In this study, to further understand the mechanism of animal growth and to develop a miniature transgenic animal model, we constructed and tested tetracycline-inducible RNAi system using shRNA targeting the mRNA of mouse insulin-like growth factor (mIGF-1) or mouse growth hormone receptor (mGHR) gene. Quantitative real-time PCR analysis of mouse liver cell (Hepa1c1c7) cells transfected with these vectors showed 85% or 90% of expression inhi-bition effect of IGF-1 or GHR, respectively. In ELISA analysis, the protein level of IGF-1 in the cells expressing the shRNA targeting IGF-1 mRNA was reduced to 26% of non-transformed control cells. Unexpectedly, in case of using shRNA targeting GHR, the IGF-1 protein level was decreased to 75% of control cells. Further experiments are needed to explain the lower interference effect of GHR shRNA in IGF-1 protein. Accumulated knowledge of this approach could be applicable to a variety of related biological area including gene function study, gene therapy, development of miniature animals, etc.

Until recently the most popular tetracycline-inducible gene expression system has been the one developed by Gossen and Bujard. In this study, we tested the latest version of same system and the results are summarized as follows: Compared with previous one, the difference of new system are minor changes of nucleotide sequences in transactivator and tetracycline response element (TRE) regions. Sensitivity to the doxycycline (a tetracycline derivative) was improved. Leakiness of GFP marker gene expression in non-inducible condition was significantly decreased. Higher expression of the marker gene was observed when the cells were fed with doxycycline- containing medium. Optimal insertion site of woodchuck posttranscriptional regulatory element (WPRE) sequence which was known to increase gene expression was different depending on the origin of cells. In chicken embryonic fibroblast, location of WPRE sequence at 3’ end of TRE resulted in the highest GFP expression. In bovine embryonic fibroblasts, 3’ end of transactivator was the best site for the GFP expression.

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 연구에서는 돼지의 체지방을 감소시키고 성장을 촉진시키는 인자인 PGH를 cloning하여 이 유전자를 외래 유전자의 발현이 유도적으로 조절되는 Tet system에 도입하고자 하였다. 또한 유전자의 발현이 turn on되었을 때 그 발현 정도를 최대화하기 위하여 WPRE 서열을 도입하였다. 구축된 각각의 vector는 retrovirus 생산 세포주에 도입하여 virus를 생산하였으며 이를 여러 종류의 표적세포에 감염시켜서 PGH 유전자의 발현을 확인한 결과, 1×10/sup 6/ 세포에서 350∼2,100 ng의 PGH가 분비되었으며 특히 PFF 세포에서 가장 높은 발현을 나타내었다. Tet system에 도입된 PGH의 발현이 유도적으로 조절되는지를 PFF 세포에서 확인한 결과, 유도 효율이 2∼6배로 나타났으며 WPRE 서열이 rtTA 유전자의 downstream에 위치한 조건에서 가장 높은 유도 효율을 나타내었다. 이러한 PGH 유전자의 유도적인 발현의 조절은 고급육 생산의 형질전환 돼지 연구에 있어서 가장 큰 문제점이 되는 PGH 유전자의 과다한 발현에 의한 생리적인 부작용을 최소화할 수 있는 해결 방안으로 제시될 수 있을 것이다.

모유에 존재하는 유지방구의 막을 구성하는 주된 당단백질인 하나인 lactadherin(과거에는 BA46로 일컬어짐)은 rotavirus에 의한 감염증상을 예방하는 것으로 보고되고 있다. 본 연구에서는 retrovirus vector system을 이용하여 Chinese Hamster Ovary (CHO) 세포에 tetracycline에 의해 발현이 제어되는 promoter 하의 lactadherin 유전자를 전이 시킨 후 lactadherin이 tetracycline에 의해 발현이 유도되는지의 여부를 실험하였다. 먼저 기초 실험으로 대장균의 LacZ 유전자를 이용하여 tetracycline에 의한 유도 여부를 시험하였다. RevTet-On과 RevTRE-LacZ retrovirus를 동시감염시킨 NIH3T3는 doxycycline (tetracycline 유도체)에 의해 투여량에 비례하여 반응정도가 증가하는 양상을 나타내었으며 최대의 반응은 doxycycline 농도가 1 ㎍/ml 이상에서부터 관찰되었다. 이 예비실험의 결과를 바탕으로 RevTet-On과 RevTRE-Ltd retrovirus vectyor를 이용하여 사람의 lactadherin 유전자의 유도적 발현을 검정하였는데 CHO 세포에서 lactadherin 유전자의 유도적 발현을 RT-PCR 기법을 이용하여 확인하였다. 표적세포 내에서 외부에서 도입된 유전자가 지속적으로 발현될 경우 심각한 생리적 부작용을 야기시킨다는 사실을 감안할 때 본 실험의 결과는 유전자 치료와 형질전환동물의 생산에 크게 도움이 될 것으로 예상된다.