BiotechnologyGene therapy, Cloning

Heredity No 439by RNDr. Hana Zoubková, PhD

What is Biotechnology?

The application of technology to improve a biological

organism by changing or adding genes from another

organism

…………...produce Genetically modified organisms

Gene engineering, transgenosis, transgenic organism

The application of technology to improve (to heal)

a human organism by changing or adding genes

from another organism

What is Gene therapy?

What is Cloning?

The process of producing populations of genetically

identical individuals

Molecular cloning – amplification of DNA fragments

Cell cloning – an application of stem cells

Organism cloning – somatic cell nuclear-transfer

Reproductive vs. therapeutic cloningAn aim of reproductive cloning is origin of a baby

An aim of therapeutic cloning is to provide stem cells

for a patient, which requires a transplant

Technique of embryo division – old technique of formation

genetically identical individuals, division of morula or blastocyst

Technique of nucleus transfer a transfer of

somatic cell nucleus to enucleated egg

Cloning of cell or organism

Stem cellsEmbryonic stem cells – an application is forbidden

Infant and adult stem cells – an application is permitted

and they are used for therapy. They are present in small

numbers in

Bone marrow

Peripheral blood

Skin epithelium

Umbilical cord blood

Dental pulp of infant’s teeth

Stem cells may be obtained by reprogramming

somatic cells

Sources of adult and infant stem cells

Gene therapySomatic

A genome is changed, but the change is not passed to other generation. The gene in patients cells are repaired and returned back.

Germ gene therapy

A genome is changed directly in ovum or sperm cell and the change is passed to other generations

- is not proceeded for any kind of animals

Researchers may use several approaches

A transgen may be inserted into a non-specific location within

the genome to replace a function of an abnormal gene.

An abnormal gene might be swapped for a normal gene through

homologous recombination.

An abnormal gene could be repaired through selective reverse

mutation, which returns the gene to its normal function.

An gene might be turned on or off, a regulation of its

expression is altered.

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Problems:Multifactorial disorders Short-lived nature of gene therapyImmune response The genes are not expressed in intended tissues and in intended place of the genome.

Ex vivo – pathologic tissue is taken from patient. Cells accept transgen in laboratory conditions and after that are cells given back to patient

In vivo – we prepare viral vectors with transgene and we introduce (infect) patient‘s tissue with the viral vectors

Disorder Cells altered Gene therapy strategy

SCID - ADA deficiency T cells and hemopoietic stem cells

Ex vivo GAT using recombinant retroviruses containing an ADA gene

Cystic fibrosis Respiratory epithelium In vivo GAT using recombinant adenoviruses or liposomes to deliver the CFTR gene

Familial hypercholesterolemia

Liver cells Ex vivo GAT using recombinant retroviruses to deliver the LDL receptor gene (LDLR)

Gaucher's disease glucocerebrosidase

Hemopoietic stem cells Ex vivo GAT using retroviruses to deliver the gene (GBA)

Examples of gene therapy trials for inherited disorders

1. An identification and isolation of gene

2. We modify an existing gene (a new allele) or we create

gene knock-out or we modify foreign DNA or synthetic

genes to be transgene

3. The introduction of transgene into an organism for

multiplication

4. Selection of transgenic organisms and a usage of

multiplicated transgene or its protein

Gene manipulation and introduction and multiplication

Molecular cloning - steps

Molecular cloning: the principle of bacterial production of insulin, it is used a vector with transgene (insulin gene)

1. Identification, isolating unknown gene by

Homology cloning based on a similarity to known

genes. e.g. Mouse‘s gene will help to determine the

localization of human one in hybridization method

Complementary genetics we predict nucleotide

sequences due to known aminoacids sequences

Map-based cloning based on searching of genetic

markers in linkage with the unknown gene – chromosome

walking

● Gene of interest

● Selectable marker

● Insertion sequences

2. Preparation of recombinant DNA

which contains polylinker: artificially synthetized part of

DNA with specific spots for restriction endonucleases

(RE), which allow to incorporate gene of interest transgen,

also contains resistance to two or more antibiotics and

origin of replication. The whole recombinant DNA must

be short enough for vector.

Isolated gene (gene of interest) becomes transgene - the gene must be combined with other genetic elements in order to be expressed properly. The gene can also be modified at this stage for better expression or effectiveness.

Promoter Region and Terminator regionControls when, where and how much the gene is expressed - viral promotors

Transit PeptideTargets protein to correct organelle

Coding Region Encodes protein product

Promoter Coding RegionTP

Bacterial plasmids

Bacteriophage lambda (λ)

Cosmids - combination phages and plasmids

Integrated vectors

Artificial yeast chromosome, YAC

Viruses (adenoviruses, retroviruses, lentivirus) – mammals and

humans

vectors for transgenosis

Restriction endonuclease enzymes, which cut DNA

molecules at specific recognition nucleotide sequences

known as restriction sites palindroms

mostly bacterial enzymes

3. Insertion of vector to genome:

Transgenic mammals and gene therapy: introduction of

viral vectors, retroviruses, adenoviruses, lentiviruses with

recombinant DNA and transgene

Plant and bacterial transgenosis: introduction of

plasmids, phages, cosmids with recombinant DNA and

transgene by electric, heat or sound impulse, mikroinjection,

gene shooting, liposomes

4. selection of transformed organism and an

application of multiplied transgene or its protein in

a basic research of the gene and protein or

a medical, food or agricultural sciences and industry.

Transgenic plants with resistance and higher profitability.

Transgenic bacteria able to clean toxic wastes.

The application of protein generated by transgene for basic

research and for clinical use: insulin, human growth

hormon …

Application in Medicine

….also human albumin, monoclonal antibodies, anti-

hemophilic factors, interferon, vaccines, enzymes.

Agriculture: production of Golden Rice

First…Bacteria in 1973 Mice in 1974Insulin-producing bacteria in 1982 Gene therapy in 1990Genetically modified food has been sold since 1994

Trangenic animals

• are useful for research of gene therapy and human

pathologic genotypes and phenotypes

• expression of mammals genes

• model organism for testing vectors

Transgenic mice, sheeps, pigs, cattle…. hens, sheep, goats,

rabbits, pigs, cattle with resistance to diseases, to

enhanced quality of meat and milk

Methods important for medical sciences:

Production of monoclonal antibodies

VNTR variable number of tandem repeats

Single nucleotide polymorphisms SNPs

Allele specific oligonucleotide analysis (ASO)

Microarrays - Identifying sets of disease genes

Pharmacogenomics

Nanomedicine - may be used for delivery of small sensors to target

sites in body, detect and destroy cancer cells

Monoclonal antibodies

Microarrays

http://www.clinigene.eu/video-intro-gene-therapy.html

Dr. Peter Snustad, Michael J.Simmons:

Principles of Genetics, 5. edition, 2009

Thank you for your attention

Commercial cloning

Somatic nucleus transfer1 clon 32 000 USD (r.2004)

Deposition of animal cells into bank 850 USD (r.2005)

Genetic donor clones

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