초록 ▼

○ 연구결과
본 연구에서는 Tet virus vector system과 Ov virus vector system을 구축하여각 system에서 생산한 고농도의 virus를 계란의배반엽층에 미세주입하여 대리난각 방법으로 형질전환 닭을 생산하였다. 그 결과, Tet-EPO와 Tet2-EPO 형질전환 닭을 5세대(G4 세대)까지 생산함으로써 계통을 확립하였다. 이 형질전환 닭은 hEPO 유전자의 유도적인 발현이 확인되었으며 당잔기 분석과 생물학적 활성을 검정을 실시하였다. 또한 LNOvEPOW 형질전환 닭도 생산하였으며 수컷 개체의

○ 연구결과
본 연구에서는 Tet virus vector system과 Ov virus vector system을 구축하여각 system에서 생산한 고농도의 virus를 계란의배반엽층에 미세주입하여 대리난각 방법으로 형질전환 닭을 생산하였다. 그 결과, Tet-EPO와 Tet2-EPO 형질전환 닭을 5세대(G4 세대)까지 생산함으로써 계통을 확립하였다. 이 형질전환 닭은 hEPO 유전자의 유도적인 발현이 확인되었으며 당잔기 분석과 생물학적 활성을 검정을 실시하였다. 또한 LNOvEPOW 형질전환 닭도 생산하였으며 수컷 개체의 sperm에서 외래 유전자의 도입을 확인하고 G1 세대 생산을 시도 중이다. 난관조직 특이적인 promoter인 ovalbumin promoter를 확보하였으며 외래 유전자에 chicken Lysozyme signal peptide (LysSP)를 tagging하여 난관상피세포에서의 효율적인 외래 단백질의 분비 체계를 구축하였다. 세포와 닭에서 발현된 재조합 hEPO의 분리 및 정제 방법의 확립을 위한 연구도 실시하였다. hEPO를 면역학적 방법으로 정제하기 위하여 단크론성 항체를 생산하는 hybirdoma cell line을 확보하고 이를 이용하여 대량의 항체를 생산하였으며, immunoaffinity chromatography를 포함하는 단백질 정제법을 확립하고자 하였다. 또한 hEPO의 정량을 위한 ELISA 방법도 개발하였다.

Abstract ▼

Ⅱ.Objective and necessity of the research and development
The biopharmaucetical industry is rapidly growing and the world market size expanded to more than 100 billion dollars in 2010. At present, most of currently available biopharmaceutics are produced from cultured animal cells such as Chinese

Ⅱ.Objective and necessity of the research and development
The biopharmaucetical industry is rapidly growing and the world market size expanded to more than 100 billion dollars in 2010. At present, most of currently available biopharmaceutics are produced from cultured animal cells such as Chinese Hamster Ovary cells. However, one of the big problems of this producing systems is high production cost due to the difficulty of purification of pharmaceutics from animal serum in culture medium. At present, as a promising solution for this problem, use of‘bioreactors’to address the growing demand for large quantities and increasing numbers of biopharmaceuticals is of prime strategic relevance to medical advancement. Originally, the mammary gland was considered a promising bioreactor, reflecting its large milk production capacity, and some pharmaceutical proteins were produced in the milk of several species of transgenic mammals. However, disadvantages of the mammary gland as a bioreactor include long generation times for domestic mammals and difficulties in purifying recombinant proteins due to the biochemical complexity of milk prote in and fat. Use of the hen egg can circumvent these problems and has additional advantages including shorter generation time, lower expense and fecundity. Most importantly, purification of recombinant protein is predicted to be much easier because egg white protein is less biochemically complex. Moreover, in comparison to mammals, the glycosylation patterns of some chicken proteins are reported to be more similar to those of humans.
Despite these advantages, production of transgenic avian species has been hampered by the unusual features of avian reproductive biology. The ovum is fertilized within one hour of ovulation, then surrounded by several grams of albumin and eggshell. Therefore, early embryonic development is initiated in the reproductive organ of the female,resulting in an embryo which,just after laying, consists of approximately 60,000 morphologically undifferentiated pluripotent cells.
In transgenic poultry production, the target cells for foreign gene transfer are:blastodermal cells of the newly laid egg; PGCs (primordial germ cells); newly fertilized eggs; embryonic stem cells;a nd sperm. Transferring foreign genes to target cells relies on two methods- retrovirus-mediated gene transfer and direct DNA transfection. Among the multiple possible permutations and combinations of target cell and gene transfer method, targeting blastodermal cells at stage X using retrovirus-mediated gene transfer is most commonly employed, reflecting its technical ease and effectiveness of gene transfer.
The main objectofthis study is generation of transgenic chickens expressing human erythropoietin. hEPO is a 30.4 kD glycoprotein with 165 amino acids with three N-linked (Asn²⁴,Asn³⁸,andAsn⁸³) and one O-linked (Ser¹²⁶)sugar moieties required for in vivo stability and biological function, respectively. The primary physiological function of this cytokine, which is produced mainly by the kidney, is to stimulate formation of red blood cells in the bone marrow by inducing the proliferation and maturation of progenitors. Therefore, hEPO has been a standard therapy for anemia caused by dysfunctions of the kidney. In addition, hEPO is the most widely used agent to treat non-renal associated anemia in cancer patients undergoing chemotherapy. The sale price of hEPO is 670 thousand dollars and the world market size ohas been reported as much as 9 billion dollars in 2010. Even in Korea alone,the domestic market size has been estimated to b emore than 15 billion won. Most importantly, the patent for the hEPO has expired in 2007. In conclusion, the significance of this main study is that it is the first successful report on the production of transgenic chicken sexpressing this human cytokine protein.
This study consists of two parts. In the main part, we attempted to generate transgenic chickens expressing the hEPO geneby employing two gene transfer systems: replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G). In second part, we tried to apply tetracycline-mediated inducible expression system and oviduct-specific expression system. Application of these two expression system was mainly to minimize physiological disturbances due to the constitutive over-expression of the exogenous genes. In conclusion, the results obtained in this study are summarized briefly as follows:
(1) In in vitro study, the hEPO gene under the controlof CMV promoter expressed asmuch as 37,000 IU/㎖ and biological activity of the product was much higher than commercially available counterpart.
(2) Generation of G0 transgenic chickens expressing hEPO cytokine protein using tetracycline-inducible promoter.
(3)Generation ofnon-chimeric G1 transgenic chickens bred from chimeric G0 transgenic chickens.
● It was confirmed that expression of the hEPO was tetracycline dependent.
● hEPO was detected not only in the blood but also in the egg white.
(4)Establishment of a new breed chickens producing hEPO transgenic chickens by successful generation of G2, G3, and G4 hEPO transgenic chickens.
(5)Generation of G0 transgenic chickens expressing the hEPO gene under the control o fovalbumin promoter.
The results obtained from this study demonstrate the possible use of chicken as bioreactor producing human cytokine proteins. Further development of this system must contribute to increase rural household income. In addition, our results also significantly provide basic scientific knowledge in avian reproductive physiology.