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Master’s Thesis of Science in Agriculture

Biofunctional Human Cystatin-C Expressed in

Transgenic Chickens

형질 전환 닭에서 생산하는 생기능성 인간 시스타틴 C

February 2020

Seo Woo Kim

Department of International Agricultural Technology

Graduate School of International Agricultural Technology

Seoul National University

Biofunctional Human Cystatin-C Expressed in Transgenic

Chickens

A thesis

submitted in partial fulfillment of the requirements to the faculty

of Graduate School of International Agricultural Technology

for the Degree of Master of Science in Agriculture

By

Seo Woo Kim

Supervised by

Prof. Tae Sub Park

Major of International Agricultural Technology

Department of International Agricultural Technology

Graduate School of International Agricultural Technology

Seoul National University

December 2019

Approved as a qualified thesis

for the Degree of Master of Science in Agriculture

by the committee members

Chairman Byung-Chul Park, Ph.D.

Member Tae Min Kim, Ph.D.

Member Tae Sub Park, Ph.D.

i

Abstract

Bioreactor is the massive production system for bioactive substances

as well as therapeutic proteins. Although the various methods such as

microorganisms and animal cells, the efficient strategy should be

necessary for the efficient production of biofunctional proteins. In the

production of therapeutic proteins, post-translational modification is not

properly performed in microorganisms. In addition, animal cell systems

require more cost and time. To overcome these problems, a new method

has been considered with transgenic animals such as the genetically-

modified hen’s eggs. Human cystatin-C is a member of cystatin

superfamily. It is produced in the body at a constant rate found in body

fluids and secretions. Moreover, this protein acts as an inhibitor of

cysteine protease and protects against microbial and parasitic infiltration.

We aim to construct transgenic chickens that produce human cystatin-C

using a non-viral piggyBac transposon system. We generated transgenic

chicken expressing human cystatin-C protein by using non-viral piggyBac

transposon system. We analyzed the expression of human cystatin-C in

DF1 cells and PGCs, and we produced transgenic chickens using the

human cystatin-C expression vector. Expression of human cystatin-C

protein in transgenic chickens were verified through Western blot and

ELISA. Biofunctional activity of purified human cystatin-C in transgenic

ii

chickens was confirmed through in vitro assay. Thus, these data suggests

that transgenic chickens could be efficiently used as a bioreactor for

production of various bioactive materials.

Key words: transgenic chicken, bioreactor, human cystatin-C

Student Number: 2018-26749

iii

Contents

Abstract ………………………………………………………………..…………ⅰ

Contents ………………………………………………………………………….ⅲ

List of Tables ………………………………………………………………..ⅵ

List of Figures ……………………………………………………………….ⅶ

List of Abbreviation ………………………………………………………ⅸ

Introduction …………………………………………………………………….. 1

Review of Literature ……………………………………………………... 4

1. Bioreactor ……………………………………………………………… 4

1.1 Definition of bioreactor …………………………….. 4

1.2 Bacterial expression system ………………………. 5

1.3 Animal cell expression system ………………….. 7

1.4 Transgenic animal expression system ……… 12

1.4.1 Transgenic chicken expression system

…………………………………………………………………… 14

iv

2. Cystatin-C …………………………………………………………… 15

2.1 cystatin-C …………………………………………………. 16

2.2 Kidney function biomarker ………………………. 18

2.3 function of cystatin-C ……………………………… 21

3. Production of transgenic chicken using non-viral

piggyBac transposon system ……………………………….. 24

3.1 piggyBac transposon system ……………………. 24

3.2 PGCs ………………………………………………………… 25

Materials and Methods …………………………………………………. 28

1. Construction of human cystatin-C expression vector

system using piggyBac transposon .……………………. 28

2. Transfection of human cystatin-C expression vector

to DF1 cell & PGCs ……………………..……………………. 28

3. Microinjection of cultured PGCs into recipient

embryos ……………………………………………………………… 29

4. Production of transgenic chickens ……………………. 29

5. Genomic PCR ……………………………………………………… 30

6. Purification of human cystatin-C in egg white and

muscle …………………………………………………………………. 30

v

7. ELISA ………………………………………………………………….. 31

8. Western blotting …………………………………………………. 31

9. Antimicrobial activity test ………………………………….. 32

Results ………………………………………………………………………….. 33

1. Construction of human cystatin-C expression vector

……………………………………………………………………………. 33

2. Identification of vector expression in DF1 cells and

PGCs …………………………………………………………………. 35

3. Production of germ-line chimera and transgenic

offspring ……………………………………………………………. 37

4. Identification of human cystatin-C expression in

transgenic chickens …………………………………………… 39

5. Purification of human cystatin-C in transgenic

chickens’ egg white and muscle ……………………… 41

6. Antimicrobial activity of human cystatin-C in

transgenic chickens’ egg white ……………………….. 43

Discussion …………………………………………………………………….. 46

Conclusion …………………………………………………………………….. 51

References ……………………………………………………………………. 52

Abstract in Korean ……………………………………………………….. 63

vi

List of Tables

Table 1. Comparison of each expression systems producing

recombinant pharmaceutical proteins ………………………….. 11

Table 2. Total estimated costs-of-goods (COGS)

comparison between animal cell expression system and

transgenic animal expression system …………………………… 11

vii

List of Figures

Figure 1. Comparison of production systems for approved

biopharmaceuticals ……………………………………………………….. 10

Figure 2. Comparison of creatinine and cystatin-C as

kidney markers …………………………………………………………….. 20

Figure 3. Antimicrobial activity of cystatin-C …………… 22

Figure 4. Antiviral activity of cystatin-C …………………… 23

Figure 5. Construction of human cystatin-C expression

vector system using piggyBac transposon …………………… 34

Figure 6. Identification of human cystatin-C in DF1 cells

and PGCs ………………………………………………………………………. 36

Figure 7. Production of germ-line chimera and transgenic

offspring ……………………………………………………………………….. 38

viii

Figure 8. Expression of human cystatin-C in transgenic

chickens ………………………………………………………………………… 40

Figure 9. Purification of human cystatin-C in transgenic

chickens’ egg white and muscle …………………………………… 42

Figure 10. Antimicrobial activity of human cystatin-C in

transgenic chickens’ egg white against E. coli ………….. 44

ix

List of Abbreviation

CMV : Cytomegalovirus

EF1 : Elongation factor 1

PBS : Phosphate-buffered saline

PGC : Primordial germ cell

PCR : Polymerase chain reaction

ELISA : Enzyme-linked immunosorbent assay

RIPA : Radioimmunoprecipitation

ECL : Enhanced chemiluminescence

WT : Wild-type

TG : Transgenic

EW : Egg white

H cystatin : Human cystatin

1

Introduction

Bioreactor is any manufactured device or system that supports a

biologically active environment. It includes prokaryotic, eukaryotic cell

system and transgenic animal using aerobic or anaerobic condition.

Through this system, it is possible from sewage treatment, fermentation

to producing antibody, vaccine and recombination protein.

In 1980s, after recombinant DNA (rDNA) technology developed,

recombinant human insulin was approved to FDA for the treatment and

enter the market as a first recombinant pharmaceutical protein. Since

then, recombinant protein production using prokaryotic cell like E.coli and

yeast was developed (Ferrer-Miralles, Domingo-Espin et al. 2009).

However, bacteria and yeast have inaccurate post-translational

modification (Jenkins 2007). Because of this disadvantage, recombinant

pharmaceutical proteins can reduce efficacy, limit their shelf life, occur

unwanted side-effects like immune reaction. Thus, in some cases, animal

cell system is used to produce recombinant proteins. But compared to

other production systems, production level is relatively low (Houdebine

2009). If production level is similar with others, the production cost will

be increasing. Therefore, therapeutic protein production system using

transgenic animal is getting attention.

In 2009, The FDA in USA first approved recombinant human anti-

2

thrombin-α in transgenic goat(Kling 2009). recombinant human anti-

thrombin-α called ATryn produced in transgenic goat’s milk. Compared

with human plasma version of ThrombateⅢ and ATrym, both efficacy is

similar but production cost of ATryn is cheaper. Moreover, human plasma

product can cause blood-borne diseases but ATryn is safe about these

side-effect. However, there is a view that needs more research on

protein maturation or secretion.

There are various transgenic animals for bioreactor. Among them,

chickens have short generation and continue to produce eggs. So, it can

be possible to get recombinant pharmaceutical proteins continuously. Also,

it is easier to purification protein in egg white because it has no fat

unlike milk. Moreover, most mammals except human produce α1,3-

galactose but chicken has not. So there is no side effect like immune

response when we use recombinant protein in chickens (Lillico, McGrew

et al. 2005, Houdebine 2009).

Cystatin-C is single polypeptide chain structure containing 120 amino

acid, molecular weight is 13kDa. Also, it is member of cystatin super

family. In human, all cells with a nucleus produce it. So it can found in

all tissues and body fluids like blood, sperm, saliva (Zi and Xu 2018). In

addition, cystatin-C is as known as kidney function biomarker. The most

commonly used kidney marker is creatinine, but some studies have shown

that cystatin-C is a better indicator of glomerular filtration than creatinine

(Dharnidharka, Kwon et al. 2002, Stevens, Coresh et al. 2008, Bagshaw

3

and Bellomo 2010). Acute kidney injury (AKI) in children and newborns

is defined as a sudden decline in glomerular filtration rate and is most

important to detect and treat early because AKI is asymptomatic, and

the symptoms become apparent after renal function has declined

significantly. Cystatin-C is a biomarker that has been studied a lot as

one of AKI diagnostic methods (Bagshaw and Bellomo 2010, Nakhjavan-

Shahraki, Yousefifard et al. 2017, Khosravi, Zadkarami et al. 2018).

Cystatin-C plays as cysteine protease (cathepsin B, H, L, S, elastase

and papain) competitive inhibitor that blocks the cutting of peptide bonds

(Shamsi and Bano 2017). The interaction of Cathepsin with cystatin-C

inhibits angiogenesis and tumor growth (Wang, Sun et al. 2006). In addition,

because cystatin-C inhibits cathepsin, serum cystatin-C can be used as

an important marker for inflammation in COPD patients (Zhang, Li et al.

2016). Cystatin-C can protect body from virus and bacteria’s invasion,

toxicity, reproduction by inhibiting cysteine protease of the microorganism

(Sun, Liu et al. 2013, Hernandez, Marcet et al. 2014, Luthra 2015).

In this study, we produce transgenic chicken for bioreactor with human

cystatin-C expression vector system using non-viral piggyBac transposon.

We designed the cystatin-C to appear throughout the chicken’s whole

body. Therefore, our goal is to purify and use cystatin-C in all part of

chicken not only egg white but also muscle.

4

Review of Literature

1. Bioreactor

1.1 Definition of bioreactor

A bioreactor is defined that any manufactured device or system that

supports a biologically active environment. In addition, if one focuses only

on the aspect of cell culture, a device or system for culturing cells or

tissues is also called a bioreactor. This system can be in aerobic or

anaerobic state. Depending on how it works, bioreactors are divided three

types; batch cultures, fed-batch cultures, continuous cultures. Biocatalysts

used in bioreactors include enzymes, microorganisms, animal cells, and

plant cells, which are used in suspension or immobilized. In a bioreactor,

specific biochemical reactions occur by enzymes, microorganisms, animal

or plant-derived cells, or intracellular structures such as chloroplasts or

mitochondria to obtain the desired materials.

Examples of bioreactors include sewage treatment, fermentation, and

the manufacture of recombinant pharmaceuticals called pharming or

molecular farming. In the case of recombinant drug production, animal

cells are usually used. However, agitated environments such as microbial

5

bioreactor devices are disruptive to these cell types and tissues, and

require highly specialized growth media for animal cells. Thus, quite

different designs are required compared to general industrial bioreactors

used to grow protein expression systems such as yeast and bacteria.

1.2 Bacterial expression system

In 1968, Swiss microbiologist Werner Arber discovered restriction

enzymes (Piekarowicz 1979) and the following year US microbiologist

Hamilton O. Smith purified type II restriction enzymes (Kelly and Smith

1970). The enzyme has been shown to be essential for genetic engineering

due to its ability to cleave at specific sites in DNA as opposed to type I

restriction enzymes. This discovery led to the emergence of recombinant

DNA technology. Based on Smith's work, American molecular biologist

Daniel Nathans showed that type II enzymes could be useful for genetic

research in 1970-71 (Danna and Nathans 1972, Danna, Sack et al. 1973,

Danna and Nathans 1999). At the same time, American biochemist Paul

Berg developed a way to isolate DNA molecules from selected sites and

attach molecular segments to DNA in viruses or plasmids to enter

bacterial or animal cells (Jackson, Symons et al. 1972). In 1973, American

biochemists Stanley N. Cohen and Herbert W. Boyer first cloned

recombinant genes into bacterial cells and cloned them (Cohen, Chang et

al. 1973). With the development of such recombinant DNA (rDNA)

6

technology in the 1970s, attempts have been made to create new gene

combinations that are of value to science, medicine, agriculture and

industry using recombinant DNA technology.

In October 1982, E. coli-produced recombinant human insulin(called

Humulin) first became FDA approved and was produced (Quianzon and

Cheikh 2012). Previously, insulin was isolated from pigs and bovines’

pancreas to treat diabetes. It was developed by Genentech which is the

first US biotechnology company. Humulin licensed Eli Lilly and became

the first marketable product through recombinant DNA technology. Also,

it was the first recombinant drug approved by the FDA for use in the

United States. Humulin is slower than normal insulin and has a longer

activity period, making it suitable for use as a drug. Since Humulin

received FDA approval, the production of useful proteins in E. coli or

yeast has been increased.

In comparison with the expression system using microorganisms such

as E. coli and yeast and the reaction process of the previous chemical

industry, In the chemical industry, metal-based catalysts require a lot of

energy and resources, but in the case of microorganisms, necessary

materials are produced by enzymes and microbial biological functions.

Moreover, unlike other catalysts, enzymes act only on specific reactions.

Therefore, the bacterial expression systems do not produce unnecessary

substances. It is possible to save resources, save energy, and to reduce

the cost of construction and equipment since it can be a small factory.

7

In addition, it does not occur smoke, harmful substances, noise pollution,

etc. The rapid growth of microorganisms allows the production of large

quantities of material in a short time. Disadvantages include that the

expressed protein is often mis-folded and can be biologically inactive,

cannot be produced if the protein produced is toxic to microorganisms,

and lacks the enzymes responsible for post-translational modifications

compared to other species. There is no device for attaching sugar residues

of glycoproteins in bacteria. If post-translational modifications are not

carried out correctly, the useful protein may be reduced, the rate of

protein degradation may be shortened, and the shelf life may be

shortened, and when used as a drug, it is likely to cause side effects

such as an immune response.

1.3 Animal cell expression system

Animal cell expression systems are widely used in recombinant protein

production because of post-translational modifications that are not

achieved in bacterial expression systems. Cultured mammalian cell lines

such as Chinese hamster ovary (CHO), COS and human cell lines such

as HEK and HeLa can be used to produce proteins. The vector is

transfected into the cell and, in the case of stable transfection, DNA can

be integrated into the genome or transfected temporarily by homologous

recombination. Examples of mammalian expression vectors include the

8

adenoviral vectors (Berkner 1992), the pSV and the pCMV series of

plasmid vectors, vaccinia and retroviral vectors (Hruby 1990), as well as

baculovirus (Kost and Condreay 2002). The promoters for cytomegalovirus

(CMV) and SV40 are commonly used in mammalian expression vectors to

drive gene expression. Non-viral promoter, such as the elongation factor

(EF)-1 promoter, is also known (Kim, Uetsuki et al. 1990).

Examination of recombinant antibodies approved from 1989 to 2018

shows a gradual increase in products produced through mammalian cells

(Figure 1). This suggests that the post-translational modification process

is very important in the manufacture of recombinant pharmaceuticals.

However, the drawback of this animal cell expression system, which is

mainly used for protein production, is its low output for the price.

Compared to bacterial expression systems, post-translational modifications

and glycosylation are superior to mammalian cells. However, the yield is

significantly lower compared to bacterial or transgenic animal systems

(Table 1). Accordingly, there have been attempts to develop relatively

high productivity recombinant cell lines through improvements in vector

construction, selection of selectable markers and advances in gene-

targeting and high throughput screening strategies (Gurtu, Yan et al.

1996, Pu, Cashion et al. 1998). Another disadvantage is the high cost of

maintaining an animal cell culture system and the difficulty of scaling up

the system for mass translation purposes. It would take three to five

years to build a large manufacturing facility for mammalian cells and

9

cost $ 250 million (Dyck, Lacroix et al. 2003). Production costs are also

more expensive for animal cell systems when compared to transgenic

animal systems (Table 2)

10

Figure 1. Comparison of production systems for approved

biopharmaceuticals

Relative use of mammalian- versus nonmammalian-based production cell

lines in the manufacture of biopharmaceuticals approved over the

indicated periods. Each dataset is expressed as a percentage of total

biopharmaceutical product approvals for the period in question. (Figure

adapted from (Walsh 2018))

11

Table 1. Comparison of each expression systems producing recombinant

pharmaceutical proteins

(Table adapted from (Wang, Zhao et al. 2013))

Table 2. Total estimated costs-of-goods (COGS) comparison between animal

cell expression system and transgenic animal expression system

Presented by H.L. Levine, 2002 BIO International Biotechnology

Convention and Exhibition, 9–12 June, Toronto, Ontario, Canada. (Table

adapted from (Dyck, Lacroix et al. 2003))

12

1.4 Transgenic animal expression system

In February 2009, recombinant human anti-thrombin produced by

transgenic goats became the first transgenic animal-producing drug under

FDA approval and began to be produced under the name ATryn (Kling

2009). Anti-thrombin has the properties of an anticoagulant. Expression

of ATryn in milk is achieved by linking antithrombin-α transgenes to the

breast-specific promoter of milk proteins. The company that developed

ATryn, rEVO Biologics (formerly GTC Biotherapeutics), says one

transgenic goat can produce the same amount of anti-thrombin as 90,000

blood donations per year. The reason why they chose goat among many

animals was that it was able to produce more rapidly than cows and

produce more protein than rabbits and mice (Stix 2005). When comparing

THROMBATE III, an anti-thrombin product extracted from human plasma,

and ATryn, which is produced by transgenic goats, the efficacy of both

products is the same, but anti-thrombin can be produced at a lower cost

with transgenic goats. Human plasma products may also be a cause of

blood-borne diseases, but they are safe from this risk when produced

with transgenic goats.

The ability of transgenic animals to produce complex biologically active

proteins in an efficient and economical way is superior to bacteria,

mammalian cells, transgenic plants and insects. It takes three to five

years to build a large (10,000 liter bioreactor) manufacturing facility for

13

mammalian cells and costs between $ 250 and $ 500 million, while

transgenic farms with a single purification facility should not exceed $ 80

million (Dyck, Lacroix et al. 2003). Establishing a commercial production

herd of transgenic goats in a company can be accomplished at a cost of

about one tenth of the cost of building a commercial cell culture facility

(Thiel 2004).

In general, because milk is easily collected in large quantities, it has

been considered an organ of choice for expressing recombinant proteins

in transgenic animal bioreactors. Milk is currently the best bioreactor.

Foreign proteins are generally reported to be produced in the milk of

transgenic animals at the rate of several grams per liter. However, the

production of protein in milk is limited by the relatively long intervals

from birth to the first feeding experienced by domestic livestock, the

discontinuous nature of the feeding cycle, and the considerable time and

material investment required to produce genetically modified dairy animals.

In addition, certain bioactive proteins produced in milk can adversely

affect animal health. This is especially true when high concentrations are

produced and proteins are resorbed. These shortcomings have led to the

search for other types of collectable fluids. Human α-AT was successfully

obtained at high levels from the serum of transgenic rabbits (Massoud,

Bischoff et al. 1991) and human hemoglobin was produced in the

transgenic pig circulation (Swanson, Martin et al. 1992). In addition,

recombinant antibodies were produced in the blood of transgenic goats,

14

pigs, and rabbits. However, blood is not an ideal body fluid for obtaining

recombinant protein because it is invasive and can affect the health of

the animal when producing bioactive proteins.

1.4.1 Transgenic chicken expression system

In 2015, the recombinant lysosomal acid lipase produced by transgenic

chickens was approved by the FDA and began producing under the name

Kanuma. To produce this drug, transgenic chickens were constructed

using genetic modification techniques to produce recombinant LAL (rhLAL)

in chicken egg whites (Sheridan 2016). It can then be extracted and

purified from egg whites and used as a drug. At the time of FDA

approval, Kunama was the first drug manufactured from chicken eggs

and designed for human use. The first approved treatment for LAL-D

patients is administered via intravenous infusion. LAL-D is a very rare

disease and is defined as a disease that affects fewer than 20 patients

per million people in the general population. It is almost always fatal in

infants and children and adult patients are at high risk of fatal organ

damage and premature death (Reiner, Guardamagna et al. 2014, Jones,

Valayannopoulos et al. 2016). Kanuma (sebelipase alfa) is an innovative

enzyme replacement therapy that addresses the root cause of lysosomal

acid lipase deficiency (LAL-D) by reducing substrate accumulation in

cellular lysosomes throughout the body. In clinical studies, Kanuma

15

treatment improved the survival rate of infants with LAL-D, and markedly

improved lipid parameters as well as a significant reduction in ALT and

liver fat content in children and adults with LAL-D.

The advantage of the transgenic chicken system is that it is easy to

purify because the generation is shorter than other animals, and about

300 eggs are produced annually, which allows for continuous recombinant

protein production. Egg white is easy to purify because it does not

contain fat and consists only of water and protein. Also, unlike other

mammals, there is α1,3-galactose, a glycoprotein not found in humans

and apes. In humans, the gene for α1,3-galactosyl transferase is inactive

and 1% of B lymphocytes respond to intestinal bacteria. It produces anti

α1,3-galactose antibodies and contributes to the rejection of tissue

transplants in other mammals. Because of this, the presence of α1,3-

galactose epitopes in recombinant drugs produced in mammals can cause

side effects such as immune responses. However, in chickens, the

recombinant protein produced through transgenic chickens is safe from

this risk since it does not produce α1,3-galactose (Lillico, McGrew et al.

2005).

2. Cystatin-C

16

2.1 cystatin-C

Cystatin-C was first mentioned in 1961 when Jorgen Clausen described

the name "γ-CSF" in the human cerebrospinal fluid (CSF) of the

"cerebrospinal fluid specific" protein (Clausen 1961). At the same time,

Butler and Flynn described a new protein in human urine that moves in

the γ- region of the electrophoretic pattern. It is named post-γ protein

(Butler and Flynn 1961). In 1962, Hochwald and Thorbecke called γ-trace

to the ascitic and pleural fluids of human plasma, cerebrospinal fluid,

urine and proteins with γ-electrophoretic mobility (Hochwald and

Thorbecke 1962). The complete amino acid sequence of human cystatin-

C was finally determined by Anders Grubb and Helge Löfberg in 1981

(Grubb and Lofberg 1982).

Cystatin-C is a member of the cystatin superfamily. Cystatin

superfamily can be divided into four types (Mussap and Plebani 2004).

Type 1 cystatin is also called stefins. It is mainly located intracellularly

and typically human cystatin-A (stefin A), human cystatin-B (stefin B),

and rat cystatin α and β belong to this type. Type 1 cystatin is generally

considered to be a cytoplasmic protein but appears to be secreted or at

least released in certain situations. Type 2 cystatins include cystatin-C,

N, SN, SA, D, E / M, among which all the staphylostats identified as

17

ortholog have cystatin-C like proteins. Cystatin-D is considered an

additional component of the oral non-immune protective system. Cystatin-

F, also called leukocystatin, is primarily expressed in immune system cells.

Cystatin E / M is expressed in most human tissues, but expression levels

vary from tissue to tissue. In particular, abundant expression in fetal skin

and amniotic cells plays an important role in fetal development. Type 3

cystatin is a kininogen, primarily intravascular proteins, present in plasma

and secretions. Kininogens inhibit cysteine peptidase. Type 4 cystatin is

also called fetuins and is mainly synthesized in the liver. During fetal life

it has been identified as a major protein mainly in the blood and brain

and is considered a major protein in several metabolic pathways, including

the regulation of bone formation and bone resorption. Furthermore,

fetuins opsonize cationic macrophage-deactivating molecules and appear

to be a key partner in the recovery step of an acute phase response to

systemic inflammation.

The cystatin-C gene is located at the cystatin locus and comprises 3

exons, spanning 4.3 kilo-base pairs. It is found in high concentrations in

fluids and is a housekeeping gene that expresses any nucleated cell.

Cystatin-C is a non-glycosylated, basic protein (isoelectric point at pH

9.3). Cystatin-C protein has 120 amino acids and is about 13 kDa in size.

The structural feature is a single polypeptide chain structure with short

alpha helices and long alpha helices across a large antiparallel, five-

stranded beta sheet. It has two disulfide bonds and about 50% of the

18

molecule carries a hydroxylated proline.

2.2 Kidney function biomarker

Cystatin-C has a low molecular weight and is removed from the

bloodstream by glomerular filtration in the kidney. Reduced kidney

function and glomerular filtration rate increase blood levels of cystatin-

C. several studies demonstrate that serum levels of cystatin-C are more

accurate tests of kidney function than serum creatinine levels (Figure 2).

Serum creatinine is the most widely used method of indirectly measuring

glomerular filtration rate in clinical practice, but it can be influenced by

various factors such as sex, age, muscle mass, diet, physical activity, and

there is a drawback that does not reflect rapid changes in glomerular

filtration rate or abnormal initial renal function. In addition, creatinine

clearance will reflect the actual glomerular filtration rate excessively as

creatinine is secreted from the proximal tubules in addition to glomerular

filtration. Moreover, in chronic renal failure, the excretion of creatinine

through the tubules is further increased, and it is difficult to accurately

collect urine 24 hours. In comparison, the clinical utility of serum cystatin-

C has recently been known. Several studies have shown that cystatin-C

levels are more sensitive markers for measuring mortality in the elderly,

cardiovascular disease, and acute kidney injury (AKI) in children (Salminen,

Laine et al. 2016, Garcia-Carretero, Vigil-Medina et al. 2017, Nakhjavan-

Shahraki, Yousefifard et al. 2017). However, it is difficult to commercialize

19

cystatin-C assays because they are three times less expensive and 20

times more expensive than creatinine assays.

20

Figure 2. Comparison of creatinine and cystatin-C as kidney markers

Cystatin-C (Cys C) versus creatinine (Cr) as markers of glomerular

filtration rate (GFR). (A) Scatterplot of correlation coefficients (r) for

1/Cys C and 1/Cr with measured GFR. Horizontal line represents the

cumulative mean r of all studies analyzed. (B) Scatterplot of receiver

operating characteristic plot area under the curve (AUC) values for Cys

C and Cr. Horizontal line represents the cumulative mean of all 14 data

sets. Adapted and reproduced from Dharnidharka et al7 with permission

of the National Kidney Foundation. (Figure adapted from (Shlipak, Mattes

et al. 2013))

21

2.3 function of cystatin-C

Despite the diversity of structure and distribution in the body, all

members of the cystatin superfamily have in common cysteine proteinase

inhibitory properties, suggesting the preservation of physiological functions

through evolution. Their physiological role is to regulate the activity of

endogenous proteolytic enzymes that are often secreted or leaked from

lysosomes of dead or diseased cells. In addition, cystatin-C not only has

antimicrobial activity against microorganisms (Figure 3), but also enters

cells whose cell membranes have been damaged by viral attacks, which

can inhibit intracellular cysteine proteinases as an important function

(Korant, Brzin et al. 1985). It may interfere with viral replication requiring

a host or viral cysteine proteinase, such as capsid maturation (Figure 4).

Especially, human cystatin-C totally blocks replication of Herpes simplex

virus (Bjorck, Grubb et al. 1990), with an activity comparable with that

of acyclovir (Bjorck 1990). It is also an effective inhibitor of the

replication of Coronavirus (Collins and Grubb 1991), which can cause

acute gastroenteritis. In order to increase the blocking efficacy of cysteine

C's cysteine protease inhibitory activity, several peptidomimetic

compounds that mimic their structure have been synthesized (Grubb,

Abrahamson et al. 1990, Kasprzykowski, Schalen et al. 2000, Pikula,

Smuzynska et al. 2017).

22

Figure 3. Antimicrobial activity of cystatin-C

Inhibition zones of six strains of (A) Escherichia coli [02], (B) Escherichia

coli ATCC 25922, (C) Staphylococcus aureus ATCC 25923, (D) Escherichia

coli S, (E) Pseudomonas aeruginosa ATCC 27853, (F) Staphylococcus

gallinarum were spread on Muller-Hinton agar plates on which which

paper disks impregnated with 80 μg (1), 100 μg (2), 120 μg (3), and

150 μg/disk (4) of chicken cystatin were placed. In the centre of the

plate, the disk paper without cystatin added. Incubation period was 24 h

at 37℃. (Figure adapted from Ref. World Journal of Microbiology &

Biotechnology 2005 21: 59-64)

23

Figure 4. Antiviral activity of cystatin-C

Cystatin-C exhibited a dose-dependant decrease of blue-stained HIV-

infected cells as compared to virus only control. (Figure adapted from

(Vernekar, Velhal et al. 2015))

24

3. Production of transgenic chicken using non-viral

piggyBac transposon system

3.1 piggyBac transposon system

In general, when producing transgenic chickens, retroviral vectors are

used to efficiently integrate and express genes in the host genome.

However, the size of transgenes that can be introduced into retroviral

vectors is limited (Wu, Yang et al. 2010), and the vector is less efficient

at integrating the transgenes into the host chromosome (McGrew,

Sherman et al. 2004). In addition, safety issues due to the use of viral

vectors cannot be ignored. Therefore, there is a need to find ways to

make transgenic chickens safe and efficient.

DNA transposons are DNA sequences, also known as "jumping genes"

that can be moved and integrated into other locations within the genome.

DNA transposons can move in the DNA of an organism through single

or double stranded DNA intermediates (Feschotte and Pritham 2007). DNA

transposons were first discovered in maize (Mc 1950). DNA transposons

have been found in both prokaryotes and eukaryotes. In prokaryotes,

transposable elements can promote horizontal movement of other genes

related to antibiotic resistance or toxicity. Among the DNA transposons,

piggyBac transposon isolated from cabbage looper is a TTAA-specific,

short repeating element, a transposon group that shares similarity in

25

structure and properties of movement (Fraser, Smith et al. 1983). PiggyBac

has a fairly high potential efficiency and accommodates relatively large

transgenes without compromising potential efficiency (Wu, Ying et al.

2007). Previous studies have shown that the piggyBac transposon system

is efficient for the production of transgenic chickens (Park and Han 2012).

3.2 PGCs

PGCs can be identified based on the morphological features of the

large globular nuclei, the developed Golgi and endoplasmic reticulum, and

the presence of refractive lipids in the cytoplasm. PGCs, which

temporarily circulate through the bloodstream and travel to the genital

ridges, are easily distinguished from red blood cells because of their

characteristic shape and larger size. These morphological properties of

PGCs are widely preserved among bird species (for example, quails, ducks,

pigeons, pheasants, etc.). In addition, PGC can be identified through

staining (Meyer 1964) and immunological markers (Urven, Erickson et al.

1988, Maeda, Ohsako et al. 1994, Jung, Kim et al. 2005) for the cell

surface glycoproteins present in the PGC such as SSEA-1 or the chicken

vasa homolog (CVH) protein located in the cytoplasm. The main biological

characteristics of algal PGCs are as follows. First, the unique route of

passage to the genital ridges through the blood circulation, and second,

the ability to propagate long-term in vitro, is limited to the gonads. These

have led to the establishment of unique developmental techniques based

26

on PGC manipulation in algae to generate genetically modified poultry

and to conserve poultry genetic resources (Tagami, Miyahara et al. 2017).

Unlike mammals, avian PGCs use the embryo's circulatory system to

move from the embryonic crescent to the future gonads. The unique

migration characteristics of PGCs provide opportunities for identification

and isolation of cells.

In stage X chicken embryos consisting of 20,000–60,000 cells,

approximately 30-130 VASA positive PGCs are scattered in the center of

the pelvic area of the lobules. In this stage of development, most areas

of pelvic cells remain on the surface to form the upper layer, the so-

called epiblast, while other cells migrate to the diverticulum descent to

form primary hypoblasts. Immediately after primary hypoblast formation,

cell sheets derived from deep cells at the trailing edge of the brain germ

layers move forward below the surface to form a lower layer called

secondary hypoplasia. Then in the posterior marginal zone of step 2, the

epiblast thickens (6-7 hours of culture). In step 3 (12 hours of incubation),

VASA-positive PGCs are distributed in a horseshoe pattern in the anterior

region of the primitive stripes. In stage 4 (18-19 h culture), putative

amniotic cardiac vesicles of the genital crescent region develop. During

steps 7-9 (23-33 hours of incubation), most PGCs are distributed in

proamnion and conscientious vesicles. During primitive strike formation

and somitogenesis, the total PGC count of chicken PGCs increases

approximately three times as initially. At step 10 (33-38 hours of

27

incubation), the PGC begins to concentrate on the splanchnopleura

towards the embryo. At step 11 (40-45 hours of incubation), PGC begins

to migrate from the extra-embryonic zone to the intra-embryonic vascular

system. Later in stage 11, PGC begins to be found in sinusoids and the

number of PGCs decreases near the anterior empty vein. Most PGCs are

axially concentrated at the end of the sinus and prefer to be transported

axially to the embryonic circulation through the anterior tachycardia in

stages 12-14. There is a limited number of PGCs available in an embryo

during the blood circulation phase (steps 14-15). In order to effectively

use a small number of PGCs, it is essential to increase the number by

culturing these cells in vitro, and this culture technique for PGCs results

in a wide range of applications for germline manipulation.

28

Materials and Methods

1. Construction of human cystatin-C expression vector

system using piggyBac transposon

To insert the human cystatin-C gene into piggyBac transposon

transgene expression system vector (System Biosciences, Palo Alto, CA,

USA), NheⅠ and NotⅠ was used for digestion. Human cystatin-C was

regulated by the cytomegalovirus (CMV) promoter. His-tag (His6) was

added in front of stop codon of human cystatin-C. The promoter of

elongation factor 1 (EF1) was used for expression of the puromycin-

resistance gene in the transgene expression vector.

2. Transfection of human cystatin-C expression vector

to DF1 cell & PGCs.

To establish the transgene expressing cell lines, transgene expression

vector was co-transfected with piggyBac transposase using Lipofectamine

3000 (Invitrogen, USA) according to the manufacturer’s protocol. Once

29

the seeded cells reached 80% confluency on 6-well culture plates, they

were washed with phosphate-buffered saline (PBS) and refreshed with

2ml of the culture media without antibiotic-antimycotic. 7.5μl

Lipofectamine 3000 reagent in 250μl OPTI-MEM (Invitrogen, USA) and

10ul P3000 reagent with 2.5μg piggyBac transgene vector and piggyBac

transposase plasmid in 250μl OPTI-MEM was added to each well. After

3 hour incubation in 37℃, each well were washed with PBS and refreshed

with 3ml of the culture media with antibiotic-antimycotic. In next day,

10μg/ml puromycin was added to select the transfected cells.

3. Microinjection of cultured PGCs into recipient

embryos.

For PGC transplantation, a small window was made on the pointed end

of the recipient and the genetically-modified PGCs were microinjected

with a micropipette into the dorsal aorta of the recipient embryo. The

egg window of the recipient embryo was sealed with paraffin film and

then the egg was incubated with the pointed end down until further

screening and hatching.

4. Production of transgenic chickens

30

G0 chicken founders were mated through the testcross analysis and

the donor PGC-derived offspring could be identified. Subsequently,

transgenic chickens were identified by PCR analysis.

5. Genomic PCR

The DNA was extracted in feather pulp. The primers were hcyst his

F&R and CMV cyst his F&R. The hcyst his F sequence was 5’-

CACTGCTCCTCCTTGCGATC-3’; the hcyst his R sequence was 5’-

GGTGATGGTGATGGGCATCC-3’. The CMV cyst his F sequence was 5’-

GTGAACCGTCAGATCGCCTG-3’; the CMV cyst his R sequence was 5’-

GTGAGGCTGGTCGTGGAAAG-3’. The annealing temperature was 65℃.

The cycle number of amplication for PCR reaction was 30.

6. Purification of human cystatin-C in egg white and

muscle.

Egg white diluted with PBS and homogenized muscle were purified

using MagListo™ His-tagged Protein Purification kit (Bioneer). The resin

31

removed supernatant were mixed with PBS and spread in the sample.

The sample with resin were shaken for 10min and centrifuged for 90sec

at 4000 RPM, and removed supernatant. The sample with resin were

washed with 3ml of Binding/washing buffer for 4 times and eluted with

500μl of elution buffer for 2 times.

7. ELISA

All samples were analyzed using human cystatin-C Quantikine® ELISA

Kit (R&D Systems). Prepared samples (50μl) were added to each well

contained 100μl of Assay Diluent buffer. After incubation for 3 hours

at 2-8℃, each well was aspirated and washed by 400μl of Wash buffer

4 times. Each well was added 200μl of cold human cystatin-C conjugate

and incubated for 1 hour at 2-8℃. The wells were washed like previous

step and added 200μl of substrate solution and incubated for 30min at

room temperature in dark place. After incubation, each well was added

50μl of stop solution and measured by ELISA reader.

8. Western blotting

32

Feather pulp and homogenized muscle proteins were extracted with 1×

radioimmunoprecipitation (RIPA) lysis buffer and purified muscle and egg

white is in elution buffer (0.5M Imidazole). These samples separated on

a 15% polyacrylamide gel followed by transfer to a nitrocellulose

membrane. The primary antibodies used were mouse anti-β-actin (Santa

Cruz Biotechnology) or anti-human cystatin-C (Abcam). HRP-conjugated

anti-mouse IgG or anti-rabbit IgG (Bio-Rad) were used as secondary

antibodies. The blots were treated with ECL substrate solutions and

exposed in a ChemiDoc XRS System (Bio-Rad) to detect

chemiluminescence.

9. Antimicrobial activity test

E. coli was subculture on LB agar plate. E. coli colony was streaked

in four directions with a swab on LB agar plate. Make holes with 1000p

tip. Each hole have 50μl test sample. The plate incubated 16 hours at

37℃ incubator. After 16 hours, check the clear zone size.

33

Results

1. Construction of human cystatin-C expression vector

We constructed piggyBac transposon vectors that contained the human

cystatin-C gene (Figure 1). We use PB510B-1 Dual Promoter Vector

Formats. CMV promoter controlled human cystatin-C gene to overexpress

in whole chicken body. Because of CMV promoter, Human cystatin-C is

expressed throughout the body of the chicken, thus there is no need to

distinguish between male and female. Therefore, it is expected to help

increase production by reducing the budget required for discriminating

chick males and females and obtaining male cystatin from male tissue.

We insert the recombinant human cystatin-C gene using NheⅠ and Not

Ⅰ. His tag was attached to the end of the human cystatin-C gene to

make selective purification possible. We used puromycin resistance gene

for selection marker after transfection. The expected size of human

cystatin-C protein is 13kDa.

34

Figure 5. Construction of human cystatin-C expression vector system using

piggyBac transposon

(A) Expression vector of hCystatin_His-tag. The CMV promoter make

over-expression of human cystatin-C_His-tag gene. The selection marker

is puromycin resistance gene. (B) Sequences of human cystatin-C_His-tag

gene and their amino acids. The bold ATG indicates the start codon and

the blue sequences present the lysozyme signal sequences. The red

sequences are the His-tag.

35

2. Identification of vector expression in DF1 cells and

PGCs

To check human cystatin-C expression vector, we identified expression

of human cystatin-C for ELISA using transfected DF1 cells (Figure 2A).

Compared with control DF1 cells, transfected DF1 cells showed high

expression of human cystatin-C over 50ng/ml. We proceed transfection

in PGCs to use microinjection in recipient embryos. We identified human

cystatin-C vector expression with genomic PCR analysis and ELISA (Figure

2B). The results showed that human cystatin-C vector entered the PGCs

and human cystatin-C protein expression was over 50ng/ml. Both results

suggested that human cystatin-C expression vector constructed by non-

viral piggyBac transposon vector system was stably expressed the human

cystatin-C proteins.

36

Figure 6. Identification of human cystatin-C in DF1 cells and PGCs

(A) Identification of human cystatin-C expression in DF1 cells with ELISA.

DF1 with human cystatin-C expression vector successfully expressed

human cystatin-C protein. (B) Identification of human cystatin-C

expression in chicken primordial germ cells (PGCs) with genomic PCR

analysis and ELISA.

37

3. Production of germ-line chimera and transgenic

offspring

In previous study, we confirmed that piggyBac transposition into

primordial germ cells (PGCs) improved the efficiency of transgenic

chicken production and led to high-level transgene expression through

GFP expression (Park and Han 2012). The rate of germ-line transmission

was 95.2% on average. In this study, we microinjected PGCs with human

cystatin-C expression vector into recipient embryos to produce germ-line

chimera. Subsequently, transgenic chickens were produced by testcross

analysis using germ-line chimera. We extracted DNA in transgenic

offsprings’ feather pulp for PCR screening using Hcyt primer and Hcyt-

CMV primer to identify transgenic chickens. After testcross analysis, 30

of the 31 chicks, except 3285, were identified as human cystatin-C

expression vector (Figure 3). The rate of transgenic chicken production

is about 96%. This result is similar to the previous study. Therefore,

there was a high probability that transgenic chickens were produced and

human cystatin-C expression vector using non-viral piggyBac transposon

system is efficient method of transgenic chicken production.

38

Figure 7. Production of germ-line chimera and transgenic offspring

Production of transgenic chickens through the testcross analysis.

Transgenic chickens from the germline chimeras were identified by

genomic PCR analysis.

39

4. Identification of human cystatin-C expression in

transgenic chickens

After identifying the transgenic chickens to genomic PCR analysis, we

investigated human cystatin-C protein in transgenic chicken to ELISA and

western blot for identification of human cystatin-C protein expression in

whole chicken body (Figure 4). We checked feather pulp, blood, egg

white and muscle lysate. The human cystatin-C protein expression in

feather pulp is less then 6.25ng/ml in ELISA, but in Western blot result,

there was no abnormality in expression. All other tissues showed high

expression of human cystatin-C. This result confirmed that the presence

of sufficient human cystatin-C protein in other tissues. Therefore, it can

be expected that human cystatin-C purification in other tissues as well

as egg white is possible. Especially muscle has a lot of human cystatin-

C protein. Since muscles make up most of the body, if human cystatin-

C can be purified in muscles, the amount of human cystatin protein that

can be obtained per chicken will increase dramatically.

40

Figure 8. Expression of human cystatin-C in transgenic chickens

In ELISA, human cystatin-C protein was expressed in transgenic chicken’s

feather pulp (A), blood (C) and egg white (D). Additionally, Western

blotting showed that human cystatin-C was expressed in transgenic

chicken’s feather pulp (B) and muscle (E)

41

5. Purification of human cystatin-C in transgenic chickens’

egg white and muscle

Chickens can produce egg consistently. Thus, there have been attempts

to purify recombinant proteins using eggs (Lillico, McGrew et al. 2005).

However, we also purified human cystatin-C in muscle because transgenic

chickens we produced can express human cystatin-C in whole body.

Purified human cystatin-C using His tag was verified through western

blot (Figure 5A). The level of the purified sample in egg white was lower

than that in the muscle. However, in ELISA, it was found that both

purified sample in egg white and muscle had more then 100ng/ml. In

addition, when the dialysis sample for the antimicrobial activity test was

confirmed by ELISA, the results show that the concentration was not

greatly diluted. Compared TG egg white purification 1 and 2 in ELISA

data, concentration of first elution sample is higher than second one.

42

Figure 9. Purification of human cystatin-C in transgenic chickens’ egg

white and muscle

Muscle tissue was disrupted by homogenizer. Egg white was diluted 1:5

in PBS. We purified human cystatin-C with these samples and identified

using western blot (A) and ELISA (B). In ELISA, TG muscle is purified

human cystatin-C in TG muscle. TG EW is purified human cystatin-C in

TG egg white. TG EW dialysis is purified and dialysis human cystatin-C

in TG egg white.

43

6. Antimicrobial activity of human cystatin-C in transgenic

chickens’ egg white

To check the biofuctional activity of purified human cystatin-C, we tried

an antimicrobial activity experiment. First, we checked antimicrobial

activity of transgenic chickens’ egg white against E. coli. (Figure 10A).

Compared with human cystatin-C 50ng/ml, the clear zone was small but

accurate. It is assumed that the clear zone is small because the egg

white is so large that the concentration of cystatin is diluted. Next, we

examined the antimicrobial activity of the purified cystatin-C in TG egg

white (Figure 10B). Compared with human cystatin-C 100ng/ml and

50ng/ml, there were no clear zone in purified cystatin-C. Since the

antimicrobial activity experiment was conducted immediately after protein

purification, It is thought that the modification of protein is unlikely to

occur after purification. It is assumed that the cause is in the purification

method. In other studies, The egg whites are pretreated before

purification for remove ovomucin (Wang and Wu 2014). In future studies,

we will add a new pretreatment method to purify human cystatin and

then confirm the antimicrobial activity.

44

Figure 10. Antimicrobial activity of human cystatin-C in transgenic chickens’

egg white against E. coli

(A) Identification of TG egg white’s antimicrobial activity. 1 is human

cystatin-C 50ng/ml, 2 is water, 3 is TG EW. (B) Identification of purified

human cystatin-C’s antimicrobial activity. 1 is human cystatin-C 100ng/ml,

45

2 is human cystatin-C 50ng/ml, 3 is purified and dialysis human cystatin-

C in TG egg white.

46

Discussion

We constructed a vector using non-viral piggyBac transposon system

to produce transgenic chickens expressing human cystatin-C and

confirmed that the vector works normally by transfection in DF1 cells

and PGCs. PGCs transfected with the identified vectors were microinjected

into embryos to produce germline chimeras. Transgenic chickens were

selected by testcross analysis among the chimeras’ offspring. When we

identified the expression of human cystatin-C in various parts of the

transgenic chickens, it was detected in all parts. This indicates that

transgenic chickens expressing human cystatin-C have been produced.

In general, chicken is livestock that produce eggs. Thus, the males are

eliminated from the chick stage. In addition, even when used as a

bioreactor, the male chickens were excepted because the recombinant

protein produce in egg white. In this study, the CMV promoter was used

to express human cystatin-C throughout the chicken body, rather than

the egg white specific promoter. This allows human cystatin-C to be

purified not only on egg white but also on muscles and other tissues. It

has the advantage that the recombinant protein can be obtained even in

the muscles of males without eliminating the males when producing

transgenic chickens. Using CMV promoters will have a positive impact on

the productivity of recombinant proteins, reducing the effort of screening

47

male and female.

Human cystatin-C purification must be accompanied for use as

medicines or diagnostic kits. In a previous study, they introduced a method

for purifying cystatin from chicken egg whites using affinity

chromatography (Wang and Wu 2014). However, we attached His tag

back of the human cystatin-C sequence when we constructed the vector,

only human cystatin-C protein was purified selectively using Ni-NTA

magnetic nanobead. The protein was purified from egg white and

homogenized muscle. But the amount of protein purified from muscle

was higher due to differences in quantity of egg white and muscle. But

the easier form to purify was egg white. In fact, when the purification

purity was identified through SDS PAGE, purified sample in muscle had

not only human cystatin-C but also other proteins. This is considered to

be a problem of the purification method, and since the purification kit

that we use is using magnetic nanobead, it is more likely that unwashed

proteins will elute together when receiving the purification solution. In

the case of muscle, the tendency to lump together with the resin was

observed. Egg white was also agglomerated together with the resin, but

after freeze-drying, diluting in PBS and once or twice with a sieve, it

was confirmed that the resin agglomeration greatly reduced.

Cystatin is a cysteine protease inhibitor that has been known to have

antimicrobial and antiviral properties against microorganisms and viruses

through several papers (Szpak, Trziszka et al. 2014, Luthra 2015, Pikula,

48

Smuzynska et al. 2017). Using this property, we are conducting

antimicrobial activity experiment to verify the activity of purified human

cystatin-C. Antibiotics are one of the most commonly used treatments

for bacterial infections. Antibiotic resistant microorganisms continue to

emerge as the abuse of antibiotics and the ability of microorganisms to

adapt. Many studies have been conducted to prevent the production of

antibiotic resistant bacteria or to effectively eliminate them (Li, Li et al.

2018, Luna, Ershova et al. 2019, Sorbara, Dubin et al. 2019). If this

experiment shows that the purified human cystatin-C has the antimicrobial

activity, we can suggest the possibility of using the human cystatin-C

protein as a new antibiotic.

We were concerned whether cystatin’s role as cysteine protease

inhibitor would affect the basic mechanism of the transgenic chicken,

but our transgenic chickens were not particularly different from those of

normal chickens. In other papers, cystatin-C protein was never fed or

injected as a drug, but there were no cases of health abnormalities when

cystatin-C overexpressed individuals were produced and confirmed. Rather,

it has been found to be possible as a therapeutic agent for inhibiting

cancer or for neurological diseases such as Alzheimer’s disease (Kaeser,

Herzig et al. 2007, Yan, Zhou et al. 2015, Coronado, Barrios et al. 2017,

Zou, Chen et al. 2017, Kaur, Gauthier et al. 2018, Li, Wang et al. 2018).

In addition, cystatin expressed by parasites can induce the production of

IL-10, which can suppress allergy and intestinal inflammation (Schnoeller,

49

Rausch et al. 2008, Jang, Cho et al. 2011). It is also being studied as a

potential treatment for collagen-induced arthritis (CIA) (Liu, Cheng et al.

2016). Therefore, it was found that cystatin-C can be purified and used

as a therapeutic agent in human cystatin-C expressing transgenic chickens.

Cystatin-C is generally known to have a characteristic of being filtered

by glomeruli so that the glomerular filtration rate (GFR) can be calculated

from the serum concentration of cystatin-C. Cystatin-C can thus be used

as a marker for kidney disease. Traditionally, kidney function was assessed

by serum creatinine concentrations. However, various studies have

concluded that serum cystatin-C shows higher sensitivity and specificity

than serum creatinine (Onopiuk, Tokarzewicz et al. 2015) and that more

accurate data can be obtained with both creatinine and cystatin-C than

with creatinine alone (Stevens, Coresh et al. 2008). However, when

compared to creatinine, the cost of cystatin analysis is three times higher

than the enzyme creatinine test and 20 times more than the creatinine

test using the jaffe reaction (Shlipak, Mattes et al. 2013). Since the main

cost is reagents, cystatin analysis will be more common if the reagent

price can be reduced. This study is expected to contribute to the

reduction in cystatin assay reagent prices.

With the advancement of gene editing techniques into zinc-finger

nuclease, TALEN and CRISPR-Cas9, it is easier than ever to modify, add

or delete the genes we want. Methods of making transgenic animals have

also evolved to make faster and cheaper than before, which is an

50

economic advantage when using transgenic animals as bioreactors. Not

many transgenic animals will grow rapidly, be easy to manage and

produce protein continuously. Through this study, we believe that chicken

is the most suitable animal for these conditions. In addition, the transgenic

chicken production technique used in this study could be of great

commercial value in the recombinant protein market if it is incorporated

into other useful proteins.

51

Conclusion

In this study, the results can be summarized in two ways. First,

transgenic chicken production using a non-viral piggyBac transposon

system. For existing commercialized transgenic chickens, transgenic

chickens were produced using a lentiviral vector system. The way in

which viral vectors are used can pose safety concerns. In this study, we

successfully produced transgenic chickens using the non-viral piggyBac

transposon system, which we believe will help in the production of other

useful recombinant drugs.

Second, presenting a new method for the production of cystatin-C.

Cystatin-C is well known as a marker for kidney disease and is being

actively researched as a therapeutic agent for neurological diseases.

Human cystatin-C, which is on sale, is produced from animal cells and

costs about 970,000 won per 10μg. The use of transgenic chickens

continues to cost less than traditional animal cell-based production systems

for the production of recombinant pharmaceuticals. Therefore, the

production of human cystatin-C from transgenic chickens could lower the

production cost and contribute to the study of the potential of human

cystatin-C as a therapeutic agent.

52

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Abstract in Korean

형질 전환 닭에서 생산하는

생기능성 인간 시스타틴 C

김 서 우

국제농업기술학과 경제동물산업기술전공

서울대학교 국제농업기술대학원

생물 반응기는 생화학 반응을 유용 물질의 생산, 환경오염 물질의 분해,

분석, 의료 등에 응용하는 반응장치를 말한다. 미생물 및 동물 세포를

이용하는 생물 반응기도 있지만, 생기능성 단백질의 효율적인 생산을 위

해서는 효율적인 전략이 필요하다. 미생물에서는 치료 단백질 생산 시

글리코실화(포도당화)와 같은 번역 후 변형과정이 제대로 이루어지지 않

는다. 또한 동물 세포 시스템은 많은 비용과 시간이 필요하다. 이러한

문제를 해결하기 위해 형질 전환 암탉의 계란을 이용하는 새로운 방법을

고려하였다. 인간 시스타틴-C는 시스타틴 수퍼 패밀리의 구성원이다. 시

스타틴-C는 체액과 분비물에서 발견할 수 있고 일정한 속도로 체내에서

생성한다. 또한 이 단백질은 시스테인 프로테아제의 억제제로서 작용하

고 미생물 및 기생 침투로부터 몸을 보호한다. 우리는 비바이러스성

piggyBac 트랜스포손(전이인자) 시스템을 사용하여 인간 시스타틴-C 단

백질을 발현하는 형질 전환 닭을 생성했다. 우리는 체외 분석을 통해 형

64

질 전환 닭에서의 인간 시스타틴-C의 발현과 생기능성 활동을 확인했다.

따라서, 이 데이터는 형질 전환 닭이 다양한 생물 활성 물질 생산을 위

한 생물 반응기로서 효율적으로 사용될 수 있음을 시사한다

키워드: 형질 전환 닭, 생물 반응기, 인간 시스타틴-C