转sMTPGH基因小鼠胚胎的体外培养

In Vitro Culture of Mouse Embryos after Pronuclear Microinjection with sMTPGH Fusin Gene

采用纯化的羊金属硫蛋白 (s MT)启动子指导的猪生长激素 (PGH )基因 (s MTPGH)为显微注射片段 ,将其导入小鼠受精卵原核 ,比较了几种不同培养液对导入外源基因的小鼠胚胎体外发育的影响。结果表明 ,改进的 M16培养液(用 m M16表示 )能有效克服 2 -细胞阻断现象 ,并使转基因胚胎发育到桑椹胚或囊胚阶段。在 m M16培养液的基础上 ,再加入表皮生长因子 (EGF) ,可使 1-细胞胚胎发育到囊胚的比率进一步提高。在 3种培养液 (M16、m M16、m M16 +EGF)中 ,转基因胚胎突破 2 -细胞阻断期的比率分别为 12 %、6 8%、90 % ,发育到囊胚期的比率分别是 0 %、2 0 %、4 3%。采用 PCR方法检测 5 7枚经体外培养发育至囊胚的显微注射胚胎 ,4枚扩增出阳性条带 ,外源基因在囊胚期胚胎的滞留率为 7%。

The sheep metallothionein (sMT) pig growth hormone (PGH) gene was constructed, purified and microinjected into the pronucleus of fertilized mouse′s eggs. Then, the development capability of these transgenic embryos cultured in three different media was compared. In the experiment, the fertilized eggs overcame the 2 cell block and developed to blastocyst stage when cultured in the modified M16(mM16) medium. In addition, the mM16 that complemented with epidermal growth factor (EGF) further improved the blastocyst rates. The results showed that 12%, 68% and 90% of the microinjected embryos passed the 2 cell block and developed to 4 cell stage, and 0%, 20% and 43% of them developed to blastocyst stage in M16, mM16 and mM16+EGF media , respectively.Taurine may play an important role in overcoming the 2 cell block during embryo development.EGF is supposed to be involved in the elimination of the toxins came from the embryos or the media. The retained foreign gene was monitored by PCR in 57 blastocysts, 4 blastocysts were proved to be positive.The retention rate of foreign gene integrated in blastocyst was 7%. This method might provide a way to screen transgenic embryos in establishing transgenic farm animals.