体细胞核移植技术生产猪植酸酶转基因胚胎的研究

Study on the Production of Porcine Phytase Transgenic Embryos with Somatic Cell Nuclear Transfer

为获得具有植酸酶腮腺特异性表达的猪转基因克隆胚胎,本研究使用植酸酶腮腺特异性表达的DNA质粒(包含腮腺分泌蛋白(parotid secretary protein,PSP)启动子与终止子序列、Neo筛选基因、绿色荧光蛋白(EGFP)报告基因和高比活的植酸酶appA基因),采用脂质体转染和基因素418(G418)药物抗性筛选的方法获取稳转细胞系,并利用体细胞核移植技术获得植酸酶转基因胚胎。结果表明,本研究构建的DNA质粒可用于细胞筛选,且质粒越小,细胞的转染效率越高,14.89kb的YM6552仅获得了7.1%的转染率,EGFP质粒则获得了43.4%的转染效率。在单克隆形成上,较小的pYN3600也获得了更高的单克隆形成数(25个),其中表达EGFP的单克隆有14个,植酸酶PCR阳性集落有11个,高于YM6552的单克隆数(19、8和6)。转基因细胞构建重构胚胎后,所有的胚胎均能表达绿色荧光蛋白,虽其体外发育能力有所下降,但差异不显著(P>0.05)。综上所述,本研究所采用的植酸酶质粒、细胞筛选方法和核移植技术可生产植酸酶重构胚。

In order to get porcine phytase salivary glands specific express cloned embryos, this study used phytase specific expressed plasmid DNA （including the promotor and terminater sequences of parotid secretary protein （PSP） genev Neo selected gene and EGFP report gene and high enzymatic activity phytase gene （app A）） and transfected it into porcine fetal fibroblast cells with liposome. The stably transfected cell lines were screened by G418 selectionvand positive phytase transgenic embryos were constructured by somatic cell nuclear transfer. The results showed that the phytase plasmid of this experiment could be used to carryon the cell selection, and the more smaller of plasmid DNA size, the more higher transfected efficiency was got. The plasmid YM6552 with 14.89 kb size only got 7.1% transfected efficiency, while EGPF plasmid got 43. 4 %. In the formation of single cell colony, pYN3600 （more smaller DNA size） got 25 colony numbers, and 14 of them expressed green fluorescent protein, 11 of them were phytase PCR positive. They were much higher than those of YM6552 （19.8 and 6）. All the reconstructed transgenic embryoes from the positive cell lines were expressed green fluorescent protein. Though the in vitro development ability of transgenic cloned embryos were a little lower than that of control group, there were no significant differences between them （P〉 0. 05）. In conclusion, all the phytase plasmid, cell selection method and nuclear transfer techniques could be used to produce porcine phytase salivary glands specific express embryos.