原核显微注射产生转基因绵羊胚胎

体外采集绵羊卵丘卵母细胞复合体,成熟培养24 h,经过体外受精培养17 h,比较高速离心对胚胎发育的影响;高速离心可以使黑色脂滴甩到一边从而使受精卵原核清晰可见.然后将绵羊乳腺特异表达人肝细胞再生增强因子和真核细胞表达增强绿色荧光蛋白(Enhanced Green fluorescence protein,EGFP)的载体DNA显微注射于绵羊受精卵雄原核中,并将异构胚在SOF液中发育培养.结果表明:高速离心组囊胚率低于对照组,但是没有显著性差异(P》0.05);显微注射外源基因2天后在激光共聚焦显微镜下可见荧光胚胎;PCR检测5个荧光胚胎均可见特异性条带.在原核显微注射生产转基因胚胎中,绿色荧光蛋白可作为标记基因进行早期胚胎筛选,为提高转基因动物移植效率奠定实验基础.

Transgic Ovine Embryos Production by Means of Pronuclear Microinjection

Ovine cumulus-oocyte complex was matured for 24 hours and fertilised for 17 hours in the vitro culture.Blastocysts rate was compared between ultracentrifugation and the control groups.By applying the method of ultracentrifugation could move the opaque lipid droplets to the edge of the zygotes so that their pronuclears were clear visualization.The breast specific expression of ALR and the non-specific expression of EGFP gene vector DNA were injected into the pronuclear of the fertilized eggs.Blastocysts rate of control were higher than ultracentrifugation group,but no significant difference(P>0.05).After microinjection for 2 days the transgenic zygotes expressed green fluorescence.The five positive zygotes were tested by PCR.GFP can be used as a genetic marker to screen transgenic embryos.The establishment of transgenic embryo screening methods using GFP as genetic marker will greatly increase efficiency of gene transfection and the production of transgenic sheep.