Cloning of MT -hGH Gene-injected Rabbit Embryos by Nuclear Transplantation

사람성장호르몬 유전자주입 토끼수정란의 핵이식에 의한 복제

The present study was carried out to examine the efficiency of cloning of transgenic embryos by nuclear transfer(NT) using gene-injected rabbit embryos. The rabbit embryos at pronuclear stage were microinjected with methallothionein-human growth hormone(MT-hGH) gene and cultured to 8- and 16-cell in TCM-199 containing 10% FCS with a monolayer of rabbit oviductal epithelial cells in a 5% $CO_2$incubator. The recipient oocytes were collected from the oviducts 14~16 h after hCG injection. The oocytes were enucleated and activated with 5$\mu$M ionomycin and 2mM 6-dimethylaminopurine. Blastomeres form gene-injected embryos were transferred into the enucleated oocytes by micromanipulation. The nuclear transplant oocytes were electrofused and co-cultured with rabbit oviductal cells. Following 120 h of culture, blastocysts were prepared for gene analysis by polymerase chain reaction(PCR). In previous experiment, the rate of gene-positive embryos detected by the nested PCR analysis was significantly decreased while developing to blastocyst(25%)(Kang et al., 1998). The fusion rate of gene-injected blastomeres was significantly(P＜0.05) lower than non-injected blastomeres(66% vs 80%). However, the NT embryos that were derived from gene-injected donor embryos did not differ from control embryos in development to the blastocyst stage(39% vs 31%). Of the 43 NT blastocysts developed from the gene-injected donor embryos, twelve(28%) were positive for the injected DNA. The results indicate that NT with gene-injected embryos can be successfully used for cloning and multiplication of transgenic embryos, furthermore applicable to improvement of transgenic animal production.

토끼 수정란의 전핵에 MT-hGH 유전자를 주입하고 핵이식 기법으로 형질전환 복제수정란의 생산효율과 PCR 검색으로 복제수정란에서 유전자존재 여부를 조사한 바 다음과 같은 결론을 얻었다. 1. MT-hGH 유전자를 주입하여 8- 및 16- 세포기로 자란 수정란을 공핵란으로 사용하여 핵이식을 실시하였던 바, 세포융합률은 각각 60.0%, 62.8% 로 비슷하였으나 정상수정란을 공급핵으로 사용한 80.4% 보다 유의적으로 낮은 융합률을 보였다. 그러나 이들 복제수정란의 체외발달률은 처리군간에 유의적인 차이는 인정되지 않았다. 2. 유전자 주입 후 8- 및 16- 세포기로 자란 수정란의 할구를 이용하여 핵이식으로 복제하고 체외에서 배반포까지 자란 수정란을 PCR -screening으로 유전자를 검출한 결과, 각각 23% 와 33% 의 유전자 양성 수정란을 감별하였다.