Transgenic chickens have been spotlighted as an highly potent bioreactor for their fecundity, short generation time, and eggs associated with mass production of protein. In this study, we generated transgenic chickens exhibiting oviduct specific expression of human growth hormone fused to human transferrin for oral administration. Gene of the modified growth hormone located at downstream ovalbumin promoter (∼3.6 kb) was introduced to stage X blastodermal cell employing retrovirus vector system. Several transgenic chickens were successfully generated. However, genomic analyses showed unexpected deletion within the transgene. The modification of the transgene seemed to occur during germ cell formation because the deletion was detected only from the sperm DNA of the G0 founder animal. There was no evidence of deletion in the somatic cell DNA samples of the same chicken. Consequently, same pattern of the deletion was confirmed in both somatic and germ cells of the G1 progeny.

Chicken Insulin-like Growth Factor-1 (cIGF-1), one of the most important hormone for regulating physiological function includes body growth, muscle volume, bone density, chicken cell development and metabolism. In order to find in vitro Knokdown expression of cIGF-1, this study introduced tetracycline inducible RNA interference expression system (TetRNAi system). Tet system can inductively control high expression of extrinsic genes and expression of intrinsic genes. So it has advantages such as minimized physiological side-effects any cell and low cytotoxicity. RNAi system is proving to be a powerful experimental tool for inhibition of gene expression and post-transcriptional mechanism of gene silencing. RNAi is mediated by small interfering RNA (siRNA) consisting of 19- to 23- nucleotide double-stranded RNA duplexes that promote specific endonucleolytic cleavage of mRNA targets through an RNA-induced silencing. Then, this study RNAi-based gene knockdown can be achieved by retroviral-based expression systems. Stable integration of our inducible siRNA vector allowed the production of siRNA on doxycycline induction, followed by specific down regulation of chicken IGF-1 gene. Analyses of Real-time PCR to determine expression of the cIGF-1 gene showed successful from chicken embronic fibroblast (CEF) cells with the reduced rate of an approximately 92%. Our results demonstrate the successful regulation of cIGF-1 knockdown expression in CEF cells and support the application of an tetracycline inducible RNAi expression system in transgenic Mini chicken production. This research was supported by Bio-industry Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

본 연구는 vesicular stomatitis virus G glycoprotein (VSV-G)으로 피막이 형성되는 replication-defective MoMLV-based vector를 이용한 hTPO 헝질전환 닭의 생산에 관한 연구이다. 실험에 사용한 retrovirus vector의 구조는 hTPO 유전자의 발현 조절을 위해 internal promoter인 hCMV promoter를 이용하였으며 외래 유전자의 발현을 증가시키기 위해 woodchurk hepatitis virus posttranascriptional regulatory element (WPRE) 서열을 도입하였다. 재조합한 vector는 GP2 293 포장세포에 도입하여 virus를 생산하였으며 이 virus를 이용하여 감염시킨 여러 표적세포에서 hTPO의 발현과 생물학적 활성을 확인하였다. 재조합 hTPO의 생물학적 활성은 시판되고 있는 재조합 hTPO에 비해 우월한 것으로 확인되었다. hTPO 형질전환 닭의 생산을 위하여 1,000배 이상 고농도로 농축된 virus를 stage X 단계의 계란의 배반엽 층에 미세주입하여 대리난각 방법으로 배양하였다. 미세주입한 132개의 계란 중 21일 후에 11개의 계란에서 병아리가 부화하였으며 그중 4마리가 형질전환 개체로 확인되었다. 그러나 생산된 4마리 중 3마리가 부화 후 1개월 이내에 원인불명으로 사망하였다. 본 연구의 의의는 상업적 이용 가능성이 있는 생물학적 활성을 가진 사람의 cytokine 단백질의 대량 생산을 위한 생체 반응기로서의 형질전환 닭 개발의 시례를 제공하는데 있다.