add in other tube).Add 150µl of 3M

sodium acetate, pH 5.2, close the lid of the tube and vortex for seconds. Good vortexing

is critical at this point. Spin the tubes for 3 min in microcentrifuge at 10000xg.Transfer

the supernatant in fresh tube and 1 ml of chilled ethanol and keep on ice for 15 min.

Centrifuge for 10 min at 12000xg, remove the supernatant and 1 ml of 70% chilled

ethanol. Recover the plasmid DNA by centrifuge at 10000xg for 10 min. Dry the pellet

and resuspend in 40µl TE containing RNase (25µg per ml concentration) keep at room

temp for 10 min. Now DNA is ready for restrict ion enzyme digestion.

Isolation of plasmid DNA Alkali lysis method (P1, P2, & P3 method).Sambrook and

Russell [4].

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Buffer P1 ( Resuspension buffer) consisted of Tris base- 6.06 g, Na2EDTA.2H2O- 3.72 g.

Dissolve Tris-HCl and Na2EDTA.2H2O in 1 litre double distilled water. Buffer P2

( Lysis buffer) consisted of NaOH- 8.0 g,

10% SDS solution in dissolve 1 litre distilled water stored at room temperature.

Buffer P3 (Neutralization buffer) consisted of 3 M potassium acetate and stored at 40C. Took

around 1.5 ml E.coli culture in a microfuge tube and Spin at 10000 rpm/ min.

Resuspended the pellet in 300µl of P1. Add 300µl of P2, mixed gently and kept at room

temperature for 5 min. Add 300µl of chilled P3 in each tube separately and mix gently. Kept

on ice for 5 min. Centrifuge at 10000rpm for 10 min. took the supernatant in a fresh microfuge

tube. Add equal volume of Phenol: Chloroform: Isoamyl alcohol (25: 24:1) and mix well.

Centrifuge at 10000 rpm for 10 min at room temperature. Took the upper aqueous layer in a

fresh tube. Add 0.8 volume of isopropanol and mix gently. Centrifuge at 10000rpm for 15

min. at room temperature. Discard the supernatant. Washed the pellet with 1 ml 70% chilled

ethanol. Centrifuge at 10000rpm for 1 min at 40C. Air-dry the pellet. Resuspend the pellet

in 30µl Tris-EDTA (10:1) buffer.

Isolation of plasmid DNA miniprep by TELT method. He [5].

Grown a 1.5 ml E.coli culture with plasmid of interest in LB medium containing

100 mg/ml ampicillin for 16h. Pellet cells 30 sec in micro centrifuge. Resuspend in

100µl TELT solution and add an equal volume of 1:1 phenol/chloroform. Vortex

vigorously 15 sec and spin 1 min in micro centrifuge at 220C. Collect the upper phase of

nucleic acid and mix with 2 vol of 100% ethanol. After 2 minutes, spin 10 minutes in

micro centrifuge at 40C. Wash pellet with 1ml of 70% ethanol, dry under vacuum, and

resuspend in 30µl TE buffer. The pellet may be located as a smear on one side of the tube.

Dissolve the DNA on a shaker4 M NaOH- 0.5 ml, DW to 100 ml. Take 1.5 ml freshly

grown bacterial culture in 1.5 ml tube. Spin for 30 sec at 10000 rpm. Remove all

supernatant leaving around 50µl LB. (It is better drain over tissue paper to remove excess

LB after discarding supernatant). Resuspend the cells uniformly by vortexing or tapping

and add TENS in one tube at a time mix thereafter add in other tube. Add 150µl of 3 M

sodium acetate, pH 5.2; close the lid of the tube and vortex for 10 seconds. Good

vortexing is critical at this point. Spin the tubes for 3 min in micro centrifuge at 10000

rpm. Transfer the supernatant in fresh tube and add 1 ml of chilled ethanol and keep on

ice for 15 min. Centrifuge for 10 min at 120000 rpm, remove the supernatant and add 1

ml o0f 70% chilled ethanol. Recover the plasmid DNA by centrifuging at 10000 rpm for

10 min. Dry the pellet and resuspend in 40µl TE containing Rnase (25µg per ml

4

concentration) keep at room temperature for 10 min. Now DNA is ready for restriction

enzyme digestion.

Plasmid DNA was isolated and run on agarose gel electrophoresis.

Agarose gel electrophoresis: TAE (50 X for 100 ml): Tris Base 24.2 gm, Glatial Acetic

Acid- 5.71 gm, 0.5 M EDTA (pH 8.0)- 10 ml.

Distilled water was added to make the final volume up to 100 ml. A working solution of

1X was used. Loading dye was prepared by mixing 25% bromophenol blue and 40%

sucrose in d/w. Prepared 1% agarose in 1X TAE buffer and heated till it dissolved.

Cooled the solution to about 600C and then added ethidium bromide at a concentration of

0.5 µg/ml. Gel tray was sealed with tape on its open ends and placed the comb on the tray.

Then poured the above gel on the sealed tray. Gel tray was allowed to solidify. The comb and

tape was removed. The gel tray was kept in electrophoretic tank containing 1X TAE buffer.

The DNA marker and sample DNA were loaded into the slots of submerged gel, using

bromophenol dye buffer and run at 70-80 volts.

RESULTS: The plates containing recombinant E.coli culture were found to contain.

pTarget.epo.hu plasmids, from which few discrete colonies were selected randomly and

grown in LB broth containing 100µg /ml ampicillin. The recombinant plasmids were

isolated. Plasmid DNA was isolated by above three methods and was run on agarose gel

electrophoresis, which yielded good amount of DNA. Since, Contaminating RNA may

interfere with detection of DNA fragments on the agarose gel. It was destroyed by adding 1µl

of a 10 mg/ml RNase solution (DNase-free) to the DNA preparation. The DNA Bands were

visualized under UV light.

DISCUSSION

Miniprep TELT method reported so far for the isolation of plasmid DNA involve

multiple pipetting, extraction, centrifugation and changes of minifuge tubes. For

screening large number of samples, they are therefore cumbersome, time consuming and

not economical. The technical report below by He et. al., (1990) is a very fast, simple and

one step 'miniprep' procedure. According to this procedure, the bacterial culture is

directly extracted with a mixture of phenol-chloroform-isoamylalcohol and the liberated

DNA is precipitated with isopropanol. This method is now being used routinely in our

laboratory. In comparison, other methods like Isolation of plasmid DNA Alkali lysis

5

method (P1, P2, & P3 method) and Isolation of plasmid DNA by TENS method are not good

but TELT method is very good because in this method, plasmid DNA preparations are of a

purity and quality usable for most molecular biology work and the yield is about 30µg plasmid

DNA per preparation. It only needs one tube per sample.

REFFRENCE

[1].Lin, Suggs,S.,Lin,C., Browne, J., Smalling, R.,Joan ,C.,Egrie, Kenneth k.,Chen,

Gary M.,Fox,Frank, Martin, Stabinsky,Z., Badrawi,M., Por-Hsiung, L. and

Goldwasser.,E.(1985).Cloning and expression of the human erythropoietin gene.

Erythropoietin Factor. Proc.Natl. Acad.Sci,USA.,Publ.No. 82:7580-7584.

[2].Spivak,J. (1994). Recombinant Human Erythropoietin and the Anaemia of

Cancer. The American Society ofHematology.84:.997-1004.

[3].Chung, C.T., Suzanne L., Niemela. And Roger H. Miller. (1988). One- step

preparation of competent E.coli: Transformation and storage of bacterial

cells in the same solution. Proc.Natl. Acad.Sci.USA,Vol.86.pp.2172-2175.

[4].Sambrook, J. and Russell, D.W. (2001). Molecular cloning. A Laboratory

Manual, 3rd

ed.,Cold Spring Harbor Laboratory Press, edd Spring Harbor,

New York.

[5] He, M., Wilde, A. and Kaderbhai, M.A. (1990). A simple single step procedure

for small scale preparation of Escherichia Coli plasmid, Nucleic Acids Res., 18,

1660.

ADDRESS FOR CORRESPONDENCE

Usha Tiwari C/O Prof. Kusum Agarwal

School of Biotechnology

Shobhit University, Modipuram, Meerut.

E.mail: ushatiwari@rocketmail.com

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