Project Report on

Quantification of ER stress in recombinant IgG secreting Chinese Hamster (Cricetulus

griseus) Ovary (CHO) cell lines.

Submitted in partial fulfillment of the degree of

Bachelor of Technology

in

Biotechnology

Under the guidance

of

Prof. Sarika Mehra

Department of Chemical Engineering

Indian Institute of Technology, Mumbai - 400 076

By

Kritika Lakhotia

10BBT0099

Internal Guide

Prof. Abhishek Sinha

DECLARATION CERTIFICATE

I hereby declare that the thesis entitled, “Quantification of ER stress in recombinant IgG

secreting Chinese Hamster (Cricetulus griseus) Ovary (CHO) cell lines” submitted to Vellore

Institute of Technology, Vellore, Tamil Nadu, India for the award of the Degree of

Bachelor of Technology in Biotechnology is an authentic record of research work carried

out by me during the period from Dec 2013 to May 2014, under the guidance and

supervision of Professor Sarika Mehra, Chemical Engineering Department, Indian

Institute of Technology, Bombay. I also declare that this project has not been submitted to

any other Universities or Institutions for the award of any degree.

13th

May, 2014

Kritika Lakhotia

Acknowledgements

I would like to extend my deepest gratitude to Prof Sarika Mehra for giving me an

opportunity to work under her established guidance and for her constant support.

I would also like to thank my PhD mentors Mr Kamal Prashad and Vikas Chandrawanshi for

their immense patience in guiding me and for their constant encouragement. Also, I would

also like to thank my other lab members: Prasanna Sir, Minal Ma’am, Yesha, Priyanka,

Monali and Sampada for their cooperation.

I would like to thank my guide Prof Abhishek Sinha from VIT University, Vellore for his

valuable support throughout and helping me in every possible way.

Abstract

With the surge in demand for recombinant products, there is a need to enhance productivity

of the cell lines used in the biopharmaceutical industries. In order to fulfill this objective, the

biology of the mammalian cell lines that are essentially preferred for the production of

recombinant products, need to be assessed. Here, we are trying to optimize the cell culture

techniques to maximize productivity. In addition, protein secretion is one the steps in the

production pathway that is said to be connected to the high productivity status of the

culminating product. The UPR pathway that fundamentally regulates the ER homeostasis is

one of the key links in understanding the optimum conditions required for the maintaining

high growth and productivity in stable mammalian cell lines. Stress assays give us important

information regarding the misfolded proteins and their quantification in a culture through the

RFU values at certain excitation and emission spectra. Glucose and lactate consumption

rates along with IgG secretion values provide an astute comprehension in studying the cell

kinetics. As a whole, the idea is to perceive optimum production conditions and gain

information on how one can enhance and produce a highly productive and stable

mammalian cell line that can be utilized successfully at an industrial scale.

Objectives

The objective of this work is to quantify ER stress in recombinant CHO cells at high

productivity conditions. These are met through the following specific objectives.

1) To understand the molecular mechanism of protein secretion in mammalian

system

2) To perform kinetic and metabolic profiling of CHO cell lines

3) To map the key stress element sites in the upstream regions of the UPR genes

4) To quantify ER stress using biochemical assays – DCFDA and ThT

Table of Contents

Chapter 1 ............................................................................................................................................... 1

Introduction ........................................................................................................................................... 1

1.1 Cell culture technology ................................................................................................................... 2

1.2 CHO cell line ............................................................................................................................ 3

1.3 Media .............................................................................................................................................. 5

1.4 Culture conditions ........................................................................................................................... 6

1.5 Growth Kinetics .............................................................................................................................. 6

Chapter 2 Materials and Methods ......................................................................................................... 8

Chapter 3 Cell Culture Assay Results ................................................................................................. 12

3.1 Growth curve ................................................................................................................................ 12

3.2 Glucose and Lactate Assay ........................................................................................................... 14

3.3 IgG quantification ......................................................................................................................... 15

Chapter 4 Unfolded Protein Response ............................................................................................... 16

2.1 The unfolded protein response pathway ....................................................................................... 17

Chapter 5 Biochemical Assays to quantify ER Stress ........................................................................ 20

5.1 Thioflavin Assay ........................................................................................................................... 20

5.2 Thioflavin T assay Results ............................................................................................................ 21

5.2 ROS Assay .................................................................................................................................... 25

5.3 ROS assay Results ........................................................................................................................ 27

Chapter 6 ............................................................................................................................................. 30

Multiple sequence alignment and stress elements identification ........................................................ 30

6.1 Verification Tools ......................................................................................................................... 34

Chapter 7 Discussion and Conclusion ................................................................................................ 53

References ........................................................................................................................................... 55

Appendix ............................................................................................................................................. 57

List of Figures

Figure 1: Epithelial-like CHO-K1 cell line ........................................................................................... 4

Figure 2: Growth Kinetics .................................................................................................................... 7

Figure 3: Viable cell densities of MTX, No-MTX, & No-G418 treated 250-4 cells ......................... 12

Figure 4: Viability profile ................................................................................................................... 13

Figure 5: Specific growth rate profile of MTX, No-MTX and No G418 treated 250-4 cells ............. 13

Figure 6: Glucose and Lactate Standard Curve .................................................................................. 14

Figure 7: Glucose and lactate assay for control cultures..................................................................... 14

Figure 8: IgG titers and cumulative productivity. ............................................................................... 15

Figure 9: A basic outline of the protein secretion pathway ................................................................ 17

Figure 10: An outline of UPR ............................................................................................................. 19

Figure 11: RFU vs dye concentration ................................................................................................. 21

Figure 12: Standard curve with 250-4 cells ........................................................................................ 22

Figure 13: Standard curve with 250-4 MTX culture .......................................................................... 22

Figure 14: RFU vs. dilution units/µL comparison for media and supernatant .................................... 23

Figure 15: Standard curve with supernatant of 250-4 CHO cells ....................................................... 23

Figure 16: ThT comparison for MTX, No-MTX, & No-G418 culture conditions ............................. 24

Figure 17: RFU plot for suppressor of UPR ....................................................................................... 24

Figure 18: ThT comparison for Control (dark) & Ind 1 (light) treated culture ................................... 25

Figure 19: ROS Standard curves with different dye concentrations ................................................... 27

Figure 20: ROS Comparison for 1.25 X 106 cells ............................................................................... 27

Figure 21: ROS comparison for 0.5 X 106 cells.................................................................................. 28

Figure 22: Complete culture ROS profile ........................................................................................... 28

Figure 23: ROS assay for control and Tunicamycin treated cultures .................................................. 29

Figure 24: MSA for GRP78 ................................................................................................................ 37

Figure 25: Matched Stress Elements with Grp78 consensus sequence .............................................. 38

Figure 26: MSA for GRP94 ................................................................................................................ 39

Figure 27: Matched Stress Elements with Grp94 consensus sequence ............................................... 40

Figure 28: MSA for CRT .................................................................................................................... 41

Figure 29: Matched Stress Elements with CRT consensus sequence ................................................. 42

Figure 30: MSA for CNX ................................................................................................................... 43

Figure 31: Matched Stress Elements with CNX consensus sequence ................................................ 44

Figure 32: MSA for ATF4 .................................................................................................................. 45

Figure 33: Matched Stress Elements with ATF4 consensus sequence ............................................... 46

Figure 34: MSA for CHOP ................................................................................................................. 47

Figure 35: Matched Stress Elements with CHOP consensus sequence .............................................. 48

Figure 36: MSA for GADD34 ............................................................................................................ 49

Figure 37: Matched Stress Elements with GADD34 consensus sequence ......................................... 50

Figure 38: MSA for XBP1 .................................................................................................................. 51

Figure 39: Matched Stress Elements with XBP1 consensus sequence ............................................... 52

List of Tables

Table 1: Selected list of approved antibodies produced in CHO cells (Wlaschin & Yap, 1987) ......... 5

Table 2: Overview of the UPR site information for chaperones ......................................................... 32

Table 3: an overview of the UPR site information for UPR pathway and the related apoptotic

pathway ............................................................................................................................................... 33

Table 4: List of consensus positions for UPR genes ........................................................................... 35

1

Chapter 1

Introduction

A biopharmaceutical product or a ―biologic‖ essentially refers to a medicinal product which

are produced through biotechnology. It could be a vaccine or a recombinant protein or a

blood component but in totality, it can be utilized as a therapeutic for the treatment of a

disease. A majority of biologic products are obtained from life forms. There can be a spark

of a controversy here as these products can be acquired from a method that involves

transgenic organisms specifically, genetically modified plants and animals. More work is

being done on high ―content‖ assays than on ―throughput‖ assays. There is a logicality

behind this, i.e., instead of working on miniaturizing assays to reduce costs and increase

productivity, complex biology is now being transferred to 96-well formats.

The biopharmaceutical market can be categorized on the basis of the class of the medical

drug into purified proteins, monoclonal antibodies, and recombinant proteins. The United

States has the largest market for biopharmaceuticals valued at USD 90 billion and is

assessed to grow in the coming years. The major section of this can be ascribed to

monoclonal antibodies which have witnessed an upsurge since the 90s due to the exquisite

specificity it offers while tracking proteins and other chemicals. Though their effectiveness

is limited, some of the technical problems have been overcome and drugs based on

monoclonal antibodies have been routinely used.

Monoclonal antibodies have been used for diagnosis of diseases by the western blot test and

immuno dot blot test which detect the protein on a membrane. By combining monoclonal

antibodies with poison, cells have given a protein on their surface that can be tracked down

by the antibody and destroyed. This method has been successful against some types of

cancers, especially breast cancers and leukemia. In addition, monoclonal antibodies are

being exploited for treatment of autoimmune diseases such as rheumatoid arthritis. CHO cell

lines are optimal for the production of monoclonal antibodies at larger scales.

2

1.1 Cell culture technology

Cell culture technology derived products have been used as medicines to treat and prevent

cancer, viral infections, etc. The products of cell culture are said to be safe, effective, and

economical. It all began with the use of cells as viral vaccines for therapeutic purposes and

this led to the acceptance of continuous cell lines.

Cell cultures can be obtained by removal of cells from an animal or plant and ensuing

growth in a favorable environment. These cells can be removed by means of enzymatic

degradation or mechanically before cultivation. Primary culture refers to the phase of

culture that after the cells are isolated from the tissue and proliferated under suitable

conditions until they reach confluence. At this stage, the cells need to be subcultured or

passaged. Passaging or subculturing is referred to as the removal of medium and transfer

of cells from the primary culture for further propagation of the cell line. Subculturing for

mammalian cells is carried out before they reach confluency lest causing it to clump and the

solution to render turbid. Once surfeits of cells are obtained, they can be treated with

cryoprotective agents like dimethylsulfoxide (DMSO) or glycerol and carefully frozen

following storage at cryogenic temperatures (below -130ºC until needed).

Two basic cell culture systems that are used for growing cells are based upon the capability

of the cells to grow attached to a surface (Monolayer Culture Systems) or floating free in

the culture medium (Suspension Culture Systems). Of the two systems, suspension culture

was used for our mammalian cells. The suspension cultures are usually grown in Erlenmeyer

flasks in which the cells are actively suspended in the medium. The characteristics of

cultured cells depend on how ably they adapt to the culture conditions. Some characteristics

are lost or change when placed in an artificial environment. The cell lines that eventually

stop dividing are called finite cell lines. The cell lines that keep dividing infinitely are called

continuous cell lines.

Suspension cultures are easier to passage albeit it requires cell counts on a daily basis for

viability determination. They do not require enzymatic or mechanical disruption which is

beneficial as there will be minimal cell loss. These cultures are maintained in culture vessels

but require agitation for passable gas exchange on a routine basis.

The vertical laminar-flow biosafety cabinet provides a clean and sterile environment for the

worker and the product in carrying out the cell culture experiments. The successful

3

manipulation of cell culture majorly relies on the capacity to maintain aseptic conditions.

The effectiveness of laminar flow cabinets as physical barriers to contamination depends on

the cabinet design integrating high-efficiency particulate air (HEPA) filters to trap airborne

contaminants and the blowers should move the filtered air at specified velocities in a non-

mixing stream across the work area.

Incubators are another basic necessity for maintaining a constant temperature of 37ºC for the

cell culture. They are required to maintain constant culture conditions and for preserving the

viability of the cells. The humidified atmosphere is maintained to prevent the loss of

medium of unsealed culture systems. The CO2 atmosphere is for maintaining a constant

buffering system.

The popular form of culture containers that we used were multi-well plates, and culture

flasks. The multi-well plates can accommodate many replicates of small-volume cultures.

The rapid volumes can be added through multi-well pipettors especially for dyes that follow

a high reaction speed. Following this, we can read the absorbance data using a

spectrophotometer.

1.2 CHO cell line

The cells that are used to a larger extent in any cell culture process are mammalian cell lines

due to the numerous advantages that it offers. They have the ability to perform post-

translational modifications which increase the efficacy of the protein drugs targeted towards

therapeutics (Wong, Wong, Tan, Wang, & Yap, n.d.). Mammalian cell lines, at large, are

classified into three basic categories on the basis of their morphology:

1. Fibroblastic: Bipolar or multipolar cells that have elongated shapes. They grow

attached to a substrate

2. Epithelial-like: These cells are polygonal in shape and grow attached to a substrate

in detached patches

3. Lymphoblast-like: These cells are spherical in shape and grown in suspension

without attaching to a surface

CHO cell line stems from the ovary of Chinese Hamster Cricetulus griseus organism. CHO-

K1 comes under the epithelial-like cell line and is the subclone of the parent CHO cell line.

4

Figure 1: Epithelial-like CHO-K1 cell line

Basic overview of CHO mutant cell line development

CHO-K1 cell line is suitable as a transfection host and therefore, it makes the development

of a mutant cell line easier. The expression vector containing the promoter region, dhfr site

along with an antibiotic selection marker can be transfected into a CHO cell by a variety of

methods that include co-precipitation, lipofection, electroporation and microinjection. This

is grown in a media comprising antibiotics and simultaneously, deficient in glycine,

hypoxanthine, and thymidine. Post selection pressure, the transfected cell lines grow and

survive and the producing cells expand either as pools or colonies. It is then screened for

producing clones following which a scale-up step is performed in tissue culture plates or

flasks. It is then amplified via Methotrexate and one adapts the cells to grow in a serum-free

and protein-free suspension culture. A selection step is followed where top clones are

chosen based on titre, product quality and growth which is further apt for long term stability

evaluation by cell banking starting from the Master Cell Bank (MCB) to a Working Cell

Bank (WCB) and finally for production and operational processes.

5

Table 1: Selected list of approved antibodies produced in CHO cells (Wlaschin & Yap, 1987)

Product Therapeutic use Manufacturer

Rituximab Chronic lymphocytic

leukaemia

Dr. Reddy’s Laboratories

Ltd.

Vectibix Metastatic colorectal cancer Amgen

Luveris Infertility Serono

Advate Hemophilia A Baxter

Orencia Rheumatoid arthritis Bristol-Myers Squib

Xolair Moderate/severe asthma Genentech

Aranesp Anemia Amgen

1.3 Media

Cell culture media plays the most important role in the culture environment and it is one of

the most demanding aspects for recombinant CHO cell lines. Hence, it necessary to optimize

the culture components as they provide nutrients, growth factors, hormones and also,

regulate the pH and osmotic pressure of the culture.

A chemically defined media is the most suitable for in vitro cell culture and it contains a

basic class of media known as the basal media. This medium is an amalgamation of small

components (sugars, vitamins, and amino acids) and it provides balanced salt concentrations

and osmolarity to allow cell growth. Basal media formulations must be further supplemented

with serum. Serum is a vital component in a cell culture media. It is free of blood cells and

most coagulation proteins. It acts as a source of growth and adhesion factors, hormones,

lipids and minerals for culture of cells in the basal media. As much as serum is an important

component, it has its drawback which is its contamination factor and the high cost.

6

1.4 Culture conditions

Carbohydrates in the form of sugars are a major source of energy. Ideally, most media

contain glucose or galactose. The most commonly used proteins and peptides are albumin,

fibronectin, and transferrin. The binding capacity of albumin contributes in the removal of

toxic components from the media. Fibronectin is important for cell attachment whereas

transferrin is an iron transporter which is recycled in the culture broth. Vitamins are present

in modicum and are essential in the growth and proliferation of the cells. The optimal pH for

mammalian cells is 7.4 and they grow well at this pH. Nevertheless, some transformed cell

lines grow better at slightly acidic environments. Buffering of the cells is required against

changes in the pH. This is often achieved by the means of CO2-bicarbonate based buffer. pH

of the medium is dependent on the balance between dissolved CO2 and bicarbonate (HCO3-)

and thus, changes in the atmospheric CO2 can alter the pH of the medium. Most cell culture

experiments are carried out in 5-10% CO2 as this allows firm maintenance in the pH of the

medium. A drop in the pH results in the accumulation of lactic acid which is essentially a

by-product of cell metabolism. Also, lactic acid can be toxic to cells and is in probability,

sub-optimal for the growth of cells. Temperature of the incubator where mammalian cells

are grown is maintained at 36ºC to 37 ºC. In most cases, the temperature is maintained at a

slightly lower temperature than the optimal temperature as overheating poses a more serious

threat than underheating. Another essential component is the distilled water that is used for

various experiments involving mammalian cell lines. A typical water preparation involves

deionization through ion exchange followed by microfiltration to remove particulates and

bacteria and finally, reverse osmosis to reduce the conductivity. Lipids play an equally vital

role in protein secretion by the lipid bilayer membrane. The effect of lipid supply is the

medium is understated. Calcium and magnesium are responsible for cell-substrate adhesion.

Sodium and potassium help in balancing the membrane potential. Iron plays a role in

electron transfer complexes.

1.5 Growth Kinetics

An indication in the growth characteristics of a cell line can facilitate in the monitoring of

the cellular growth and if there happens to be any detrimental effect, one can know of it in

advance and prevent faulty experimental results. The cell growth curve is typically ramified

7

into four different growth phases: Lag phase, Logarithmic growth phase, Plateau phase

and Decline phase. A classic growth curve displays a sigmoid pattern of proliferation.

The time following subculture and reseeding is a phase where there is little or no increase in

the cell number. The cells in the lag phase adapt to the culture conditions by replacing the

elements of the glycoprotein lost during trypsinization following which they attach to the

substrate and spread out. The length of this period depends upon the seeding density and the

growth profile of the cell line during the time of subculture.

The cell population is said to be the most viable in the log or the exponential phase where

the cells actively proliferate and an increase in the cell density arises. The culture is in its

most reproducible form as the growth fraction is as high as 90 to 100%. This phase is the

finest period for sampling and to determine the population doubling time. Suspension cells

should be passaged in the log phase growth before they reach confluency.

As confluency is reached at the end of log phase, the cellular proliferation slows down.

Consequently, the plateau phase is observed where the growth rate of the culture is reduced

as all the available growth surface is occupied. The growth fraction plummets to 0 to 10 %

and the cells are the most disposed to injury.

With the reduction in the number of viable cells, cell death predominates in the decline

phase. The cell death is not due to reduction in the nutrients but a natural occurrence in the

path of the cellular cycle.

Figure 2: Growth Kinetics

0

0.5

1

1.5

2

2.5

3

3.5

4

0 1 2 3 4 5 6 7 8 9

Via

ble

cell

den

sity

(m

illi

on

cell

s/m

L)

Culture age (Days)

8

Chapter 2

Materials and Methods

Cell Culturing & Cell line

CHO cell lines secreting anti-rhesus IgG were obtained from BTI, Singapore. The cells were

cryopreserved with 10% DMSO (v/v) in a liquid nitrogen container (-196 ºC) at 107

cells/mL in 1mL vials.

Anti-rhesus IgG secreting CHO cells were cultured in a media encompassing 50% PF-CHO

(Thermo-Hyclone) and 50% CD CHO (Gibco-Invitrogen) supplemented with 2.0 g/L

sodium carbonate (sigma-Aldrich), 6mM L-Glutamine (Sigma-Aldrich), 0.10% Pluronic

(Himedia), 600 ug/mL G418 (Sigma-Aldrich) and 250 nM Methotrexate (Sigma-Aldrich) at

37 ºC in 20 mL Erlenmeyer flasks (Corning) in duplicates.

Cell counting

A Neubauer haemocytometer was used for counting the number of live and dead cells by a

dye exclusion method. Trypan Blue (HiMedia) dye is used to stain dead cells. Due to the

specific permeability of this dye, it can penetrate only through dead cells. Dilution factors

were maintained appropriately to obtain a minimum of 10 cells/square of haemocytometer.

Various growth parameters using formulae given below:

Viable cell density: VCD = 𝐿𝑖𝑣𝑒 𝑐𝑒𝑙𝑙𝑠 ∗ 𝐷𝐹

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 ∗10000

Dead cell density: DCD = 𝐷𝑒𝑎𝑑 𝑐𝑒𝑙𝑙𝑠 ∗𝐷𝐹

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 ∗10000

Total cell density: TCD = 𝑇𝑜𝑡𝑎𝑙 𝑐𝑒𝑙𝑙𝑠 ∗𝐷𝐹

𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 ∗10000

Integral viable cell count: ∆ IVCC (i) = [ 𝑋 𝑡ᵢ + 𝑋 𝑡ᵢ₋₁ ]

2∗ (𝑡ᵢ − 𝑡ᵢ₋₁)

9

IVCC (i) = ᵢ₌₀∆𝐼𝑉𝐶𝐶₍ᵢ ₎

Specific growth rate: µspecific = 𝑋𝑡𝑜𝑡𝑎𝑙 𝑡2 − 𝑋𝑡𝑜𝑡𝑎𝑙 (𝑡₁)

𝑋𝑣 𝑡2 ∶ 𝑋𝑣 𝑡1 ∗(𝑡2− 𝑡1)

Cumulative growth rate: µcumulative (ti) = 𝑋𝑡𝑜𝑡𝑎𝑙 𝑡𝑖 − 𝑋𝑡𝑜𝑡𝑎𝑙 𝑡0

𝐼𝑉𝐶𝐶𝑖

Specific death rate: Kd, specific = 𝑋𝑑𝑒𝑎𝑑 𝑡2 − 𝑋𝑑𝑒𝑎𝑑 (𝑡₁)

𝑋𝑣 𝑡2 ∶ 𝑋𝑣 𝑡1 ∗(𝑡2− 𝑡1)

Cumulative death rate: Kd, cumulative (ti) = 𝑋𝑑𝑒𝑎𝑑 𝑡𝑖 − 𝑋𝑑𝑒𝑎𝑑 𝑡0

𝐼𝑉𝐶𝐶𝑖

Glucose Assay

Glucose has to be regularly monitored in order to measure the substrate consumption rates

and for feed addition planning in fed-batch operations. The glucose estimation was

performed using GOD-PAP Glucose Estimation Kit (Biolab Diagnostics). The principle of

this experiment is shown below.

Glucose + O2 + H2O ------GOD---->Gluconic acid +H2O2

2 H2O2 + PAP ------POD----> Quinoneimine + 4H2O

Glucose is oxidized by Glucose Oxidase (GOD) to Gluconic acid with the simultaneous

formation of Hydrogen peroxide. The newly formed hydrogen peroxide reacts with Phenol

and 4-amino antipyrene) reagent in the presence of peroxidase (POD) enzyme coalescing

into a pinkish red dye Quinoneimine with λmax at 500 nm. Dextrose was used as a standard

starting from 10mg/mL serially diluted to 0.16 mg/mL

10

Lactate Assay

Lactate levels need to be regularly assessed in a cell culture process in order to keep a track

of the cell viability. The estimation of lactate was done using lactate dehydrogenase enzyme

(Sigma) and the principle is summarized below.

Lactate + NAD <--------LDH---------> Pyruvate + NADH

Pyruvate + Hydrazone ------------> Pyruvate hydrazone

Here, lactate is oxidized to Pyruvate in the presence of lactate dehydrogenase (LDH)

enzyme. The hydrazone formation is triggered by hydrazine to prevent the reverse reaction

by LDH. The concentration of lactate present in the sample is commensurate to the increase

in absorbance at 340 nm as NAD+

is reduced to NADH.

The stock LDH (4250 u/mL) is diluted to a working concentration of 12.5 u/mL A fresh

stock of NAD solution (17 mg/mL) and lactate buffer (pH = 9.0) containing 0.5M glycine

(Himedia) was prepared for this assay. Standard is run with a fresh lactic acid solution

(Sigma) starting from 16mM serially diluted to 0.25 mM.

The calculations performed are shown below:

Specific Productivity (qp, specific) = 𝑃𝑟𝑜𝑑𝑢𝑐𝑡 𝑡2 − 𝑃𝑟𝑜𝑑𝑢𝑐𝑡 (𝑡1)

𝐴𝑣𝑒𝑟𝑎𝑔𝑒 𝑜𝑓 𝑋𝑣𝑖𝑎𝑏𝑙𝑒 𝑡2 : 𝑋𝑣𝑖𝑎𝑏𝑙𝑒 𝑡1 ∗ [𝑡2− 𝑡1 ]

Cumulative productivity (qp, cumulative) = 𝑃𝑟𝑜𝑑𝑢𝑐𝑡 𝑡𝑖 − 𝑃𝑟𝑜𝑑𝑢𝑐𝑡 (𝑡0)

𝐼𝑉𝐶𝐶𝑖

Enzyme Linked Immunosorbent Assay (ELISA)

Antibody titres in the culture supernatant were measured by sandwich ELISA using the

protocol ascribed by Chuainow et. al (2009). 10 µg/mL Goat Anti-human IgG + IgA + IgM

(H + L) (KPL, USA) was used as the primary coating antibody. Dilution of 1:200 Alkaline

Phosphatase conjugated with anti-human IgG (Fc specific) was used as a secondary antibody

(Sigma-Aldrich, St. Louis, MO) was used as the substrate. The absorbance was read at 405

nm using a multi plate reader (Spectramax M5e, Molecular Devices, USA). Human IgG

(Sigma-Aldrich, St. Louis, MO) was used as a standard.

11

The calculations followed are shown below.

Specific Productivity (qp, specific) = 𝑃 𝑡2 − 𝑃(𝑡1)

𝑋𝑣 𝑡2 : 𝑋𝑣 𝑡1 ∗ (𝑡2− 𝑡1)

Cumulative productivity (qp, cumulative) = 𝑃 𝑡𝑖 − 𝑃(𝑡0)

𝑋𝑣𝑡𝑡0

𝑑𝑇

P(t) is the concentration of IgG at time t determined by ELISA.

Thioflavin T Assay

This assay is basically to quantify the presence of misfolded protein aggregates by

measuring the change in fluorescence intensity of Thioflavin T (Sigma). Thioflavin T (4-(3,

6-dimethyl-1, 3-benzothiazol-3-ium-2-yl)-N, N-dimethylaniline chloride) is a benzothiazole

dye that exhibits enhanced fluorescence upon binding to proteins that are rich in β-sheet

structures. ThT portrays fluorescence intensity upon binding to these structures at an

emission wavelength of 482 nm and an excitation wavelength of 450 nm. Cell

concentrations in the range of 105

to 2 X 106 have been used as the initial concentration

following which it was serially diluted to 0.015624 dilution units/µL. This assay has also

been performed with supernatant to quantify the presence of misfolded aggregates in the IgG

titres.

Reactive Oxygen Species Assay

ROS assay is typical method to measure the ROS activity within the cell. The major source

of ROS is complex I and Complex II which is a part of the mitochondrial electron transport

chain. This assay uses a cell permanent reagent, 2, 7 – dichlorofluorescein diacetate

(DCFDA, Sigma). DCFDA is converted to a non-fluorescent compound in the presence of

deacetylated cellular esterases which then leads to the formation of 2, 7 – difluorescein

(DCF) by oxidation of the reactive oxygen species. Lower levels of ROS play an important

role in signalling pathways and hence, it can give us information regarding the extent to

which cell is damaged due to apoptosis and necrosis.

12

Chapter 3

Cell Culture Assay Results

3.1 Growth curve

The cells were daily maintained at 37 oC, 85 % R.H., 8% CO2 and 110 rpm culture

conditions with periodic sub-culturing on day3 or 4. These cells were then grown in three

different conditions mentioned below.

Culture Methotrexate Gentamycin Media nutrients

MTX YES YES YES

NO MTX NO YES YES

NO G418 NO NO YES

A detailed comparison of growth and death parameters was performed for the passage

number 51. VCD reached a maximum of 7.05 x 106

cells/mL on Day 5 for No MTX

containing culture whereas the other two cultures remained around 5.1 x 106 cells/mL.

Figure 3: Viable cell densities of MTX, No-MTX, & No-G418 treated 250-4 cells

0

1

2

3

4

5

6

7

8

0 1 2 3 4 5 6 7

Mil

lio

n c

ell

s/m

L

Time (Day)

MTX

No MTX

No G418

13

Figure 4 shows the viability comparison for the different treated cultures. The viability

profile is similar for all the conditions throughout the culture duration.

Figure 4: Viability profile

The specific growth rate is highest for the culture where Methotrexate is absent. This could

lead to a possibility that when Methotrexate is present the growth is slowed to an extent as

more resources are diverted towards IgG production. By day 6, the growth rate substantially

decreases.

Figure 5: Specific growth rate profile of MTX, No-MTX and No G418 treated 250-4 cells

0

20

40

60

80

100

120

0 2 4 6 8

Per

cen

tag

e (%

)

Time (Day)

MTX

No MTX

No G418

0

0.2

0.4

0.6

0.8

1

1.2

1 2 3 4 5 6

Sp

ecif

ic g

row

th r

ate

(D

ay

-1

)

Time (Day)

MTX

No MTX

No G418

14

3.2 Glucose and Lactate Assay

A glucose standard was run using the serial dilution method starting from a concentration of

10 mg/ml. It helps us in monitoring the substrate consumption rate and planning for nutrient

addition time points.

Similarly, a lactate standard was run using a serial dilution technique beginning with a

concentration of 16mM lactic acid solution. Higher lactate levels are toxic to the cells as

they reduce the culture pH significantly.

Figure 6: Glucose and Lactate Standard Curve

Figure 7: Glucose and lactate assay for control cultures.

y = 0.173x - 0.199

R² = 0.975

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

0.00 2.00 4.00 6.00 8.00 10.00 12.00

Ab

sorb

an

ce a

t 500 n

m

Glucose concentration (mg/mL)

y = 0.056x + 0.078

R² = 0.985

0

0.2

0.4

0.6

0.8

1

1.2

0 5 10 15 20

Ab

sorb

an

ce a

t 340 n

m

Lactate concentration (mM)

15

A glucose and lactate assay was performed on control samples (MTX) for two biological

replicates and results are plotted as an average. The glucose levels during inoculation are

around 6 g/L. At the end of the culture on day 8, the glucose levels are as low as 1g/L.

Initially the lactate levels up to day 4 are very low but reach considerable high levels of 16

mM by day 9.

3.3 IgG quantification

The IgG levels in the culture were quantified using sandwich ELISA. The IgG levels in

control culture reached to 1.35 mg/mL by day 8. The cumulative productivity started to

increase from day 4 onwards reaching an maximum value of 90 pg/cell-day.

Figure 8: IgG titers and cumulative productivity.

0

300

600

900

1200

1500

0 2 4 6 8

IgG

(µ

g/m

L)

Time (days)

0

10

20

30

40

50

60

70

80

90

100

1 2 3 4 5 6 7 8

pg

/(ce

ll-d

ay

)

Time (days)

16

Chapter 4

Unfolded Protein Response

The endoplasmic reticulum is the cardinal membrane of a complex process, Protein Folding,

where the secretary and the transmembrane proteins conform in their native state. Like in

several biochemical pathways, the early steps in the secretary pathway are controlled and the

transit from the endoplasmic reticulum to the Golgi complex is rate-limiting (Schroder &

Kaufman, 2005). The Golgi becomes another primary site as the post-translational

modifications of the protein occur in this organelle which is essentially important for its

activity and structure. Factors like nutrient deprivation and overloading of cholesterol and

genetic mutations lead to perturbations in the ER and disrupt the normal functioning of the

ER (Kraskiewicz & FitzGerald, 2012). In such an instance, when the protein folding is

encumbered, the signal transduction pathways play a key role in bringing the endoplasmic

reticulum to homeostasis. In a simple way, it can be said that if the influx of the newly

formed polypeptides is much greater in comparison to the folding capacity of the protein,

there is bound to be a certain perturbation which causes distress to the endoplasmic

reticulum. The signal transduction pathway increases the biosynthetic capability whilst

decreasing the biosynthetic burden of the endoplasmic reticulum (Schroder & Kaufman,

2005). Consequently, the unfolded protein response (UPR) is activated to return the

endoplasmic reticulum back to its normal state. This is an example of what is called as

―endoplasmic reticulum stress‖. The endoplasmic reticulum quality control and the ERAD

(endoplasmic reticulum associated degradation) machinery guarantees some credence to the

folding mechanism (van Anken & Braakman, 2005).

17

Figure 9: A basic outline of the protein secretion pathway

2.1 The unfolded protein response pathway

The UPR is basically a way of managing the secretion pathway by attenuating protein

translation and increasing the synthesis of molecular chaperones(Schroder & Kaufman,

2005). As a result, the endoplasmic reticulum increases in size to dilute the increased protein

load. Binding immunoglobulin protein (BiP) or GRP78 is the most critical member of the

HSP70 (Heat Shock Protein 70) family of chaperones which ensures that incorrectly folded

proteins do not exit the endoplasmic reticulum. The transduction of unfolded protein signals

occur when the folding protein binds to a molecular chaperone. This, in turn activates three

transmembrane proteins namely:

(i) ATF6 - Activating Transcription Factor

(ii) PERK – Protein Kinase RNA-like Endoplasmic Reticulum Kinase

(iii) IRE1 – Inositol Requiring Kinase 1

18

ATF6 is a membrane spanning protein containing two homologs (ATF6α and ATF6β) with

an unfolded protein sensor domain and an effector domain in the cytosol (Schroder &

Kaufman, 2005; van Anken & Braakman, 2005). It ultimately leads to the up regulation of

the pro-survival transcriptional program in the presence of unfolded or misfolded proteins

(Szegezdi, Logue, Gorman, & Samali, 2006). ATF6 contains two N-terminal Golgi

localization sequences (GLS1 and GLS2) which are apparently involved in the regulation of

BiP (Schroder & Kaufman, 2005). When BiP dissociates from the N-terminal, ATF6 is

translocated to the Golgi where it is cleaved by regulated intramembrane proteolysis with

the help of serine protease (S1P) and metalloprotease site-2 protease (S1P). This cleaved

ATF6 initiates a gene expression program synergistically with bZIP (basic leucine zipper)

factors for example; Nuclear Factor-Y which is responsible for degradation of unfolded

proteins and an increase in the chaperone activity (Schroder & Kaufman, 2005). ATF6

induces the expression of X-box binding protein (XBP1) which essentially activates various

chaperones and control elements. XBP1 has two versions of which one is the unspliced form

(XBP1u) and the other is the spliced form (XBP1s).

PERK is a type I endoplasmic reticulum transmembrane kinase and it has an ER luminal

stress sensor and cytosolic protein kinase domain (Oslowski & Urano, 2011; Schroder &

Kaufman, 2005). As BiP dissociates from the N-terminal of the kinase domain, it causes the

initiation of dimerization and autophosphorylation of the kinase domain. It is of concern

that the C terminal of the cytosolic domain shares homology with the eif2α (eukaryotic

translation initiation factor) (Schroder & Kaufman, 2005). Activated PERK phosophorylates

eIF2α following which there are marked downstream effects of importance to the UPR.

First, the phosphorylated eIF2α attenuates translation resulting in the decrease of protein

entrance to the ER and consequently, it decreases the folding load to a reasonable extent

(Oslowski & Urano, 2011). In actuality, the attenuation of translation isn’t universal and

some genes don’t succumb to this translational block (Szegezdi et al., 2006). ATF4 is one

such gene and it is responsible for driving the expression of pro survival functions. ATF4

gives rise to the expression of CHOP (C/EBP homologous protein), also known as GADD34

(Growth-arrest and DNA damage-inducible gene), which is a transcriptional factor. CHOP is

said to be associated with apoptotic cell death by suppression of BCl2 expression and

sensitization of cells to endoplasmic reticulum stress inducing agents (Szegezdi et al., 2006).

IRE1 is a type I transmembrane protein kinase that is comprised of an endoribonuclease

domain and a Serine-Threonine kinase domain (Oslowski & Urano, 2011; Schroder &

19

Kaufman, 2005). The N-terminal domain of IRE1 recognizes unfolded or misfolded

proteins by BiP interaction. Post dissociation of BiP from this domain, there is IRE1

dimerization followed b7y autophosophorylation of the endoribonuclease and the kinase

domains. This endoribonuclease activity cleaves an intron from XBP-1 mRNA leading to a

spliced form of XBP-1. This is accountable for regulating the expression of ER chaperones

and ER associated degradation (ERAD). In addition, the cytosolic IRE1 dimers interact

with adaptors like TRAF2 (Tumour necrosis factor receptor associated factor 2) and drive

the expression of signal regulating kinase (ASK1) which initiates apoptosis.

Figure 10: An outline of UPR

APOPTOSIS

20

Chapter 5

Biochemical Assays to quantify ER Stress

5.1 Thioflavin Assay

Thioflavin T, also known as Basic Yellow, is a dye with a yellow component that is actually

responsible for staining amyloid fibrils in solution. It was suggested that the positive charges

of the dye was involved in micelle formation (Khurana et al., 2005). The basic conclusion

that could be drawn from this information is that increased fluorescence of amyloid

(essentially known to bind to Thioflavin for detection) causing it to be selectively brighter

than the background as a result of the increased fluorescence of the micelles attaching to it.

The increase in the fluorescence quantum yield can be ascribed to the restriction of torsion

oscillations of the ThT fragments when the dye incorporates in the amyloid fibril

(Kuznetsova, Sulatskaya, Uversky, & Turoverov, 2012). It was revealed that when ThT

binds to fibrils, it displayed a striking shift of its excitation maximum from 385 nm to 450

nm and emission maximum from 445 nm to 485 nm (Picken MD, PhD, FASN, Dogan,

M.D., Ph.D., & Herrera, M.D., 2012). Researchers are still ambiguous when it comes to

high-resolution characterization because of the insolubility and the heterogeneous nature of

the amyloid fibrils (Groenning, 2010). Despite the shortcomings of Thioflavin T as a dye, it

has been used for estimation of misfolded aggregates as it provides a broad staining

capacity, an extraordinary sensitivity and ease of use.

We indulged in obtaining RFUs for the supernatant culture as this can give us information

about the presence of misfolded aggregates in the IgG titers and consequently, we can gain

crucial information on the stress quantification in these supernatant samples of differently

treated cultures.

21

5.2 Thioflavin T assay Results

The Thioflavin assay requires one to optimize the dye concentration and hence, an

experiment was performed to check the optimal concentration range of dye that should be

used. As seen in the figure, saturation was observed at higher concentrations of the dye

suggesting that lower concentrations should be considered for conclusive results.

Figure 11: RFU vs dye concentration

To verify that RFU increases with concentration of cells or with decrease of diluted

supernatant solutions, we ran a standard with supernatant and cells of 250-4 CHO cell lines.

A standard curve was obtained with increasing concentration of cells to verify that RFU

increases with increase in the concentration. The final working dye concentration that was

used is 20 µM.

0

100

200

300

400

500

600

700

0 50 100 150 200 250 300

RF

U

Dye concentration (µM)

22

Figure 12: Standard curve with 250-4 cells

A standard curve was obtained with 250-4 Methotrexate containing culture cells which were

serially diluted. The initial concentration of the cells was 1.5 X 106 cells. This again verified

that RFU increases with increase in the concentration. The final working dye concentration

that was used is 20 µM.

Figure 13: Standard curve with 250-4 MTX culture

y = 0.003x + 75.49

R² = 0.990

0

500

1000

1500

2000

2500

0 0.2 0.4 0.6 0.8

Flu

ore

scen

ce

Million cells

y = 0.000x + 326.7

R² = 0.994

0

200

400

600

800

1000

1200

0 0.5 1 1.5 2

Flu

ore

scen

ce

Million cells

23

A ThT assay was done for media (Day 0) and MTX supernatant (Day 1) with a dye

concentration of 25µM. End point results were plotted. The RFU for media has shown a

significantly lower value as compared to the MTX supernatant. This validates the

functionality of the assay.

Figure 14: RFU vs. dilution units/µL comparison for media and supernatant

Likewise, a standard curve was run with the culture supernatant that was serially diluted.

With an increase in the dilutions, RFU showed a proportional decrease.

Figure 15: Standard curve with supernatant of 250-4 CHO cells

0

20

40

60

80

100

120

140

160

0 0.2 0.4 0.6 0.8 1 1.2

Flu

ore

scen

ce

Dilution units/µL

Media

Supernatant

y = 494.2x + 46.72

R² = 0.984

0

100

200

300

400

500

600

0 0.2 0.4 0.6 0.8 1 1.2

Flu

ore

scen

ce

Dilution units/µL

24

ThT assay was performed with Day 5 supernatant samples for three different conditions i.e.

MTX, No MTX and No G418. The final working concentration for the dye was 25µM and

the incubation period was 30 minutes. At the point where there is zero-dilution, the No-

G418 culture showed the highest RFU suggesting higher amounts of misfolded proteins in

the culture.

Figure 16: ThT comparison for MTX, No-MTX, & No-G418 culture conditions

ThT assay was performed for supernatants of the culture treated with different conditions;

Control (Con), Suppressor (Sup 1 and 2). The dye concentration followed was 20µM. The

increase in misfolded proteins was evident from the increasing RFUs in the cultures treated

with suppressors of UPR pathway.

Figure 17: RFU plot for suppressor of UPR

0

100

200

300

400

500

600

MTX No MTX No G

Flu

ore

scen

ce

0

50

100

150

200

250

300

350

Con Sup 1 Sup 2

RF

U

Tht assay for suppressor of UPR

25

Another ThT assay was performed with supernatant of 250-4 cells of which one is Control

and the other is treated with an inducer resulting in higher productivity (Ind 1). The final

working dye concentration was 20µM. An increase in the RFU values for Ind 1 treated

culture on Day 2 and Day 3 showed that the misfolded aggregates are higher in the Ind 1

treated cultures.

Figure 18: ThT comparison for Control (dark) & Ind 1 (light) treated culture

This assay suggests that the suppression of UPR pathway leads to the formation of

misfolded aggregates as the suppressor block one of the arms of UPR pathway leading to

constraint n the availability of folding resources. But contrary to that, treatment with an

strong inducer (Ind 1) of overall protein synthesis pathway too resulted in aggregate

formation again suggesting limitation of folding resources. In order to achieve a high quality

and quantity of titers, there needs to be balance between unfolded proteins and folding

machinery.

5.2 ROS Assay

Specific production rate is high for proteins such as monoclonal antibodies in mammalian

cells as they grow more rapidly after the cell growth phase than during the growth. In any

case, it becomes difficult for the cell activity to be maintained in the protein production

phase which can be due to the poor nutritional conditions surfacing from the low-serum or

serum-free environment. As such, from this information, one can say that death of

0

50

100

150

200

250

Day 2 Day 3

RF

U

26

mammalian cells including CHO cells is mainly via the apoptotic pathway. Owing to this, it

is necessary to optimize strategies to increase protein productivity by downregulating the

apoptotic pathway (Yun, Takagi, & Yoshida, 2003). Reactive oxidation species (ROS) such

as superoxide, hydrogen peroxide, hydroxyl radical, and singlet oxygen are shown to induce

apoptosis by suppressing the association of cytochrome c which causes the loss of

mitochondrial transmembrane potential (Yun et al., 2003). Naturally, the viability of cells

decreases when the ROS production increases. The two major sources of ROS are said to be

complex I and complex III which is a part of the mitochondrial electron transport chain.

They generate ROS when the electron transport is slowed down by high mitochondrial

membrane potential. Alterations in the ROS or the redox status directly or indirectly affect

ER homeostasis and protein folding (Malhotra & Kaufman, 2007). The major enzymatic

components of UPR that contribute to ROS production are protein disulfide isomerase

(PDI), NADPH Oxidase complexes, and endoplasmic reticulum oxidoreductin (ERO-1)

(Bhandary, Marahatta, Kim, & Chae, 2012). It is said that by way of depletion of

Glutathione (which essentially decreases ROS) during protein misfolding, ROS is produced

during disulfide bond formation. It is being said that ER stress and ROS production are

linked to one another in the UPR pathway and is the cause of a few pathological diseases.

We have performed the ROS assay to quantify these species in differently treated cultures.

Also, as we quantify the ROS, we can gain information regarding the amount of ROS

responsible for apoptosis and thereby, the contribution of ROS to the ER stress. The dye

used was 2’, 7’ – dichlorofluorescein diacetate (DCFDA) as it rapidly and efficiently

diffuses into the cells as a colorless probe (Pogue et al., 2012). A kinetic is run for an ROS

assay and the values are plotted at the 60th

minute.

27

5.3 ROS assay Results

A standard curve with different dye concentrations and cell concentrations was run to check for the

sensitivity of the assay. It was found out that, at the lower concentrations of dye the assay is linear.

So concentrations ranging 5-25 µM of DCFDA were used depending on the available numbers of

cells for analysis.

Figure 19: ROS Standard curves with different dye concentrations

ROS assay was performed with Day 3 samples of 250-4 cells for three different conditions

i.e. MTX, No MTX and No G418. DCFDA dye concentration used was 5µM. The excitation

and emission wavelengths are 485 nm and 525 nm respectively. Initial number of cells that

was considered is 2.5 X 106 following serial dilution. It can be inferred that the MTX culture

has a higher RFU at the second lowest dilution suggesting that the amount of ROS is the

highest in the culture treated with Methotrexate.

Figure 20: ROS Comparison for 1.25 X 106 cells

0

50

100

150

200

250

0 100 200 300

RF

U

Dye conc. (µM)

ROS standard curve (0.5 million

cells)

0

200

400

600

800

1000

1200

1400

0 100 200 300

RF

U

Dye conc. (µM)

ROS standard curve (1 million cells)

0

500

1000

1500

2000

2500

3000

0 100 200 300

RF

U

Dye conc. (µM)

ROS standard curve (5 million cells)

0

10

20

30

40

50

60

70

80

MTX No MTX No G

RF

U

28

ROS assay was performed with Day 5 samples of 250-4 CHO cells for three different

conditions i.e. MTX, No MTX and No G418. The DCFDA dye concentration was changed

to 10µM to check the sensitivity of the dye and the effect it has on the treated cells. Initial

number of the cells was 106 following serial dilution. As shown, Methotrexate containing

culture still maintains a higher RFU than the respective cultures suggesting that ROS is

present in an increased amount in this culture. The dye concentration seems to have not had

an effect to a large extent when used in the range of 2 to 10 µM.

Figure 21: ROS comparison for 0.5 X 106 cells

In order to see the generation of ROS pattern throughout the culture, daily ROS assay was

done with 0.5 million cells. It was observed that the latter half of the culture (Day 4

onwards) had higher ROS concentration as compared to the early stages. We had also seen

an increase in cumulative productivity from day 4 onwards suggesting that higher

productivity conditions leads to higher ROS formation.

Figure 22: Complete culture ROS profile

0

500

1000

1500

2000

2500

MTX No MTX No G418

Flu

ore

scen

ce

0

40

80

120

160

200

240

280

1 2 3 4 5 6 7

RF

U

Time (Day)

Day-wise ROS profile

29

ROS Assay was performed for Control and Tunicamycin (inhibitor of glycosylation) treated

cultures. It was evident from the results that, from the time-point of addition of

Tunicamycin, there was a significant increase in ROS levels as compared to control.

Tunicamycin treatment is known to increase productivity but such high levels of ROS levels

lead to formation of misfolded proteins.

Figure 23: ROS assay for control and Tunicamycin treated cultures

0

100

200

300

400

500

600

1 2 3

RF

U

Time (Day)

ROS assay (Con vs Tun)

Control

Tun

30

Chapter 6

Multiple sequence alignment and stress elements identification

Recombinant antibodies are presently the most significant biologics in mammalian cell

culture. Owing to this, their demand has increased manifold and it has become essential to

employ methods that improve antibody-titer in bio-production. It is essential to study and

locate these stress element sequences like ERSE and UPRE, in various genes that are

directly related to the protein processing pathway as it will help us in identifying genes that

are likely to get induced under stress conditions like excess protein production.

Consequently, we can gain information on various methods and components that affect the

production of the biopharmaceutical products.

Promoter regions are crucial regions that work synergistically with other regulatory regions

to direct the transcription of a gene. The promoter is located in a region upstream of the

gene. The promoter length can vary from 100-1000 bp but for the purpose of easy analysis,

we have considered locating these sites in a 500 bp region. Specific and short DNA

sequences called binding sites are located in this region. The ER Stress Response Element

(ERSE) has a consensus sequence CCAAT-N9-CCACG which is essential and adequate for

the induction of at least three major chaperones GRP78, GRP94, and calreticulin. The

Homocysteine-responsive endoplasmic reticulum-resident ubiquitin-like domain member 1

protein (HERP) which is one of the most highly inducible genes during the UPR, contains

not only the ERSE I but also the cis-acting element ERSE II having consensus sequence of

ATTGG-N-CCACG (Samali, Fitzgerald, Deegan, & Gupta, 2010). The Unfolded Protein

Response Element (UPRE) containing a consensus sequence of TGACGTGG/A was

initially considered as a DNA sequence bound by ATF6. The CCACG domain in the ERSE

I and ERSE II elements is considered the primary binding site (Samali et al., 2010). For

example, XBP1s binds to this domain without NF-Y/CBF factor while A|TF6 requires this

nuclear factor to bind at the same site (Kokame, Kato, & Miyata, 2001). The GC box has a

consensus sequence if GGGCGG and is usually located 100 bp upstream to the transcription

site. The TATAA box is located approximately 70 bp upstream of the start site. It is said to

associate with the transcription process by RNA polymerase.

31

The following flow chart explains the methodology used for identifying the stress element

sites in genomic DNA sequences of a particular chaperone or gene.

Mapping the upstream regions of the UPR genes involves an extensive protocol as shown in

the flow chart. The CHO genome database provides upstream sequences of some genes

involved in the UPR and the connecting apoptotic pathway. After entering the desired name

of the gene in this database, the mRNA of the respective gene shows a symbol representing

the gene. From here on, one can acquire an external link to the National Center for

Biotechnology Information (NCBI) site with a unique gene ID. The genomic location on the

NCBI website leads to a choice for downloading sequences of which GenBank provides the

necessary information for the given protocol. This opens up an entire page of information

regarding coding DNA regions and the upstream sites. Locate the start ATG codon site and

extract the FASTA sequence of the promoter sequences from the CDS region. From the 500

32

bp of nucleotides that one obtains from this region, ERSE, UPRE, GC box, and TATA box

sites can be located and marked successfully. For the genes in human, rat and mouse; the

protocol differs slightly. In this case, one can directly enter the gene name on the NCBI gene

database and choose the required gene from the gene ID and the remaining protocol stands

the same as explained. It is important that the reference sequence number be noted for future

work.

Table 2: Overview of the UPR site information for chaperones

Sites/Gene ERSE I ERSE II UPRE TATA box GC box

GRP78 (CHO) Y* Y* N Y Y

GRP78

(Human)

Y N N N Y

GRP78 (Mouse) Y Y* Y* Y Y

GRP78 (Rat) Y Y* Y* N Y

GRP94 (CHO) Y Y* N N N

GRP94

(Human)

N Y* Y* N Y

GRP94 (Mouse) Y N Y N Y

GRP94 (Rat) Y* Y* Y* Y* N

ERDJ4 (Human) N Y* Y* Y N

ERDJ4 (Mouse) N Y* Y* Y N

CNX (CHO) Y* N Y* N N

CNX (Human) Y* Y* N N N

CNX (Mouse) Y* Y* N N N

CNX (Rat) N Y* N N N

CRT(CHO) Y* Y* Y* Y N

CRT (Human) Y* N Y* Y Y

CRT (Mouse) N Y* Y* N Y

CRT (Rat) N Y* N Y Y

PDI (CHO) N Y* Y* N N

33

Table 3: an overview of the UPR site information for UPR pathway and the related apoptotic pathway

Sites/Gene ERSE I ERSE II UPRE TATA box GC box

ATF4 (Human) Y* Y* Y* N Y

ATF4 (Mouse) N Y* Y* N Y

ATF4 (Rat) N Y* Y* N Y

CHOP (Human) Y* Y* Y* Y N

CHOP (Mouse) Y* Y* N N N

CHOP (Rat) Y* Y* N N N

GADD34 (Mouse) Y* Y* Y* N N

GADD34 (Human) Y* Y* Y* N N

GADD34 (Rat) Y* N Y* N N

XBP1s (Mouse) Y* Y* Y* N N

XBP1s (Human) Y* Y* Y* N N

XBP1s (Rat) Y* Y* Y* N Y

ATF6 (Mouse) Y* N Y* Y N

EDEM (Mouse) Y* N Y* N Y

PERK (Mouse) Y* Y* N Y N

HIFa (Mouse) Y* Y* Y* N N

B Actin (CHO) Y* Y* Y* N N

CASP3 (CHO) N Y* Y* Y N

FADD (Mouse) Y* Y* Y* N Y

BAX (CHO) N Y* Y* N N

BCl2 (Mouse) Y* Y* Y* N Y

BAK1 (CHO) Y* Y* N N N

CASP8 (CHO) Y* Y* Y* N N

JNK1/MAPK8

(CHO)

N Y* N N N

TRAF2 (CHO) Y* Y* Y* N N

BID (Mouse) Y* Y* Y* N N

TRIB3 (Mouse) Y* Y* N N N

ASK1/MAP3K5

(Mouse)

Y* Y* N Y N

Cyclin D1 (CHO) Y* Y* N N N

CDK2 (CHO) Y* Y* N N N

34

APAF1 (CHO) N Y* N N N

6.1 Verification Tools

Multiple Em for Motif Elicitation (MEME) is a tool for identifying motifs in groups of

nucleotide or protein sequences. The input to MEME is a set of unaligned sequences in the

FASTA format. In this particular case, the aim was to match the endoplasmic reticulum

stress element (ERSE I and ERSE II) and the UPRE sites from the promoter regions of the

unfolded protein response related genes. The basic aim was to check the occurrences of the

required sites and the consensus sequences. Further, when the FASTA sequences were

added in the input site of MEME, it was found that the sites that were being probed were

highly conserved and hence, the motif sites did not give a conclusive result. Therefore, the

FIMO (Find Individual Motif Occurrences) tool provided a clinching output result. The

protocol that was followed is:

Go to www.meme.ncbr.net which gives a MEME Suite webpage

↓

Choose ―Discover New Motifs Using MEME‖ from the ―Submit A Job‖ menu

↓

In the Data Submission Form, provide the email address for result submission along

with FASTA sequences containing a repetition of the sequence for the required site

↓

The minimum and maximum width of the resulting sequence can be inserted as per

the requirement (here: 5) with a choice of repetitions

↓

Click on the link containing the results in various output formats. In this case,

HTML output provides a conclusive output

↓

The motif result page gives you a detailed summary of the sites. The site that is

aimed at, for e.g.: CCACG is shown along with the start position

↓

An option for further analysis provides a link to ―FIMO‖

↓

FIMO will search the site using the previously provided motif in MEME. Paste the

desired sequences in the FASTA format and choose the p-value output threshold =1

↓

Start the search

35

↓

View the results in the FIMO HTML output

↓

The results are shown in a tabular format for the high-scoring motif occurrences

along with the start and the end site

Further, Multiple Sequence Alignment from MultiAlin by Florence Corpet was performed

for the sequences of different organisms to check whether they share a common ancestry.

This helps in determining the extent to which the sequences of the same gene among

different organisms are related. We can obtain a set of aligned sequences and then locate the

UPR sites. This saves the time-consuming process of manually aligning each sequence and

also, eases out the process of analyzing the data sequences. The FIMO results that were

obtained in a tabular format aids in locating the sites from the aligned gene sequence data.

After obtaining this aligned data, the consensus sequence was matched for the various sites.

For every gene whose sequences were available for three or more organisms, a table was

deduced with the consensus positions as shown in the following pages.

Table 4: List of consensus positions for UPR genes

Gene/Sites ERSE I ERSE II UPRE AARE I AARE II TATA

Box

CAAT

Box

GC

box

GRP78 -317 to -

336

-22 to -31 -409 to -

418

(partial)

-323 to -

332

(partial)

-323 to -332

(partial)

-278 to

-283

-353 to

-362

-

GRP94 -302 to -

321

-274 to -283

(partial)

-430 to -

436

(partial)

-398 to -

407

(partial)

-398 to -407

(partial)

-

-

-105

to -

111

CRT -321 to –

340

(partial)

-282 to -

291(partial)

-94 to -

103

(partial)

-396 to -

405

(partial)

-396 to -

405(partial)

-101 to

-106

- -

36

CNX -75 to -

94

(partial)

-48 to -57

(partial)

-257 to -

266

(partial)

-183 to -

192

(partial)

-183 to -192

(partial)

- - -

ATF4 -202 to -

221

(partial)

-446 to -455

(partial)

-322 to -

331

(partial)

-21 to -

30

(partial)

-21 to -30

(partial)

- - -

CHOP -853 to -

872

(partial)

-559 to -568

(partial)

-117 to -

126

(partial)

-546 to -

555

(partial)

-546 to -555

(partial)

- - -

GADD34 -87 to -

106

(partial)

-248 to -257

(partial)

-209 to -

218

(partial)

-28 to -

37

(partial)

-28 to -37

(partial)

- - -

XBP1 -97 to -

116

-165 to -174

(partial)

-422 to -

431

(partial)

-441 to -

450

(partial)

-441 to -450

(partial)

- - -42

to -

48

37

GRP78

Figure 24: MSA for GRP78

38

Figure 25: Matched Stress Elements with Grp78 consensus sequence

39

GRP94

Figure 26: MSA for GRP94

40

Figure 27: Matched Stress Elements with Grp94 consensus sequence

41

CRT

Figure 28: MSA for CRT

42

Figure 29: Matched Stress Elements with CRT consensus sequence

43

CNX

Figure 30: MSA for CNX

44

Figure 31: Matched Stress Elements with CNX consensus sequence

45

ATF4

Figure 32: MSA for ATF4

46

Figure 33: Matched Stress Elements with ATF4 consensus sequence

47

CHOP

Figure 34: MSA for CHOP

48

Figure 35: Matched Stress Elements with CHOP consensus sequence

49

GADD34

Figure 36: MSA for GADD34

50

Figure 37: Matched Stress Elements with GADD34 consensus sequence

51

XBP1

Figure 38: MSA for XBP1

52

Figure 39: Matched Stress Elements with XBP1 consensus sequence

53

Chapter 7

Discussion and Conclusion

Mammalian cell productivity has become a primary topic of research. Cell culture

technology has become a powerful medium through which one can alter process parameters

and assess the effects they have on the productivity profile of a particular cell line. CHO cell

lines have become this valuable tool to monitor and control these processes due to the ease

of post-translational modifications and glycosylation. The UPR pathway is the cardinal

pathway which links the overall productivity of recombinant proteins and the folding

capacity of the protein. The UPR, which is responsible for bringing the endoplasmic

reticulum to homeostasis in events of misfolding of proteins, provides an insight into the

mechanism of action that takes place in order to allow the secretion of correctly folded

proteins. An essential understanding of the three UPR transmembrane sensors namely

ATF6, PERK and IRE-1, helped us in overlooking the modifications that we can assume in

performing the experiments.

Metabolic assays, on the other hand, give us vital details regarding the consumption rate and

the productivity profile as a whole. Accumulation of lactate at the end of glycolysis causes

disturbance in the environment of the mammalian cell culture system and hence, it is a

critical limiting factor especially when cell density is high. Thus, the lactate levels of a

particular culture act as an indicator of deteriorating culture. The glucose consumption rate

acts as an indicator of the amount of substrate being consumed. When the cells are reaching

its decline phase, the glucose levels substantially decrease.

Tunicamycin is an antibiotic that inhibits N-linked glycosylation which consequently cause

the accumulation of unfolded proteins in the ER. The treatment of cells with tunicamycin

increased the overall IgG titers (data not shown) but accordingly led to increase in ROS and

misfolded proteins. Similarly treating with other inducers and suppressors resulted in

misfolded protein formation validated by Thioflavin T assay. The ease and sensitivity of the

Thioflavin T assay can be employed in screening large sets of inducers and suppressors

without using costlier and labor intensive techniques. The ROS data indicated that at higher

productivity stages or conditions, there is a significant increase in the ROS concentrations

inside the cell.

54

The computational data gives an altogether different dimension to studying the UPR

pathway and applying it in the productivity profile. Here, the aim is to locate the ERSE and

the UPRE sites in the coding DNA sequences of the transcription factors involved in UPR.

This way, one can determine if there are genes linked to the UPR pathway containing the

primary binding ERSE sites. And from this information, it can give an idea if there are

certain genes that have an effect on ER stress and the mechanism by which they have a

substantial effect, if at all.

To conclude, various parameters have been studied that are said to have an effect on ER

stress and consequently, the productivity. The growth kinetics of CHO cells showed a

variable effect and we could study the effect it eventually had on the culminating days of the

culture profile. The computational data that were obtained for various UPR genes provided

a way to focus closely on the sites and their consensus sequences.

55

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57

Appendix

The location of the stress elements of the chaperones and genes in the UPR are shown

below.

GRP78/HSPA (Human) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA) NCBI Reference Sequence: NG_027761.1; >gi|307746866:4501-11540 Homo sapiens heat shock 70kDa protein 5 (glucose-regulated protein, 78kDa) (HSPA5),

RefSeqGene on chromosome 9

GAGTGGGTTGCCACAGTAGGGAGGGGACTCAGAGCTGGAGGCAATTCCTTTGGCCGGGCTTGTCCTGCGACTTACCGTGGGGCAGCGCAATGT

GGAGAGGCCTGGTAAAATGGCTGGGCAAGGGTGCGGAGGGGACATAACTGGCAGGAAGGAGTCATGATTCGTGGTCGAACAGAGTCCAGACCAGCTCGACCTGTGAGCAACGAACGGCCCTGAGACTCGCATACCCCAATACCGGTAGTGGCCGTGAAGGGCAAAGAAATGTGTTCTGAGGCGATCCCAGCA

TCTAAGCTGCGACTGGTCTACTCAGAGACTGGATGGAAGCTGGGAAGAGAAAGCTGCTTCCCGCTTCGGGGTGAGGGATGGAGGAAGGGAGAACAAGCA

GTAGAGAAGAAAAAGTTTCAGATCCCACAGCCCCGGGGGGTCACTCCTGCTGGACCTACTCCGACCCCCTAGGGCCGGGAGTGAAGGCGGGACTTGTGC

GGTTACCAGCGGAAATGCCTCGGGGTCAGAAGTCGCAGGAGAGATAGACAGCTGCTGAACCAATGGGACCAGCGGATGGGGCGGATGTTATCTACCATT

GGTGAACGTTAGAAACGAATAGCAGCCAATGAATCAGCTGGGGGGGGCGGAGCAGTGACGTTTATTGCGGAGGGGGCCGCTTCGAATCGGCGGCGGCC

AGCTTGGTGGCCTGGGCCAATGAACGGCCTCCAACGAGCAGGGCCTTCACCAATCGGCGGCCTCCACGAcggggctggg

ggagggtatataagccgagtaggcgacggtgaggtcgacgccggccaagacagcacagacagattgacctattggggtgtttcgcgagtgtgagagggaagcgccgcggcctgtatttctagacctgcccttcgcctggttcgtggcgccttgtga

ccccgggcccctgccgcctgcaagtcggaaattgcgctgtgctcctgtgctacggcctgtggctggactgcctgctgctgcccaactggctggcaagATGAAGCTCTCCCTGGTGGCCGCGATGCTGCTGCTGC

TCAGCGCGGCGCGGGCCGAGGAGGAGGACAAGAAGGAGGACGTGGGCACGGTGGTCGGCATCGACCTGGGGACCACCTACTCCTGGTAAGTGGGGTTGC

GGATGCAGGGGGACGGGGCGTGGCCGCCTGGCCTGGCGTGAGAAGTGCGGTGCTGATGTCCCT

GRP78/HSPA (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA) NCBI Reference Sequence: NC_000068.7; >gi|372099108:34771590-34776529 Mus musculus strain C57BL/6J chromosome 2, GRCm38.p2 C57BL/6J

GAAGATTCGAAAGGCCTGGAAAGACACATACGGCTAGCCTTGGGGTGAAGGAGAAACACGGTTAGCTGAGAAGCACCAGGATTCTCAGCGAGGCAGAAT

CCAGATCAGGCCCCAGCTCGAGACGTGCAGGCCGGGCGAGTAACAGGGCCTGGACTCTGGGACATCCGAGAACGTGTGGAGGCTGGGGAGGGCGATCAC

AGCTGAGGCCGGGCAGCTCAGGACGCGGGGAATCGAGGAGGAGAAAGGCCGCGTACTTCTTCAGAGTGAGAGACAGAAAAGGAGACCCCGAGGGAACGA

CAGGCAGCTGCTGAACCAATAGGACCAGCGCTCAGGGCGGATGCTGCCTCTCATTGGTGGCCGTTAAGAATGACCAGTAGCCAATGAGTCAGCCCG

GGGGGCGTAGCAATGACGTGAGTTGCGGAGGAGGCCGCTTCGAATCGGCAGCAGCCAGCTTGGTGGCATGGACCAATCAGCGGCC

TCCAACGAGTAGCGACTTCACCAATCGGAGGCCTCCACGACGGGGCTGTGGGGAGGGTATATAAGGCGAGTCGGCGACGGCGCGCtcgatactggccgagacaacactgacctggacacttgggcttctgcgtgtgtgtgagGTAAGCGCCGCGGCCTGCTGCTAGGCCTGCTCCGAGTCTGCTTCGTGTCTCCTCCTGACC

CCGAGGCCCCTGTCGCCCTCAGACCAGAACCGTCGTCGCGTTTCGGGGCCACAGCCTGTTGCTGGACTCCTAAGACTCCTGCCTGACTGCTG

AGCGACTGGTCCTCAGCGCCGGCATGATGAAGTTCACTGTGGTGGCGGCGGCGTTGCTGCTGCTGGGCGCGGTGCGGGCCGAGGAGGAGGACAAGAA

GGAGGATGTGGGCACGGTGGTCGGCATCGACTTGGGGACCACCTATTCCTGGTAAGTGGTATCCGTCGAAGGAGGAGGGGGTGGGGAGGAGTGG

58

GRP78/HSPA (Rat) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA) NCBI Reference Sequence: NC_005102.3; >gi|389675126:19157374-19162333 Rattus norvegicus strain BN/SsNHsdMCW chromosome 3, Rnor_5.0

GAGAAAAGTGCCGAGGCTGGGAAGGGTGATCACAGCATCACAGCTGAGGCCGGGCAGCTGAAGACATGAGTGAATCTAGGAGAAGAAAGGCAGCGTACT

TCTTCCGAGTGAGAGACAGAAAGAGAGGACCCGAGTCTCACAGCCCTGAGGGAACTGACACGCAGACCCCACTCCAGTCCCCGGGGGCCCAA

CGTGAGGGGAGGACCTGGACGGTTACCGGCGGAAACGGTTTCCAGGTGAGAGGTCACCCGAGGGACAGGCAGCTGCTCAACCAATAGGACCAGCTCTCAGGGCGGATGCTGCCTCTCATTGGCGGCCGTTAAGAATGACCAGTAGCCAATGAGTCGGCCTGGGGGGCGTACCAGTGACGTGAGTTGCGGA

GGAGGCCGCTTCGAATCGGCAGCGGCCAGCTTGGTGGCATGAACCAACCAGCGGCCTCCAACGAGTAGCGAGTTCACCAATCG

GAGGCCTCCACGACGGGGCTGCGGGGAGGATATATAagccgagtcggcgaccggcgcgctcgatactggctgtgactacactgacttggacacttggccttttgcgggtttgagagGTAAGC

GTCGCGGCCTGCTTCCAGGCCTACCCTGATTTTGGTTCGTGGCTCCTCCTGACCCTGAGACCTCTGTCGCCCTCAGATCAGAACCGTCGTCGCGTTTCG

GGGCTACAGCCTGTTGCTGGACTCTGTGAGACACCTGACCGACCGCTGAGCGACTGACTGGTCCACAGCGCCGGCAAGATGAAGTTCACTGTGGTGGCGGCGGCGCTGCTGCTGCTGTGTGCGGTGCGGGCGGAGGAGGAGGACAAGAAGGAGGA

GRP78 (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351516441:303874-308120 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold260, whole genome shotgun sequence; NCBI Reference Sequence: NW_003615108.1

GGCAGAGATGCGTTCCCAGGCGACCACAGCATCTATGCTGAGGCTGAGCAGCTCGGGACCCGAGGGGACTTAGGAGGAGAAAAGGCCGCATACTGCTTC

GGGGTAAGGGACAGACCGGGGAAGGACCCAAGTCCCACCGCCCAGAGGGAACTGACACGCAGACCCCGCAGCAGTCCCCGGGGGCCGGGTGA

CGGGAGGACCTGGACGGTTACCGGCGGAAACGGTCTCGGGTTGAGAGGTCACCTGAGGGACAGGCAGCTGCTGAACCAATAGGACCGGCGCACAGGG

CGGATGCTGCCTCTCATTGGCGGCCGTTGAGAGTAACCAGTAGCCAATGAGTCAGCCCGGGGGGCGTAGCGGTGACGTAAGTTGCGGAGGAGGCCGCT

TCGAATCGGCAGCGGCCAGCTTGGTGGCATGGACCAATCAGCGTCCTCCAACGAGAAGCGCCTTCACCAATCGGAGGCC

TCCACGACGGGGCTGGGGGGAGGGTATATAAGCCAAGTCGGCGGCGGCGCGCTCCacactggccaagacaacagtgaccggaggacctgcctttgcggctccgaga

GGTAAGCGCCGCGGCCTGCTCTTGCCAGACCTCCTTTGAGCCTGTCTCGTGGCTCCTCCTGACCCGGGGGGCTTCTGTCGCCCTCAGAtcggaacgccgccg

cgctccgggactacagcctgttgctggacttcgagactgcagacggaccgaccgctgagcactggcccacagcgccggcaagATGaagttccctatggtggcggcgg

59

GRP94/HSP90B1 (Human) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA) NCBI Reference Sequence: NC_000012.12; >gi|568815586:103939834-103947930 Homo sapiens chromosome 12, GRCh38 Primary Assembly

ACGTTGCCATGGCTACCGTTTCCCCGGTCACGGAATAAACGCTCTCTAGGATCCGGAAGTAGTTCCGCCGCGACCTCTCTAAAAGGATGGATGTGTTCT

CTGCTTACATTCATTGGACGTTTTCCCTTAGAGGCCAAGGCCGCCCAGGCAAAGGGGCGGTCCCACGTGTGAGGGGCCCGCGGAGCCATTTG

ATTGGAGAAAAGCTGCAAACCCTGACCAATCGGAAGGAGCCACGCTTCGGGCATCGGTCACCGCACCTGGACAGCTCCGATTGGTGG

ACTTCCGCCCCCCCTCACGAATCCTCATTGGGTGCCGTGGGTGCGTGGTGCGGCGCGattggtgggttcatgtttcccgtcccccgcccgcgggaagtgggggtgaaaagcggcc

cgacctgcttgcggtgtagtgggcggaccgcgcggctggaggtgtgaggatccgaacccaggggtggggggtggaggcggctcctgcgatcgaaggggacttgagactcaccggccgcacgccATGAGGGCCCTGTGGGT

GCTGGGCCTCTGCTGCGTCCTGCTGACCTTCGGTGAGTGATTCTGGAGGAGCAGACGTCCCCCCTC

GRP94/HSP90B1 (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA) NCBI Reference Sequence: NC_000076.6; >gi|372099100:c86705444-86690341 Mus musculus strain C57BL/6J chromosome 10, GRCm38.p2 C57BL/6J

AGGTGACGGCGAACGTAGCGCTGAAAGGACTCGTAACGTGACCCGCGTCGTAGACGAGAAAAGGGTAAAGGACGCATTGTCTTGGCTACCGTTTCCCCT

AGTCACGGACTAAACGTTCGCTAGAAGCCGGAAGTGGTTCCCCGGGACCTCTAGGAATGGACAGACGTGCTATGCGCCTACGTTCATTGGACGGTTTTC

CTCAGGGACCAAGGCTTCCCAGGCCAAAGGGTGGCCCGGTGTGTGAGGGCCCGCGGAGCCATCTGATTGGAGGAAAGCCGCTGGACAAGCCCAAT

CGCAAGGAGCCACGCTTCGGGCATCGGGCACCGCACCTGGACAGTTCCGATTGGCGGGCTGCGGTCCCCCCCCATGCGTCTCCATTGGGT

GCAGAGAGTGCGTGGTGAGGCACGATTGGTGAGTTCGTGTTTCCCGTCCCCCGCCCGCAAGCAGTGGGGTGAAAAGCGGCCCGACCTGCGCGCGGCTTAGTGGGCGGACCGCGCTGCtggaggtgtgaggagcttagactcgggattgggggggtggaggcggctcctgagaccgaaaaggacttgcgactcgccggccacgcaccATGAGGGTCCTGTGGGTGTTGGGCCTCTGCTGTGTCCTGCTGACCTTCGGTGAGTGACCGGGCGGCAGTGGGCGCCCTCCCCTTCCTGTGTGGCCGCTTCTCGAACGTTCTTGG

GGCGTTGAACCTGGGTT

GRP94/HSP90B1 (Rat) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA) NCBI Reference Sequence: NC_005106.3; >gi|389675122:c27359757-27345230 Rattus norvegicus strain BN/SsNHsdMCW chromosome 7, Rnor_5.0

AATTTCTCTTTTGCGAAAAGAAACGCCCAAAAGAAAGGTGACGGCGAACGTAGCGCTGAAAGGGCTCGTAACGTGACCCACGTCGTAGACGG

GAAAAGGGTATAAACCACATTGTCTTGGCTACGGTTTCCCCTAGTCACGGAACAAACGTTCTCTAAGAGCCGGAAGTGGTTCCCCGGGACCTCTAGG

AAAGGACAGACGTGCTATGCGCCTACATTCATTGGACGGTTTTCCTCAGAGACCAAGGCTTCCCAGGCCAAGGGGTGGCCCGGTGTGTGAGGGGCCCGC

GGAGCCATTTGATTGGAGAAAAGCTGCTGGACAAACCCAATCGAAAGGAGCCACGCTTCGGGCATCGGGCACCGCACCTGGACAGTT

CCGATTGGCGAGTTGCGGTCCCCCCCATGCGTCCCCATTGGGTGCAGAGAGTGCGTGGTGAGGCACGATTGGTGGGTTCGTGTTTCCCGTCCCCCGCCCGCAAGCTGTGGGGTGAAAAGCGGCCCgacctgcgcgcggtttagtgggcggaccgcgctgctggaggtgtgaggacctgagactccgggttgggggggtggaggcggctcctgcgaccgaaaaggacttgc

gactctccggccacgcaccATGagggtcctgtgggtgctgggcctctgctgcgtcctgctgaccttcTCCAGAGCGTGTTTCTGTTTTCTAACGCCCGACTCGCGAGCGTGGGC

60

GRP94 (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351517354:c640360-626072 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold3191, whole genome shotgun sequence; NCBI Reference Sequence:

NW_003614195.1

CTCACTACCATCCATACGCACCCAGGAAGAGTGTTCTACCCTTTACATATTTCCCTTTTTCGAAAAGCGATAACGAACAGAAAGGTGACGGCGAGCGTA

GCGGAAACGGCTCCCAACATTACCCTCACCCCGTCGTAGACGGGAAAAGGGTAAAAAACGCGTTGTCTTAGCTACCGTTTCCCCTAGTCACGGACTAAA

CGTTCTGTAGGAACCGGAAGTGGTTCCCCGGGACCTCTAGGAAAAGACAGACGTGCTATGCGCTGACGTTCATTGGACGGTTTTCCTCAGAGGC

CACGGCTTCCCAGGCCAGGGGGTGGCCCTGCGTGTGAGAGGCCCGCGGAGCCATGTGATTGGAGGACAGCTGCTGGCCGAGCCCAATCGGA

AGGAGCCACGCTTCGGGCATCGGGCACCGCACCTGGACAGTTCCGATTGGTGGGCTGCGGTCCCCCCCGGGCGTCCCCATTGGGTGCGGGGAG

TGCGTGGTGAGGTGCGATTGGTGTGTTCGTGTTTCCCGTCCCCCgcccgcaagccgtgcggtgaaaagcagcccgacctgcgcgcgggttagtgggcggaccgcgcggctggagg

tgtgaggacctgaggctcggggtgggggcggaggcggctcctgcgaccgaagaggacttgcgactcgccgtccgcgcaccATGagggtcctgtgggtgttgggcctctgctgcgtcctgctgaccttcg

ERDJ4 (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA) NCBI Reference Sequence: NC_000078.6; >gi|372099098:c44210068-44205397 Mus musculus strain C57BL/6J chromosome 12, GRCm38.p2 C57BL/6J

TTTCTGAAGTATTTGGGAAGTTAAATTTATGCAAACAGACTATTTTAACCACTTTAAGATCAAATAGATTTTACAGATTTGAGAAAAATCTTTCCT

TCCCCACCTTTGCCTTTCTTCCTGCGGTTCTAGCCAAACACAGAAAAGACAGATTTCTTTTTCAGTAATTGGTTTATATTCTGAAATTAAATGT

GGTAATGAAGACAGCGCTGAGGAAGCTGGGGTAGATCAGGAGCCACCTCAGGAAAATGCAGTATTAAATAATAATATAAACAAGTAATAA

TACCTTTTGTATCACAGGCAGACAAGTTCACCATCAAGTAGTAGAAATCCTAAGCCTTCCTAAGAAACTATCAGTTTTATCTTTCCAGTAGAT

AAGAAAAGCCTTGCTAATAGACTCTAATATCAGAAGTACAAGAGCGTGACTAATGTGATACTATGTGCATAACAGCTTGATGCTGCTGTCTCA

ACACCAGAGCTTTATTGAGTTGGATTTTTTTTTTAAGTCTTAAAATTTGTTCCTTGGACTTAAAAAGACACTATGTTTTTCTTTCTTAGGT tattagaaAT

Ggctactccacagtcagttttcgtctttgcaatctgcattttaatgataacagaattaatcctggcctccaaaagctactatgatatcttagg

ERDJ4 (Human) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA)

NCBI Reference Sequence: NC_000007.14; >gi|568815591:108569245-108574850 Homo sapiens chromosome 7, GRCh38 Primary Assembly

ATTTGCAGTCCTTGGCTCACCTGCTGAAATGAAAGCTGAATTTTTGGAGCGAGATTAATAATACTAATTTAGATATACTGGGCTATTTACCTTGGT

GCGCCACCTTTTTTTTTTAATGTTTTTTTTTTCTGTGAAGTTAAATACATTATTACTTTCAGGATATGAAGTCTTGAAAGGAAAGTTGGGATCTTATATTATTTGGTTTTCTTTTTTTTTCTTACAATGCTTGCATATGATGAATCTTCAATATAAACTGGTTAAAAGTCAGTTATTGATAATTTTCTATCTC

61

CTCTGTGTATGGCCAGAGTATTAGTAGACAGACAAAGCCAAGAATTATTTTTGTTCTCAATAAGGTTAAGCCAGTTTGAGAGTTATAAAACTAAAAG

TGAAATTACATGTGGACAGGTAATAGATACTGTATGCTTACTATTTTGATACTGCATTAGGTTTACCTTGTGTTGGATTTCTTTTAAAAGAAAAAC

GTTTTAAGATTAGCTCCATCCATAACATTTTTTTTTCTTTTCTGTTTTAGGATattagaaATGgctactccccagtcaattttcatctttgcaatctgcattttaatgataacagaattaatt

ctggcctcaaaaagctactatgatatcttaggtgtgccaaaatcggcatcagagc

CNX (Human) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA)

NCBI Reference Sequence: NC_000005.10; >gi|568815593:179698429-179731641 Homo sapiens chromosome 5, GRCh38 Primary Assembly

AGAGAAGAGTTTCGCCATGTTGGCCAGGCTGGTTGTGAACTCCTGACCTAGTGAGCCACCTGCCTCGGCCTCCCAAAGTGC

TGGGATTACAGGCGTGAGCCACGGCGCCCGGCCATCTCTGTTATTTCATTGTAATGTTTTAACGGGTACCCCCTGTAAATTAGTGTATGAAA

GGGCTTTTGTCTGTTGTAAAGCACTTTACAAATGCAAAAGTTTGTTGGAAATTGTATTTGAATGCCCCACATCTGTGTAACCAATCCTCTCTATATGAGGATGTTGTGTATGTTTCAGTTTTATTTTGAAATTTTCCAGAAATGGAATCTTTTAACTGATTTTAGGGAAACCCTTTAATCTCTCTA

GGCTGCCTTTCTTTATCTATGAAATGAAATAGTAAGTTCTTTTTAGCTCTGCGATTTAAAAGCTTTAATTTTAAATGAGAGAGTGGTTAGTGATCTTCA

TGAAGTTTGATAGGTGGCAATACATTTAACTGATTTTGCTCTTTATGTGTAgatcATGgaagggaagtggttgctgtgtatgttactggtgcttggaactgctattgttgaggctcatgatggaca

tgatgatgatgtgatt

CNX (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA)

NCBI Reference Sequence: NC_000077.6; >gi|372099099:c50325673-50293457 Mus musculus strain C57BL/6J chromosome 11, GRCm38.p2 C57BL/6J

TTATTTTTTGATAGCACTTCACAGTTCAGACAGGCCTTTAATTCAGGGTTTTCCTGTCTCAGTCACCCAAGTACTAACTAGTACAGGTATGCACCAGAATACTCGGTTTACATGGTATACTTAGATATGCTTTTGAATGTTGCTTTTAAGTGACTGAATCTAGGAAAATCTTTTCATCTCCCAGGG

TTCACTTTCCCTTTTGTATAGGTTGAAAATGCATTTGTTGTTGTTGTTCTGTGATTTGAAACCCTGATTAACAAGGAACAGACTAAGGTTAGTGGTTTT

ATGGAATTTAAAATGGGAAACAGTACATTTCACATTCCATTCTTTATTTTTCTTTTTCTTTTTTTGGTTTTTTGAGACAGGGTTTCTCTGTGCAGTCCT

GGCTCTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCGAACTCAGAAATCCTGTGCCTTCCAAATGCTGGGATTACAGGCATGCGCCAGC

ACTGCCCGGCTTCACATTCCATTCTTTTTGCAGATAGAtcATGgaagggaagtggttactgtgtttgctgctggtccttggaactgcagctgttgaggctcatgatggacatgatgatgacgcg

62

CNX (Rat) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA)

NCBI Reference Sequence: NC_005109.3; >gi|389675119:c35595530-35562108 Rattus norvegicus strain BN/SsNHsdMCW chromosome 10, Rnor_5.0

AGGTCACACATAATAAATAAGTATAATGGGACTAATTAATTAAATATCCTTGGGCTATGTATTGTTTGATACTGTATGTGAGAGAAAGTGACAGACAGG

GTTTCCTGTGGTTTTGAGATTTTACTCTGTAGCCAAAGACAACATTGAACTTCTGATCTCCTACCTGTGCTTCCTGAGTGGAGCACTGGTGTAATTTTA

TTAATTTTTGATAGCACTTCACAGTTCAGACAGGCCTTTAATTCAGGGTTTTTCTGTTTCAGCCACCCAAGTACTAGTACACGTATGCACCA

GAATACCCAGTTTATATGGTGTCATTAGATATTCTTTTGACTTTTACTTTTAATTAACTGACTTTAGGAAAGTCCTTTCATCTCCCAGGGTT

CACTTTTCGTTTTTTATAGGCTGAAAATTAAAAAAAAAAATTTTTTTTTTTTGTTCCATGATTTGAAACCCTGATTTTAAACAAGGAGGCTA

AGGTTAGTGGCTTTATGGAATTTAAAATGGGAAACAGCACATTGTACATTCCATTCTTTTTGCAGAtagatcATGgaagggaagtggttactgtgtttgctactggtccttggaact

gcagctattcaggctcatgatggacatgatgatgacatgattgatattgaagatgatcttgatgatgtt

CNX (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351517404:c768221-753373 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold1501, whole genome shotgun sequence; NCBI Reference Sequence:

NW_003614145.1

ACAGGGTTTGTCTGTGGTATTGGAGGCTGTCCTGGAACTAGGTCTTGTAGACCAGGCCGGTCTCAAACTCGCAGAGATCCGCCTGCC

TCTGCCTCCCGAGTGTTGGGATTAAAGGCATGTGCCACCAACGCCCGGCCATTTTAGGGGATTTTTAGTAGTGTGTTAGATCCCCTAGACTCAGGTGGTAGAAGGAAAGAATCTTGAAAGTTGTCCCTTTTTTTTTTTTTTTTTTCCCCTAGTTTTTCAGACAGGGTTTCTCTGTGTAACA

TCCCTGGCTGTCCTGGAACTTGCTTTGTAGTCCAGGGTGTCCTCAAACTCAAAGAGATCTGCTTGCCTCTTCCCCCAAGTGCTGGGATTAAAGGTATGC

ACACCATACCCAGCTCACTTTTCCTTTTCTATAGGCTGAAGTATAGTATTTCTTTTGTTCTTTGATTTTTAAACAAGGAGTAGGTAAAGGTTTTATGGG

ATTTTAAAATGGGACACAGCACATCTTACATTCTATTCTTTTTGCAGGTAGATCATGGAAGGGAAGTGGTTACTGTGTTTACTCCTGGTCCTTG

CRT (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA) NCBI Reference Sequence: NC_000074.6; >gi|372099102:c84846931-84841588 Mus musculus strain C57BL/6J chromosome 8, GRCm38.p2 C57BL/6J

TCAGGATCCTGGCTGGCCCTTGACCTTATCCTGAATAGGAAACGCTCGCCATCGGTGGGCGTTCCCTAGGTGCAGGACAGACGGAACGTGAAAGTTGCA

AATAATCCTTACTTCTTCCCTCTGACCAGAGAGGATGGGAAAGGGCCGAAGCTAAGGACCCGTCTCGGTCCCGCACCGCACGGTTAACACCTGGTACCG

CTCGCGCGGATTCTTTAAACGACTCCTAGCGAGCCAGAGACTCTCAGCAGCAAGGGCGGGGTTGGGCTGAGGTTCAGTCACGTGACCGTGCCTGAGTGG

GCTAGCGGCCCCCACCCCACCAGGGGGCGTCCCCCACAACGCGTGGTCGACCCTCATTGGCCCATAGTGCGACCAATAGAAATCAGCCATCTG

GGATCCCAGCGTTCCGAGCCACAGCCTAACTTGCTGAGCCAACTGGGAAGCAATGGAAAGGGACAGCTGTAGGTCTAAACCAGTCAAAAGGACCGAGGGGCGGGCTCAGCggctgtgtcaggttcgggtgagaggtaggtgaatataaattgaagcggcggtggccgcgtccgtcaataccgcagagccgctgcctgaagatcgtcttaaaaggcctgtgtgccgccgccccct

cggcccgccATGctcctttcggtgccgctcctgcttggcctcctcggcctggccgccgcagaccctgccatctatttcaaagagcagttcttggac

63

CRT (Human) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA) NCBI Reference Sequence: NG_029662.1; >gi|343098520:4501-10891 Homo sapiens calreticulin (CALR), RefSeqGene on chromosome 19

GGCAGGGGTGGGGGAGCAGCAGTGGGGTGCTGGTTCTCAAATGCAAGATAAGAGCTGGCTAAGAAAGCCTTGCCCAGCCCCTCCACCTAGAGGGAATGG

GAGGGAGAGAAGCTGAGGGCAGGGTCCCGGTCCCGCGTGGAGACAGCTGCGCTCCCGCGGTTTCTTTAAACGCCCAGATGGGCAACGACGCGC

GCGGACGAGGGCGGGGTTGGGTTCAGGTCTGGTCACATGACCTGGCCTGAGGTGCTCGCGGCCCCCACCCCACCAGTGGGCGTCCCCCC

CACGCGTGGTCGACCATCATTGGTCGGTGGTGAGGCCAATAGAAATCGGCCATCTGGGAACCCAGCGTTCCGAGGCGCAGCCTAACATAGTGAACC

GACGAAGGTCCAATGGAAAAAGACGGCCATGGGCATAGACCAATGACAAAGTGGCAGGGGCGGGCCCAAGGGCTGGGTCAGGTTGGTTTGAGAGGCG

GGTGGGTATAAAAGTGCAAGGCGGGCggcggcgtccgtccgtactgcagagccgctgccggagggtcgttttaaagggcccgcgcgttgccgccccctcggcccgccATGctgctatccgtgccgctgctgctcgg

cctcctcggcctggccgtcgccgagcctgccgtctacttcaaggagcagt

CRT (Rat) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) CAAT box (GGCCAATCT) GC box (GGGCGG) TATA box (TATAA)

NCBI Reference Sequence: NC_005118.3; >gi|389675110:c36937289-36931894 Rattus norvegicus strain BN/SsNHsdMCW chromosome 19, Rnor_5.0

GCAGTATAGATGGAACATCAAAGTTGCAAAGAATCCTTGCTTCTTCCCTCTGACCAGAAAGGATGGAAAAAGGCCGAGACGAGACGGAGGCCCAGTCTC

GGTCCCGCACGGTTAACACCCGGTACTGCTCGCGCGGATTCTTTAAACGACTTCATGGCGAGCAAGGGACTCTCACCAGCAAGGGCGGGGTTGGGCTGA

GGCTCAGTCACGTGACCGCGCCTGAGTGGGCTCGCGGCCCCCACCCCAACAGGGGGCGTCCCCTACAACGCGTGGTCGACCCTGATT

GGCCCAGGGTGCGGCCAATAGAAATCAGCCATCTGGGATCCCAGCGTTCCGAGCCACAGCCTAACTTGCTGAGGCGACTAGGACGCAATGAGAAGGGAC

AGCTGTAGGTCTAAACCAGTCAGAAGGACCGAGGGGCGGGCTCAGCGGTCGTGTCAGGTTGGGATGAGAGGTAGGTGGATATAAATTGGAGCAGC

GGCGGCCGCGTCCGTCAATACcgcagagccgctgcttgaagatcgttttaaagggccagtgtgccgccgccccctcggcccgccATGctcctttcggtgccgctcctgc

ttggcctcctcggcctggctgccgcagaccctgccatctatttcaaagagcagttcttggacggaggtaagg

CRT (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351517684:390174-394530 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold1102, whole genome shotgun sequence; NCBI Reference Sequence:

NW_003613865.1

CCTCCGGCCCTGTGTCCGGAGGGGATGGGAAGTGGGCGAAGCTGAGCCCGGGTGTGGGTCCCTCACGGCTGACACCTGGGCTGGCTCGCGCGGATTCTT

TAAACGACTCGAAGCAGAGCCAGGGAGTCGCACTAGCAAGGGCGGGGTTGAGCGGAGGTCCCGTCACGTGACCGTATCTGAGTGGGCT

CGCGGCCCCCACCCCACCAGGGGGCGTCCCCCACAACGCGTGGTCGACCCTCATTGGCCCGTGGTGTGACCAAT

AGAAATCGGCCATCTGGGATCCCAGCGTTCCGAGCCACAGCCTAACGTGCTGAGCCGGCTAGGATGCAATGAGAAGGGACGGCTGTG

GGGCTAAACCAGTCAAAAGGGCCGAGGGGCGGGCTCAGCGGCTGTGTCAGGTTGGTGTGAGAGGTGGGTGCATATAAATCGGAGCCGCGGCGGCC

64

GCATCCGTCAGTACCGCAGAGCAGCTGCCTGAGGATCGTTTTAAAGGGCCCGTGCGCCGCCGCCCCCGCGGCCCGCCATGCTCCTCTCCGTGCCGCTC

CTGCTCGGCCTCCTCGGCCTGGCCGCCGCGGAACCTGCCGTCTATTTCAAAGAGCAGT

ATF4 (Human) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|568815576:39520564-39522686 Homo sapiens chromosome 22, GRCh38 Primary Assembly; NCBI Reference Sequence: NC_000022.11

GCGGGTCGTCCAGCTGTGCTCCTGGGGCCGGCGCGGGTTTTGGATTGGTGGGGTGCGGCCTGGGGCCAGGGCGGTGCCGCCAAGGGGGAAGCGATTTAACGAGCGCCCGGGACGCGTGGTCTTTGCTTGGGTGTCCCCGAGACGCTCGCGTGCCTGGGATCGGGAAAGCGTAGTCGGGTGCCCGGACTGCTTCCCCAGGAGCCCTACAGCCCTCGGACCCCGAGCCCCGCAAGGGTCCCAGGGGTCTTGGCTGTTGCCCCACGAAACGTGGCAGGAACCAAGATGGCGGCGGCAGGGCGGCGGCGCGGGCGTGAGTCAAGGGCGGGCGGTGGGCGGGGCGCGGCCGCCCTGGCCGTATTTGGACGTGGGGACGGAGCGCTTTCCTCTTGGCGGCCGGTGGAAGAATCCCCTGGTCTCCGTGAGCGTCCATTTTGTGGAACCTGAGTTGCAAGCAGGGAGGGGCAAATACAACTGCCCTGTTCCCGATTCTCTAGATGGCCGATCTAGAGAAGTCCCGCCTCATAAGTGGAAGGATGAAATTCTCAGAACA

GCTAACCTCTAATGGGAGTTGGCTTCTGATTCTCATTCAGgcttctcacggcattcagcagcagcgttgctgtaaccgacaaagacaccttcgaattaagcacattcctcgattccagcaa

agcaccgcaacATGaccgaaatgagcttcctgagcagcgaggtgttggtgggggacttgatgtcccccttcgaccagtcgggtttgggggctgaagaaagcctaggtctcttagatgattacctggaggtggc

caagcacttcaaacctcatgggttctccagcgacaaggctaaggcgggctcctccgaatggctggctgtggatgggttggtcagtccctccaacaacagcaagggt

ATF4 (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|372099095:80254684-80257545 Mus musculus strain C57BL/6J chromosome 15, GRCm38.p2 C57BL/6J; NCBI Reference Sequence: NC_000081.6

GCGTGAGTATGGGGCCGGCCGCGGAGGGCGGGGGCCTCGCTGTGGTTGGGTGCGGCCCGGGCGCGGTGGCCGGCACACGCGGTTTTACAAGCGGCCGGA

CGCGTCGGCCTTGTTTGCGTTGCCTGCGACGCCGGCGCTCCCGGGCAGAGCTGGGCGGAGGAGTGTCTAAAGCGCTACTGCTGCCCCTTCGTCCTGTCT

TAGCTTAATCATCTCGGGCTCACCGGGGTCCCCGTGTCATCCTGCGAACGTGGCGATGCCCAAGATGGCGGTGGGGCGGGGGTGTGA

GTCACGGGGGCGGGGCGCGGCGGCCTTGGCCGTATTAGGACGCGAGGACAAGCTGCTTCCTCTGGGTGGCCGGTGAAGCAAAGCTAAGCCTCCATCT

TGTGCAACCCGAGCTGGCGGCCGGGGAGGCTTACACAATGGCCTTGGGCCCGCGTGCTCTCCCTGTAGACGCTTCTGGGATTTGGCCATCCGGCATCTT

AGATAGAAAGATGACTGGACTTGCTTTTGGGTCCCCATCCAGgctcttcacgaaatccagcagcagtgttgctgtaacggacaaagataccttcgagtt

aagcacattcctcgaatccagcaaagccccacaacATGaccgagatgagcttcctgaacagcgaagtgttggcgggggacttgatgtcccccttcgac

cagtcgggtttgggggctgaaGAAAGCCTAGGTCTCTTAGATGACTATCTGGAGGTGGCCAAGCACTTGAAACCTCATGGGTTCTCCAGCGACAAGGCG

GGCTCCTCGGAATGGCCGGCTAT ATF6 (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|372099109:c170868484-170704457 Mus musculus strain C57BL/6J chromosome 1, GRCm38.p2 C57BL/6J; NCBI Reference Sequence: NC_000067.6

TATTGCGAGTTTTAGAAACTCTTCTTTAGGAGGTAAGTGCGGACATGGAGAATTTATCTTATATATTTTCTTTGTTTAAATTTGTTTGAAGGTGGGTTT

TCTACAAGTATTATTTGTGAGAAAGTTTACCTCGGATTCTGTCCCAGTAAAGGAGTGGTTCTAACACATGTCGGAAATTTGTATTTTTCTTTATTTACC

GTGTACGTGGAAAATTTTTACAGCGATTGAAAAGCATTTATAAATACAGAATCAGGTTTACTGTCAATTTCTAATTTCGTTCTGAGATGTCT

ATATCGTAGTATTAAGCACAAAGAACAAGCCACTATGATCAGGAGACTTTTCAGTTTTAGTACTATCAGCGAATTTTAACAAAGCGG

TAAAATCTGCGTGCTCTTCGCTCAATTTAAAAAAAAAAAGAGAGAGAGGGAGAGAGAAACAAAAAAGAACAACCACAAAACCCCACAGCGGGACAGAAAGTGCTGAAATCCTCGTAGGGAAATATTTACTCACAAGTCTATTGAGTTTGCTTATCTGCTGACGTCTCCTTAGctttggatcccagttcccgc

65

gtgcggtgagatagtttgcctccgcccggccaccgtccgtgtcagcgttcagcttattttgtcctccggccgccgccgtttcaggttactcacccatccgagttgtgagggagaggtgtctgtttcggggaagccggctt

gtgttgccggcgccATGgagtcgccttttagtccggttcttcctcatggaccaga

ATF4 (Rat) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|389675122:121467775-121470587 Rattus norvegicus strain BN/SsNHsdMCW chromosome 7, Rnor_5.0; NCBI Reference Sequence: NC_005106.3

TTGCTGTGTTTGGGTGCGGCCTGGGCGCGTTGACCAGCATACGCGGCTTTACAAGCGGCCAGACGCGTCGGCCTTGTTTGGGTGGCCCCGCAACGCCGG

CGCACACGGGGAGGGTTGGGCAGGCGGCGTGGAGGGAGTAGTGCCTAAAACCCTGCTACTTCCTCTTCGTCCTCTCTTAACTTAGTCGTCTCG

CGCCCCCCCGGGTTCCCGGTGTCATTCTGCGAACGTGGCGAGGCCAAATGGCGGTGGGGCGGGGGGGGGGTGTGAGTCACGGGGGCGGGGC

GCGACGGCCTTGGCCGTATTAGGACTTGAGGACAAGCTGCTTCCTCCGGGTGGCCGGTGAAGCAAAGCTAAGCCTCCATCTTGTGCAACCCGAGCTGGC

GGCTGGGGAAGCTTACACAATGGCCTTGAGCCCACGTGCTCTCCATTTAGAAGCTTTTGGGATTTTGTCATCCAGCATCCTAGATAGAAAGA

TGATTGAACTTGCTTTTGGATCCTTATGCAGGctcttcacgaaacccagcagcagcgttgctgtaacggacaaagataccttcgagttaagcacattcc

tcgataccagcaaatccctacaacATGaccgagatgagcttcctgaacagcgaagt

CHOP (Human) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|568815586:c57520517-57516588 Homo sapiens chromosome 12, GRCh38 Primary Assembly; NCBI Reference Sequence: NC_000012.12

GCTGAGTTGGCCAGGACTTTACTATTATGTAACCAGGACTACAAATGTCAGCAACTAAAAATAAAGAAAGTCAGGCCCTCTTCTGCCCTTCGAAATGGC

TACAGGGACCAAGTATGCATACCCCACAAGACCAGAAGTAAGGAAGGACCAGTAGGAGGCTGGAGGTAAAAGAAAAATAAGGGCCCAGCACGGTAGCTCATGCCTATAATCCCAGCACTTTGGGAAGCGATGGATCACAAGGTTAAGAGATGGAGACCATCCTGGCCAACATAGTGAAACCCTATCTCT

GCTAAAAACACAAAAATTAGCTGGGCGTGGTGGCACGCGCCTGTAGTCCCAGCTACTCGGGAGGCCGAGGCAGAAGAATCACTTGAACCGAGG

AGGCAGAGGTTGCAGTGAGCCGAGATCGCACCACTGCACTTCAGCCTGGCAACAGAGCAAGACTTGGTCTCAAAAAAAAAAAAAGAAAGAAA

AAAAGAAAAAGAAAAGTAAGTTGCCTCTCCCCCTTCCAAAAATGGCTGACATTTCTCTTTGTTGCCCACAGtgttcaagaaggaagtgtatcttc

atacatcaccacacctgaaagcagGTAAACTTAACCTACCCTTTTCCAAAAATTTTAAACGGCAGGACAGTAAATATTTTAGATGTTAAAAGTCCTATAGTCTCT

AGCGTGACTCTTCATCTctgccactgtagcaccaaagcagccataaacaatatgtaaataaacagatgtggctgtattccagtacaactttacctacaaaaacaggcatcagaccagcttgccaacttgt

ggcatagactgtttgctacATGgagcttgttccagccactccccattatcctgcaga

66

CHOP (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|372099100:127290256-127296288 Mus musculus strain C57BL/6J chromosome 10, GRCm38.p2 C57BL/6J; NCBI Reference Sequence: NC_000076.6

TGGTAATTGCCCCTGGAAATTACCAGTAGTGTTCCCAAGAGAGTTGAATACTTTTACTGTAATCCTGTAAGAATATATATGTATAGCCAAGCCCAGTGACTGTTCCTCTCTCAGAACTTGAGAGGATCCTGAAGTTATCTGTAGTAAATCCTTCTCAATAAAGGGTATGTTCTATCAGGTGTAGTGGTGCA

TACCTTTAGTCCCAGCACTTGGGAGGCAGAGGAAGGTAAATGTCTGAGTCAAAGCCAGCCTATTTTGCAGAGTGAGTTCCAGGGAAGCCAGA

ACTATATATACAGAGAAACCCTTTCTTGAAAATACACCCCCCACCCTCCAAGTTTTGTTTCCAGGAGCTAGAGAGATGACTCAGTGGTTAAGAGCACGTGCTCTTCTTGTAGAGGATCTAGTCTAACTCCCAGTACCCAAATGGCAGTTCAAAACCATCTATGACTCTAGTTCCCAGGGAATCCAT

TCCCTCTTGTGGCCTCCATGTCTACCAGGAACACACACACAGCACACATTCAGGCACTCACTTATATATGCAtagataataaaataaatatatctttgggaaaaaaaaa

aagatatttaaggttaggtagccagcctggattaagcttggtagtgaccagcatataaatgaaaacaaaacaaaaagttggaaagctgtgtgggggtggtgcatacttttaatcccagccttcaggaggctgaggc

aggttgatctctgaggccagtgtgagttctaggacaggcagggctacacagagagaccctgtcttgaacaaacaccaaaaagaatggcaatgagagcccggagaaagcctatcagttccacacccatgctgcc

tgtgtgccgtacctgagtcaggtttccagcagccacagaaggtggctcacatggcctggacctccagctccaggagagccaatgaatgctgctggcccccagacactgaattacatccgtttcagggtcctggcca

tggtgtgcatgtgatcatctggacaacttttgagagttggatctggcagggtcaaagtcaaggctgctaggcttgagaggcagccatctccccatcccgacacaccatcattagtgtgtgtgcaggtcagagaacaa

cttgtgcgagttgactcttcacctccaccctctgccaatgtagccttcaaggagtgacaacccatgcccttacctatcgtgcaagaccagtaaattttaaattctacgtgttagaaaagggacaaggtcagctcaccg

actgtggtgaatggaatgtATGtcctttccagaacctggtccacgtgcagtcatggcagctgagtccctgcctttcaccttggagac

CHOP (Rat) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|389675122:70753300-70759220 Rattus norvegicus strain BN/SsNHsdMCW chromosome 7, Rnor_5.0; NCBI Reference Sequence: NC_005106.3

TTATCAGTTATGCATGCTTGCTGCTTCCTGCTGTACCTAAATCAGGCTTCCTGCAGCCACATCAGGTGGTTCACATAGCCTGGAACTCCAGTTCCAGGAGAGCCAAAGCCTCTGGCCCCAAACACTGATCATGTATACACATGCAAATTAAAAGCTTGAAACAAAATAGTAGAGATAACTAAATAA

TATTGACATTTACTGATTTAGTTCTTAAATTACATTTATTTGTTTGGGGTTTGTCTGCTGGCCATGGTGTGTATGTGATCATCTGGACATTGAGAGTTGAATCTGGCAGTATTAAACCCAGACTGCTGGGCTTTGGTGGTAGCCATCTCCCCATCCCAACACTCATTTAGTTTGTGTGCAGTGGTGTGCAG

GTCAGAGAACAACTTGTGGGAGTTGATTCTCCACCCCCACCCTATTCCAATGTGGCTTTCAAGGAGTGACAACCTATGTATGCCCTTACCTG

TTGTGTAAGACCAGTGCACTTTAAATTCTATATGTTAAAACAGGCATGAGATCAGTTCACCAACTGTGGTGAATGGAATGTATGTCCTTTTTCAGaaacc

ggtccaattacagtcATGgcagctgagtctctgcctttcgc

GADD34 (Human) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA) >gi|224589810:49375149-49379319 Homo sapiens chromosome 19, GRCh37.p13 Primary Assembly; NCBI Reference Sequence: NC_000019.10

TTTAGAAAGGAGAAGGGGTTGGGAGCCTGGAGTCCTGAGCCTGAGGGAGGAGGGATCTGGAAGCCAGATTCTTGGGTCCCCCGTGAAGGAATCATC

TGCCAAGTAGGGGGTCGGGTCAGAATGTTTCAGTCTGCGAGGGAGGAGGGTGTGAGGGTTTGATTTGTCAATCTTCAGCAGAATGAGAGAGCTAG

GGACTATGACTTTGTCGCCAAGGGAAGCAAGTAAAGACTTTTGTTCTTGGTTCTTGGGACTGGGAGTTCTGCGTCTGAAGAAAGAGA

GGGCTGGGGGCTTGGACCCTTGGATCTAAGGCAGAACTAGGGGCTCAGACTCCTGGCTATTGAGAGATAAGAACAGAGCCAAGGGACAGAGA

TGGGCGTGGCCGAGATCAGAAAGGAATTTGGGACTCTCGCGTTGCTATTTACAATAGTTGTGTTACTATTTCCGTTGCTATGACTCATAGTCACGCCCGGATGCCATCCTCTAAATGGCCCCTAAACTTTATTTTTTTCTCCCCCTTTTCCAGcccagacacATGgccccaggccaagca

67

GADD34 (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA) >gi|372099103:c45526268-45522917 Mus musculus strain C57BL/6J chromosome 7, GRCm38.p2 C57BL/6J NC_000073.6 Reference GRCm38.p2 C57BL/6J

AATCTGAGGGGGCAAGCTGGAGGGCGATTCCAGGTTTGGGGAAAAGGCACAGAAGCCCAGATGGCTTATTTGGTCTGAGAGAGGAGAGCCCAC

GTCTACACTCTTCGTTTAAAGGGAAAGGGGCCGGGAGGCTCGACCGGCAATATCTGTATAGCGCCAAATAAAGGGAAGGACCCCAGGAGGAATAACCAGGGTCCGAGGGGTAGGCAGAGGGAGGTTTTAGGGGCATGAACACAAGCTTGAGGAACGAAAGGTCACTAAAAAGCCAGACTCTTACAG

GTCTGAGAAGGGCTGGCCAGGAACTGTGACTGGGGCTAAGGGAAGGGTTTAAGAGTGTGGGCTCTTGGTTCTAAGGATGTAGAGCTTGGGTGTTGAGAG

AGATTAACCAAGTGGCAGAGTTGGGTGTGAGCAATGTTATTTGGAATTCGAGGCAGGCATTCACGTGTTGCTGCTGTGACTATTTGT

TACCATGACTCAGTGCTGTGACCCGAGACTGTTTCCCAGAGCGACTTCTAAACAAATTTCCCCCCTTTCTAGcccagacacATGgccccgagcccaagacccca

gcatgtcctgcactggagggacgcccacaacttctatctcctgtccccactgatgggcttgctcagtcgggcctggagccgcctgaggggcccagaagtcccagaggcatg

GADD34 (Rat) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|389675128:c102593806-102590677 Rattus norvegicus strain BN/SsNHsdMCW chromosome 1, Rnor_5.0; NCBI Reference Sequence: NC_005100.3

CAGACTGGGGAGGAACCCAGATTTGAGGAAACGGCACTGAAAGCCGGATGCTTTATTTGGTCCGAGAGAGGAGAGCCCAGGTCTAGT

CTCTACATTGAAGGGCAGGGGTCCTGAACTAGAACTGCAGTACTTGTACATTGCTAAATAAAGAGAGGGACTCCAGGAGGAGCAGCCTGGGTCTAAGAGGTAGGCAGGAGGAGGTTTTAGGGGCCTGAGCACAAGCTTGAGGAGAGAAAGGTTATTAAAAAGCCAGACGCTTACAGGTCTCAGAAG

GGCTAGCCAGAAACTGTGGCTGGGGTTAAGGAAAGGGTTTAAGAGTGTGGGCTTTTGGTTCTGAGGATGTAGAACGTGAATGTTGAGAGAAGAACCAAGTGGCGGAGTTGGGTGTGAGCAATGCTATTAGGAATTTGAGGCAGGGATTCACGCGCTGCTGTGACTATTTTTTAACAATGACTCAGTGCTGTG

ACCTGATACTGTTTCCAGAGCGACTTCTAAACAAATTCCCCCTTTCTAGgccagacacATGgccccaagcccaag

XBP1s (Human) ERSE I (CCAAT-N9-CCACG)

UPRE (TGACGTGGA)

ERSE II (ATTGG-N-CCACG) GC box (GGGCGG)

TATA box (TATAA)

>gi|568815576:c28800572-28794560 Homo sapiens chromosome 22, GRCh38 Primary Assembly; NCBI Reference Sequence: NC_000022.11

TTTTTCTTTTTCTTTTTTTGAGACGGAGTCTCGCTCTGTCGCCCAGGCCGGGGTGCAGTGGCGCGATCTCGGCTCACTGAAACCTCTGTCCAGTCTTTT

CGAACCCAAGGCCCAACTGCGCTCTATCTCGACTTTCGGCTCCACTCGGATCCCGAAGTGGCGCACGAGATAAAATGTTGTCAGGCTGAGGTAATTCTC

TGTTAGTCCCGGTAAAAATTCGTCAGTCTGGAAAGCTCTCGGTTTGGAATTAAATTCTGTCACTCCGGATGGAAATAAGTCCGCTTAAGGGGGGAAAAT

CCG

TTTGTGGAGGACACGCTCCCGCACGTAACCCCCCGCGGAAAATGACCCCAAGTACCTTTGGCCAGGGATTGCCGCTGCCACGCCGGAC

TCCATAGCCACGGTCCTGAAACGCCCCGCCGGGCAGGCCGGACCAATGGACGCCGAGCTCGGCCGTGCGTCACGCGACGCTGGCCA

68

ATCGCGGAGGGCCACGACCGTAGAAAGGCCGGGCGCGGCGAGGCTGggcgctgggcggctgcggcgcgcggtgcgcggtgcgtagtctggagctATGgtggtggtg

gcagccgcgccgaacccggccgacgggacccctaaagttctgcttctgtcggggcagcccgcctccgccgccggagcccc

XBP1s (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|372099099:5520141-5525993 Mus musculus strain C57BL/6J chromosome 11, GRCm38.p2 C57BL/6J; NCBI Reference Sequence: NC_000077.6

TGGAGTGCTAGAGCATTAGGTTCTGGGGTCTGGCTCCAGCACAGCAAAACAAAGACAACAGTGATAAAAGCTCATACCAGAAGAGATCAGAAATAAGA

AGAAAAACGAAACCACATGTATTCTCACTATGCAGACTTTATTTACTAATCCCTTACTACATGCTAGCCAAGGCTCT

AGTGTTTTCTTTTAAAGTTATTAATTTATATCAATTAATTTATATGAAATAGGGTTTGATCTATAGCCCCAGCTGGCCTAGCTAGACCTCCCTATGT

AGCCCAGGCTGCTCTCAAACTGGCAATAAAGTGCTGAGATTACAAGTGAGTACTACCCTGCTACATTTAGTTTTGCAATCATCTCATTTTCTAA

AGACTCATTTTGTGAAAATTTCCCAGGTGTGTGTGTGTGTCCGCTAACCCTAAGCCGGATATGCCACCAGTTCAATTTTCT

GCTGTACTGGGAAGTGGGGGGGGGGGGGGGACGGGACGGGGGACGTAGCGCCGGACATTAAAATGGGGTCGGGctagggtaaaaccgtgagactcggtctg

gaaatctggcctgagaggacagcctggcaatcctcagccggggtggggacgtctgccgaagatccttggactccagcaaccagtggtcgccaccgtccatccaccctaaggcccagtttgcacggcggagaac

agctgtgcagccacgctggacactcaccccgcccgagttgagcccgcccccgggactacaggaccaataagtgatgaatatacccgcgcgtcacggagcaccggccaatcgcggacggccacgaccctaga

aaggctgggcgcggcaggaggccacggggcggtggcggcgctggcgtagacgtttcctggctATGgtggtggtggcagcggcgccgagcgcggcca

XBP1s (Rat) ERSE I (CCAAT-N9-CCACG)

UPRE (TGACGTGGA)

ERSE II (ATTGG-N-CCACG) GC box (GGGCGG)

TATA box (TATAA)

>gi|389675115:86423339-86428923 Rattus norvegicus strain BN/SsNHsdMCW chromosome 14, Rnor_5.0; NCBI Reference Sequence: NC_005113.3

CACCACCCTACTACATTTCGTTTTGCAATAATCTCATTTCCTAAACACTCATTTTGTGAAATTCCCCAGGTCTGTGTGTATGTGTTCGCTAACTCTAAA

CCGGATATGCCACCAGTTTGATTTTCGGCTGTACTAGGGACCGATGTGGCGCCGGACATTATAACGTGGTCGGGCTATGGTAACGATCTGTGAGACTCGGTTTGGAAATCTGGCCTGAGAGGAAAGCCTGGCATTCCGGGTAAAAGTCTCAGCTGTGTGGGGACGCGTCTGCCGAGGACCCTGGACCCTGGA

CTTCAGCAACCGGCTGTCTCTACCGTCCACCCACCCTAAGGCCCAGCTCGCACGGCGAACAGCTGGGCAGCCACGCTGGACACTCAC

CCCGCCCGCGTTGAGCCCGCCCCCGGGCCTGCAGGACCAATAAACGATGAATACAGCCGCGCGTCACGCAGCACAGGCCAATCGCAGACGGCC

ACGACCCTAGAAAGGCTGGGCGCGGCAGGCGGCCACGGGGCGGTGGCGGcgctggcgtagacgtttcctggctATGgtggtggtggcagcggcgccgagc

gcggcctcggcggcccccaaagtgctactcctatctggtcagcccgcct

PDI (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA)

69

ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351517697:86110-89160 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold1806, whole genome shotgun sequence; NCBI Reference Sequence:

NW_003613852.1

CTTAATGCACTTCCTTCCCACAGTGGACAAGGCCATCTTCATGGTGGGCAGCTATGGCCCCAGGGCCCACGAGTATGAATTTGTGACGACAG

TGGAGGAAGCACCAAGGGGCGCACTGGCACGTGGCCTCTACGTGGTCAGGTCCCTCTTCACTGATGATGACAGGCTTGACCACCTGTCCTGGGAGTGGTGCCTTCACGTCTGCCAGGACTGGAAGGACTGAACCTACTTGGGGCCTGCTTTCCCAATTTCTGTCAGTTGCAGAAGGACCTGCAAGCA

TTCCCTAGACCTCCCAGTTGGTGACCAGATGTCCCCCACTCTGCTGTGTCCTGCCCCCCTGCCTGGTGCCCATTAAATGTTACCCTGTCTCGCTGGCCT

TGTGTTGCCTCTTTCTGCCTATCTCCACCCTCTGGGGCCTTTCAGGCTTTATCTTCCCTGAGACCCAGCCAGGTGCCACGTGGCTGTTGCTG

CTTGGTCCCCAATCCCTTTATCTACTCCAGCCTTCAGGGCCCAGCACACCATGGATGATCGGCTCCTGACAGTGTTGCTGCTCCTGCTGG

EDEM (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|372099104:108827757-108859356 Mus musculus strain C57BL/6J chromosome 6, GRCm38.p2 C57BL/6J; NCBI Reference Sequence: NC_000072.6

TTTCGTGCGGTCTGGACCAGGGAGTCCCACTCCCACCAAGTTAAAAACCTTTCCTAGATATTCGTAAGTACTTCCTTTGCCTAAAGGCCCTGGCCCTTA

CCTTTCTCACACCTACTACTCCATACCTGGACGGGCTTTCTCCCATCCATCTTCTGTTCAGGGACACCATTTACCCGGGCTAGGGCGGGTCAGAGGCCA

TGTCTGTGTCCGGCTCCCGGAGCTCCCGTGCCGCGCTCCTATTGGCTGGAAGTTCTTTCCCCAAAAGCCAGAGCGACCTAGGCTCGCCA

CGCCAGCTAGACCAGACTCGGAACCGCCCAGGGGGCGTGGCCCAGTGGCAGGGCGGGGCCTTGCAGTCCGGGCGGCCTATAGAGCCGGAGGG

AGTCCCCGGCCGCGCCCCTCGAGGCCGGGCCGGTGGAGGCCAATCCCCGGCGGCCGGCGGGCGAACGCGGGGCCAGGCGGGCCGGTCCCGTCCGCGGG

GCGGGGGACAATGGACCAGTTCCTGGTGCGTCACGAGGGAGTTCCTTaaaggggaagtgagcgggctcccggcggacgcgcggggaggcggtcggctggggacgcgcccgg

cgcttcaaaataatgcccgcggcggacgcgcgaccATGcaatggcgagcgcttgtcctggggctggtgctgc

PERK/Eif2ak3 (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|372099104:70844527-70905240 Mus musculus strain C57BL/6J chromosome 6, GRCm38.p2 C57BL/6J; NCBI Reference Sequence: NC_000072.6

CCCCAACTCTCATCCCTTCTTTCCAAATAAATTCGGCTTGGGAATAGCAAAAGCATCTATCCCTTTAAGATCAGCCACCTCCCGGCC

ACCCGGGACTCCTTTCCACTCTTCTTCCTTTCACAGGGGACGGTCCTCTTCACACTCAGAATCGGCCACGTACTAAGTGCCGCTTCCAACCA

ATCAAGAAGCAGTTAGCTCAGGCCTTTGAGGAGCATCCACTTTCACCAATAATCTCCAAGCTTTTCCCAGCGCCTCACTTTGAGGGGCGAGGCCAATCAGGGAATGGACCAATCCGTTCCAGAAGTGGCATTAGGGAACTGAAATGATTCACCAATCGCCTAAGAAGACTCTGGGGAGGGGCATTT

CTCATTGGCCGTCGTTCCCGGAAAAGGGCCGGACTGCCACAGGGAAAACTACAATTTGATTGGTAGGTATAATGTTGACAACAAGGAACGCGTCTGC

CTGGCTCGGAGTGGCAAGGCGCGCAACCAATCGCAGAGCAGACGCACGGAACCCTCGctcaatgggcgatgtctgcacaaggctgtcactcaggtggcagtggctgagacgtgg

ccgggcagctctgctgctgcggcgcgaagtcgagaggcggcggggtccgtggcgcgcgctcgcagtgctccgaggctccgagcggcgagacgggcgggcgccgacggcagggtctccatgcccgcgcgtg

gggcgggccgctgATGgagcgcgccacccggcccgggccgcgcgcgctgctgct

HIFa (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA)

70

ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|372099098:73907867-73947530 Mus musculus strain C57BL/6J chromosome 12, GRCm38.p2 C57BL/6J; NCBI Reference Sequence: NC_000078.6

GCGGAGACTGGAGACTGAGTGGTGTCGGTCACCTGGGAAGGAGGGACCCCACTCTAACATCAGGGGGGCTGGCTGCCAGAACGCAAAGTTGGCTTTTTT

TTTTTCAACTGGAAACTCGGGCGGGATGGGCGGCGACCGGGTCCAGCCACGCAGCTAAGGAGTAAGCGAGCCTCAGAGAACTCTATA

GAGAGCCGGGGCGGTGCGAGCCAGGCTCGGACCCAAGCTTGGCAGCCCACTCGCTCCAGCAGCGCCTCCCAAAGCCAGACGCAGCTTTCCATTGAAGGTGCAGAGACCCCCCCACCCCCAGACCTGGGCTCAGCCGCACCCGTCGCTCGCCCCTTTTTCTTCCCTCCCTAGCTGCACACA

GACAAGCACGTGAGCGGGGCAGCTCGTCCCAGCTGGGGCTCAGCTGACCTCCTCCTGATTGGCTGAGAGCAACGTGGGCTGGGGTGGGGCCTG

GCCGCCTGCGTCCTTCACCATTGGCTCTCCGGGAACCCGCCTCCGCTCAGGTGAGGCgggcccgcgggcgcgcgcgttgggtgctgagcgggcgcgcgcacccctcggcttttc

cctcccctcgccgcgcgcccgagcgcgcctccgcccttgcccgccccctgccgctgcttcagcgcctcagtgcacagagcctcctcggctgaggggacgcgaggactgtcctcgccgccgtcgcgggcagtgtct

agccaggccttgacaagctagccggaggagcgcctaggaacccgagccggagctcagcgagcgcagcctgcagctcccgcctcgccgtcccggggggcgtcccgcctcccaccccgcctctggacttgtctct

ttctccgcgcgcgcggacagagccggcgtttaggcccgagcgagcccgggggccgccggccgggaagacaacgcgggcaccgattcgccATGgagggcgccggcggcgagaacgagaag

B Actin (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG)

TATA box (TATAA)

>gi|351517931:939564-941969 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold393, whole genome shotgun sequence; NCBI Reference Sequence: NW_003613618.1

TGGGGGCTGGGTTGCCACTGCGCTTGCGCGCTCTATGGCTGGGTATTGGGGCGCGTGCACGCTGGGGAGGGAGCCCTTCCTCTTCCCCCTC

TCCCAAGTTAAACTTGCGCGTGCGTATTGAGACTTGGAGCGCGGCCACCGGGGTTGGGCGAGGGCGGGGCCGTTGTCCGGAAGGGGCGGGGTCGCAGCGGCTTCGGGGCGCCTGCTCGCGCTTCCTGCTGGGTGTGGTCGCCTCCCGCGCGCGCACTAGCCGCCCGCCGGCGGGGCGAAGGCGGGG

CTTGCGCCCGTTTGGGGAGGGGGCGGAGGCCTGGCTTCCTGCCGTGGGGCCGCCTCCGGACCAGCGTTTGCCTCTTATGGTAATAACGCGGCCGGCCTGGGCTTCCTTTGTCCCCTGAGTTTGGGCGCGCGCCCCCTGGCGGCCCGAGGCCGCGGCTTGCCGGAAGTGGGCAGGGCGGCAGCG

GCTGCGCCTAGTGGCCCGCCAGTGACCGCGACCCTCTTTTGTGCCCTGATATAGTTCGCCATGGATGACGATATCGCTGC

CASP3 (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351517956:c3064906-3056127 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold903, whole genome shotgun sequence; NCBI Reference Sequence: NW_003613593.1

AGACAGGAAGGAACAGATGTGGAGAGGTGCACATGTCTGGTATTTGGAGACAAAGTCACTTAGAATTAGAAGTCAAGTTTCTGAGTAGGATACTGTGGA

GGTTTGGAAGTTCCTGGGGCTCTCACCATTGTGGTGTAGTATTATGTGAGCTTTACTTCGTGGTTAGTGTTTGGGTTTTGTTTTAGTTATGTT

GAGTTGGAAGCTGAGTTCCAGTAAATAACTAGATTGGTCGGGTGACCAAGGAAAGAAGGTGTTACCTGTTTTGATTGGCAGGGGTATACAAATGGGGGAAAGAGACTGAGCTGCTTTACACTCTGTTCTTGATATAATTCATAGCTAGAAGATCAAGGAAGGAGAAGGATGGTGCTATTGCAAAGGACCAT

TCTCTAGACATCTGACTCATGGGACCAATAGCGATGGAAACAATGAATATGGCATTGCTAATATATTGCAGTGGTTCTATAATGGGTAACATCTCTC

TTTTTTTTTTCCTTCTATATTCTAGTTAAGAAAAGTGACCATGGAGAACAATGAAACCTCAGTAGATTCC

FADD (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA)

71

ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|372099103:c144582436-144578323 Mus musculus strain C57BL/6J chromosome 7, GRCm38.p2 C57BL/6J; NCBI Reference Sequence: NC_000073.6

AAGAGGATGTCAAAGCCCCTGGAACTGGAATTACAGATGGTTGTAAGCCATCATGTGAGTGCTGGGAATTGAATTCGTGAAGGTCCTCTGTAA

GAATTGCCAGTGCTCTTAACCCCTGAACTGTCTCTCCAGCCCATAAGTAACTCCATCCATCTCTCTCTCTCTCTCTCTCTCTCTCTCT

CTCTCTCTCTCTCTCTCTCTCTCTCTTTCCTTCTTTCCGGTGATAGAACATGATCTGGCAGCCGTAGGCATCTGCAGCTGCTCTTCCAGCCC

CCCCCCACACAGTAGGGCTGCGCAGCTCCTCAGAGCCCCAAGAGGATCCACGTTACACTTCTGGTGAACAAGATCTGTTCTGCCTCG

GGGTGAGAAACGCGCGCGTAAGCTGAAGTCAACGCAGCTCAGCTGTCAAAGCTACTACAACAGCGGATGCCAGCCCGGGCGGAAGCGGTTAC

AGCCACATCCGAAGCGCATGCGTCTGATTTGGACATCCCGCGAGACGCTGGGCGGGAAAGCCTAAGGTgatttcctatgtgggatcgctgaggggcagcccg

gcgtaccactgccATGgacccattcctggtgctgctgcactcgctg

BAX (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351516979:279750-286195 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold1718, whole genome shotgun sequence; NCBI Reference Sequence:

NW_003614570.1

AACAATTAACTAAAATATTGCACATACAATTAATTACAGTGATGTGAAACATTATAAAGCCCAACAAAGAAAAAAAAAAAAAAGCTGTCCAT

GTCAGACATCACATTAATCCCAGTGCAGAAGCAGACGTGGGCAAATCTCTGGTTTGAGGCCAACCTGGTCAATAAAGCAAGTTTCAGGATAGTCGGGCTACACAGAAAAACCCGGTCATGAAAAAACACCAATGACTCATAGCAATGAATACTTTCAAGTCAAGAAGTGTAGACTGAAGGGTACACAGTGCA

TGAGCTCGAGGCAATACTTTCTCCTGCCTCAGCCTCCTAATACTGCATAACGAAGTGACACCATTCCTGGTAGCGGTTTTTGGGGACAAGGCTTTGTTG

AATATCGTAAGCAGGCTTTGAACTCGGAGTAATCCTTCCGCCTCAGCCTCAGTAGCACTTCTACTACGGAACTATGGAAAAGGCAATAAATAATGGAAC

CTCTAGGTCCACGTTCGGCCTCAGGCAGAACTGCGCACGCGCACTCTTGTGACCTAGGCCCCGCCCGGATAGGCCGGACAGTCACGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN

NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNAGGGTTATGAGCCTCCCTA

BCL2 (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|372099109:c106714290-106538178 Mus musculus strain C57BL/6J chromosome 1, GRCm38.p2 C57BL/6J; NCBI Reference Sequence: NC_000067.6

GGGTCCATGTGACCTGCACATGCTACATAGAAAAAGTCCATGCGGTCGTGCACACTGTACACGTTGAAAAAGATCCA

TGCAATTGTGCCCATCCTGTTAGAAAAGATCCAGGTGCCTCGCCACAGCCTGGCCCTGGCTATAGGCACGTCCAGCCAGCAGCCAG

CCCCGCAGGCCCGAGGAGGCCCCTCACACGCCCACTGAGCCACTGCCGCCCGGGAGGCCCGCGCCACCCACGGTCCGGGGCCCAGCGTAGCCCGCGGGTGGCCGCCACCCAGGCCAGCGGCCGCGGCCGGGCGCGCGCTCCCCGGGGCGGCCGCGGCGCCTTTAACCCGGGCGGCTGGCGGGCG

GCGGGAGGAGGCGGCGGGCGGGCGCTCAGAGGAGGGCTTTCTTTCTTCTTTTTTTTGAATGAACGGTGACGTACGTACAGGAAACCA

GGCGCTCCGGCGGAGAATGAAGTAAGAGGCCGGGCCCCCCGCCCCGCGCCCGCCCCCTTCCTCCCCGGCCCGTCCTCCCGCCCCGCgtccgggtccccgccgag

ccgccgccgccaacaccgaggtgcccaggccctccccagccgcagccgtcaccgtggaacgggctgcgcggcgcaagcacgaggaggcggccgccagcgccgcccgcagcggaggaggagaaagggtgcgcagcccggaggcgggg

tgcgccggtggggtgcagcggaagaggggaccagggggggacttcgtagcagtcatcctttttaggaaaaagagggggggcaaaccctcccccaccacctccttctcccgcagcgcaccacacacagtgcgcgggctgctccttgggcacccg

cggccccagcgcgtccgtgcctgcatttagcaagctgctttttttcccgtttcggaaagcgcgttggcccttcggagtttaatcagaagaggattccggtcccggtcccccagctcctccatcgtccccttggcgtgtctctctgccctggaggtctgaagc

ggtccggtggatacggatccatgcctgcgctccagcgtgtgtgtgcaagtgtaaattgccgagaagaagggagaatcacaggacttctgcaaatgctggactgaaaaattgtaattcatctgccgccgccgccgctgcctttttgccccgctgcggtg

72

ctcttgagatctctggttgggattcctacggattgacattctcagtgaagccggagtgtgaggacccaatctggaaaccctcctgatttttcctccacctagcccccagaccccaactcccgattcattgcaagttgtaaagaagcttatacaaggagact

tctgaagatcgatggtgtggttgccttatgtatttgtttgggttttaccaaaaaagggtaaacttgacagaagatcatgccgtccttagaaaatacagtaagttctttgcacaggaattttgtttaatataactttccatggacgcgtttgaaatatttttttacttc

aagtgcattcaagcaaatttcatttccagacagtttaatgcatttttaaacgtgtaacttgtagcggatatacctttcttaccctaaatatataaaggaaaacacacctgattttaacttcctaggtcgtcccgcctcttcacctttcagcattgcggaggaagt

agactgatattaacaaagcttaataaataatgtacctcatgaaataaaaagcagaaaggaatttgaataaaaatttcctgcatctcatgccaacggggaaacaccagaatcaagtgttcggtgtaactaaagacaccccttcatccaagaatgcaa

agcacatccaataaaagagctggattataactattcttttttttttttttttctttcggggccgtggggcgggagtcgggacttgaagtgccattggtacctgcagcttcttttcggggaaggATGgcgcaag

BAK1 (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351516577:224325-227955 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold5584, whole genome shotgun sequence; NCBI Reference Sequence:

NW_003614972.1

CTGAGGAGAGAGTGGGGAAGCGTCAGAAACAGCAGAAAGGCCCCCTGGTCTGTGAAAGCCGCTTCACCCTATCTAAGAGAAACTGTCAAGGGAATCAAG

AGGTTTCATGGCGAGGCTGTGTGGAGGCCTCGACCTCACTCACCTGGTCTCTGCCTGATCTCCTCAACCTCTCTTGTGGTGAGTTCCCA

TCGCTCTCAGGAGATACCACCAGGCCCCCAGAGGATTGCAAAGTCTTCCTGGCAGTGGGGTAACAGGGTCCCCTCCCTCTCGGGGTTCTCCAGCCAGATGTCCCTGCTCTATCTCTGCTCACTGTCTTCTTTCCTTCCTTTTTTTCTTGAGACAGGGTCTCTCTGTGCAGCCCTAACTGTCCTGGAATTCACTCTG

TAGAGATCCACCTGCCTCTGCCTCTGTCTCCCGAGAGCTGGGATTAGTCATGCCGCCATGCCCAGCCCAGCCTCACCTCTGCTTTT

TTCTCACCTGCTTCCCCAGGGTGACAGTGCTGCCAACCAAGGCCTGAAAGATGgcgtctggacaaggacc

CASP8 (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351517229:c225025-197835 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold880, whole genome shotgun sequence; NCBI Reference Sequence:

NW_003614320.1

GAGTCTCAACGTCAGGAGATAGGTAGCCTTTCTACTCCTCACTTTACACACAAAGACTCTTGCAATACTTCTAGCAGCTTCCCAAGG

CAGAGTGGCCTATGGCACAGCCAGGCTTTGATCAAGGTGGAGCCCATGTTCCTAGCCATGACACTTGTTGTCCTTGTATAGAGTTGCTCTACTGAAGTCTGTGTCTGTCCAGTTTGCCTCTACTTTTTGCATGCCCCTTTTCTTCAAGAACTGCTTTTCTAGCCAACTCCCCAAAGGTCT

CTTGCCTGTAGGAACCCCTATTCCCAAGACTGTTACTGCACCAAGTGGAGACCGTCTGACCAGTGACTCTCTGTGTATCCACCTCCCTCCAAAAAGCTCCTGAGAAGTAGGAACTTGTTGTTTTCTTTCCCATTATTCTCCATTTCTTCCCTCCAAAGTGTCTGGAACATGGTAGATGCAATTAG

TACTTGACAAGAAACAAGCTTGTAGATGGCACTTTACTTCCTCACTTGATCATGGTATCGTGTGTTGACCTAGGtcaccactctcctgcctcttgaaagga

ATGgatttcagaaactg

JNK1/MAPK8 (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA)

73

ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351517960:c2937392-2905781 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold945, whole genome shotgun sequence; NCBI Reference Sequence:

NW_003613589.1

GTCAGCCCCAGGTGTTCCTTGGGAGCTAGCCACTGTATTTTGAGACATCATCTTGCGGGGGTGTGGGGCTTACTGATATAGTTAAGCTTGATGGCCAGT

GAGCCTGGGTATCCTCCTATGCCCATCTTCCCAACACTGGACATGGTGCTCCACTTAACCCAGCTTTTTATGTGGGTGCTAGGGCTTGAATTCAGGCCCTCATATTTATGTGCTAAGGTCTTTACTGGCTATCTCATCTCCCTACCCCCTTTTGGTTTTGTTTTTTGTTAACTAAGAATGCATTAATACTA

GTGACTTAAGGATCAGAGAAACTGTTCATCATAATAAAAAGAACCAGTCAGTATTTTCTTACATGTAACATGTACTGAACAATGCTCTTTGGTGGTTTT

GTTTTTATGATGCACTCTTCTTAATGATGATGACCTAGCAGTCTGTGTTCCTATGGTGAATAGAGTAAGGACTTAGATGTTAATACTACACAAACTCAT

TTTCTTATATCACGTTTTCTTTTATTTTGTAGccttggtgaatatttggatgaagccattaaactaattgcttgtcatcATGagcagaagca

TRAF2 (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351515226:89683-109223 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold7511, whole genome shotgun sequence; NCBI Reference Sequence:

NW_003616323.1

ATGCCAGAAGGTAGAAAGCTCATGAATTGGATAGTTACTGTGGGTGCCACCAGGTCGCTGTCACCCTTATTGACATCTCTACCCAC

ATTTGTAGCAGGAGATTTGTTTGACTTACCTATCTTCAAGGCTTCCCAGAACTCTAAAGTCTTGTTTCTTAGATCCTCTTTCACAATCAGTGTAGT

GACCCAGGCACCTCACCATGTGCCTCTGTTAGACTCCGTCACCTTCCTTTTCCCTCTAGAGTAGTGGTTCTCAACCCGTGGCTTGTGACCCCTTTGGGCCTGGAATGACTCACATGGGCTGCTTAAGACCATTGGAAAACAGATAGTTGCATTATAGTTCATAACAGTAACAAAATTACAATTATGAA

GTGGTAACAAAAATACTTTATGATTGGGGGTCACCACAACACGAGGAATTGTATTAAGGGACTGAAGCATTAGGAAGGTTGAGA

ACCACTGCTCTAGAGGATTATTCTGAATTGAGGTGTGATATGATCTGTGTTTTTCTTTAGGGCTTTgtggtgtgtgggggttataactcacATGgctgcagcc

agt

BID (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|372099104:c120917141-120893119 Mus musculus strain C57BL/6J chromosome 6, GRCm38.p2 C57BL/6J; NCBI Reference Sequence: NC_000072.6

GTCCTGGCTGCCCTGGAACTTGGTTTGTAGACCAGGCTAGCCGAAAACTCAGGGATCCACCTCCTTCTGCCTCCAGGTGCT

GGAATTAAAGTTCTATACCACCAAGCCTGGCTGTACTGAAACTTATAATTTCTAAATTCAAATGCACAAATGGTTTTAGTGTAGAGT

AATACCATTAGTGCCTACGGGAAATTTAGGCTGAAGAACGGAGACCATGTGTGGGCTTGAGTCTTTTCTGGATCAAAAAGAGTATGGTCATCTTTCAGCTGCTTGCCTGTAACGATGAGCGTCTGCTGGGTGGGGTGGGAGGTGCCCTCCTAATCCTGGGTCTTACCCTTCACATTCTCTGTGGTA

TCAGTGGGCTCTACCTCAGGGTCTGGGTCTTCACAAAGATTCACATCTTTTTTGGGGGAGGGGGTGCGTTGAGACAGCGTTTCTCTGTGTAGT

CCTGGCTGTCCTGGAACTCACTTTGTAGACCAGGCTGGCCTTGAACTCAGAAATCTGCCTGCCTTTGTCTCCTgagtgctgggattaaaggcgtgtgcc

atcatgcccggcaagactcacatcttaacctgttaatgaagggattaaagtgcaaagttcaaagcacatcagggcacctagttataagagcctctgcactggacaaagctgctcgtctggacatcctcaatgaagt

tcttcaatgactttggtccagtcagctatggtagatcagaagacttgcatggcgggcacgttttaccagccaagctgccttgccggctcctccagatgacatcttcttcccattaagttggaatacatactgtgtgctttgc

ctcatcgtgtggaaagaggaagtggttggtggtttgggggcactgtggtcctgtagtgtagatgccctgcagtcttgcaggagtgtgtgactagctgggaaacccactaaccagtgtgaggattagcagcagcagtt

cttgtgggaagcaccagttggcctgatcagacttactgaacatgggaagaaagctgagctctggagaactggcctggggatgcccaggtcagtgccagcggaggcttcaaggaggaagactgcagacctgact

cactgggtctgtgtggagagcaaacaaatgagccaaagccagcggtgtggctgggtgtgcctcagctgcaggtgtgacagtgtcctgtatcccgcggggccccgcagaggcattgctttagggaacagccaccc

atggcttgtatatgtcctttttcaggtgattccctggactctgtgagctggcagtgcttggagctacacagcttgtgccATGGACTCTGAGG

74

TRIB3 (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|372099108:c152344060-152337425 Mus musculus strain C57BL/6J chromosome 2, GRCm38.p2 C57BL/6J; NCBI Reference Sequence: NC_000068.7

CAAGGTACCCAGTAGGGAGCTGCGAAGGGGGATTTACCCGGGTCTGGCAAGAGGGTCACTGGAAGTCCAGGCGTATTCATGAATATTTCTATTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCGTTCTCTCTCTCTCTCGTTCTCTCCCTCTCGCTCTCGCT

CTCTTTTACTCACTCTCACTCTTCTCACTCTCACTCTCTCGCTCCTTTACTTCGCCGTCTGGGGGCGCCCTCAGCCTGAGCTGTCGACCCCAGCTT

ATTATGCAGCCCAGGCTGACTGGCAATTCACCAGTCAGCCCCCAGGGTGCTGGCGAAGCAGGGGTGCGTCCCTAAGCTCAGCTCTCCTTATTGCGTTATTAAACCTTTCATTCGTCGCGGGTCCCTTCCTTCCCTAGCAGTCCTTGCATTTGAGAGCTTCGGAAAACCCAGTGTGGT

GTTTCTCTGTTCGGAAGGTCCTAGCAGAGAAGACAGGATACCAAACAGTCTTTCTTCCTTGCTCTCTAGATGCGAGCTACACCTCTGGCTGCTTCTGCTGATGTTTCCTGCAGGAA

ASK1/MAP3K5 (Mouse) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351517922:2476090-2675047 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold430, whole genome shotgun sequence; NCBI Reference Sequence:

NW_003613627.1

ATCCACAGGCAGACACAAGTGAAATACATCTTAAAAAAGAAAGTTTTATCTTTGTCCTATTTTTAGAAACTCAAATATCAGTCTCAGTTGTGATATCAG

TCTCAGATAACATTTTATTTGTCTACTTTACACTACATTTGCCATGGAAAGATTTTGTTATCAAGTTTATGATTTTTTCCTTTTAGATTTTTGAATACCAATCTTGGACTTATTTCCATTCCTATGTTGTAAAATAGTTGCCCTTATATGTATTAAGTATTCACTTAGTTCCATTACTAATAGGTA

AGGAAGCTTATTTGGTGTTATGGTGTGGGGAATGAATTCAGTTCTGTCTTTGTGTTGATCTTGGGTAGCTTGTCTCCTATGTACTCATCGAAACATGGG

AGACTTGAGAAGCATTTGCTTAGCAGGCTGGCAATGTGTGCTTAGTACTTATAAACTGGAAGTGACCATGCCATTCAGGAGAAAATA

CACTTAGTGACTACTTCCTAAGAACTTGAGTGGCAGACTAGTCTCCCAGACTTCCACGGATGGGGAGTACAGGGAAGTC

Cyt c sequence is not available on CHO genome and also, nothing concrete in the NCBI gene database (either has isoforms or oxidases..).

TRB3

Cyclin D1/CCND1/CDK4 (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA) >gi|351516727:c315096-308625 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold4342, whole genome shotgun sequence; NCBI Reference Sequence:

NW_003614822.1

TCTGGCTTGCGTGTGGCCTGGCCTCCCTCCTAGCTGTCCTGTCCAGAGCTCCCCTCCCCCGCCCCCTCCACAGATCTCAGAGGA

GCCACTTCTGAGGAAAGTGGGGAAAGAAGACACCCCACCACTACCACCCTCGCACCGCTTTTTCCCAGCTTGGAGAGAAGCAGTCCGAGCGATTTGCATATCTACGAAGGTCGAGCTGGAGGGGGGGCGCTTTGGGCTTGTCCCCTAGCCGTTTGCAGCTCCCGAGCCCCCTCCCCCTGCGCC

CGCCCCGTCCCTCCCTCCTCCCTTTCTCTGCCTAGCTTTGATCTCTGCTTAACAACAGTAACGTCACACGGACTACAGGGGAGTTTTGTTGAAGTTGCA

AAGTCCTGCAGCCTCCAGAGGGCTGTCGGCGCAGTAGCAGAGAGCAGCAGACTCCGCGCGCTCCAGAGACCGGCAGTAGAGCGCGAGGCAGCGCGC

GCCAGCAGCAGCCACCGGAGCCCAACCAGGACCACAGCCCTCCCCAGGCGGCCGCGCCATGGAACACCAGCTCCTGTGCTGCGAAGT

75

CDK2 (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351517332:c747134-741463 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold4812, whole genome shotgun sequence; NCBI Reference Sequence: NW_003614217.1

TGATAGTACACGGAGGAAGTGCCAGACGATCTCTGCGAGTTCGAAGTCAACCTCAATTTTTATTTTATTTTTAACGCATTGCAGCCGGC

CAGGCTGCAATATGAGATACTGTCTCAAAAAGGGGGTGGGAGTGCTGGAGAGACCGCTCAGGAGTTAAGAGCCTATGTAGCTCTTGCAGAAGATCAGAGCTGGGCTCCAGCGTCAGCTCACAACTGCCTATAACTTCAGCTCCAGACGATCTGCTGCTTCTGTGAAAAAAACACACACAGACACACACATAACTG

ATATATATATATATATATATATATATATATATATATATATATGATATGTATGTAGATATGATTTCCCAGGTTTCTGCTTTCCAAAGG

AGGCTCTCCTGGCCAGCTAGAATTTAATTTCACTTAAGGGATGGTGAAAATTGTGAGCGGAAAGTAAGGAAACCCTTAGACCACAGACAACA

CCAGTTCCGGCCGCCGGAGCCCAAACCAGGAACTGCGGGAAAGTTGGAAnncggcaacattgtttcaagttggataaattgtcaagggctgagctttgcttgcgttccatccagagctggggctgcggtgctcaccttggcccccaggtcctccgccccgagtgttgggcccgctttcttcagggt

tcccgggccccgctcccgggggcctgggcagccacactagtgctccATGGAGAACTTCCA

APAF1 (CHO genome) ERSE I (CCAAT-N9-CCACG) UPRE (TGACGTGGA) ERSE II (ATTGG-N-CCACG) GC box (GGGCGG) TATA box (TATAA)

>gi|351516830:c37235-474 Cricetulus griseus unplaced genomic scaffold, CriGri_1.0 scaffold4981, whole genome shotgun sequence; NCBI Reference Sequence:

NW_003614719.1

TTTTAAAACATGAGGAATTAGGTATGGTAACTTTTTCAGAACTTTAACTTAACAAAAGGAAGAATTTTTACTGTAAAAAAAAAGGACACTATAAAATTG

CAAAAGAAGAAAGTTGAAATCTTGCTACTCAGTCTTCCAAACACTTTGATAAACAAACTTTAAGCATATTCTTATACATTTTTGAGATATAACACATTT

TGTCTTATTTGGGGTTCTGTTTCTTTTTGAAATCAGCAGTGTCAGAGACACCTTTCCTTGTCAGTATATACACTGCATAGAGATATGTGCTGTCTAATT

TTAAATGGCTATGTAGTATTCCATTTTAAATATAAACATAGTTAAAAATAATAAGCCGGGTGATGGTGGCAAACACCTTTAATCTTAGCACTTGAAAAG

TAGAGGCAGGTGGATCTCTGAGATCCCTGAGTTTGAGGCCAGCCTGGTCTACAGAGTGAGTTCCAGGACAACCAAGGCTACACAGAG

AAAACTTGTCTTGAAAAGCAAAAATAAATAAATAAATAAACAAACAAACAAATGAATAAATCTTTTTTTTTTCCATATTTCTtgattccttgtcatgacaaatatgt

tgacactttgcctgtagctcatgtttgacagtgaagagagaaaaaacctgaaggacaATGgatgcaaagg