摘要
研究外源DNA转染对牦牛精子体外受精(IVF)、早期胚胎发育以及绿色荧光蛋白(GFP)基因在早期胚胎表达的影响。用构建融合人乳铁蛋白基因绿色荧光蛋白表达载体(pAcGFP1-hLF)转染的牦牛精子IVF黄牛卵母细胞,生产的胚胎在荧光显微镜下观察GFP表达情况。结果显示,用环形质粒、线性质粒pAcGFP1-hLF转染的精子及未转染精子IVF后的卵裂率分别为56.4%、54.8%和61.0%,线性转染的囊胚率显著低于环形转染(17.7%与35.7%)。环形质粒转染的精子用DNaseⅠ消化、不消化及未转染的精子IVF后卵裂率分别为44.8%、55.2%和56.3%,囊胚率分别为33.3%、33.3%和30.2%。未经DNaseⅠ消化洗涤的环形质粒组GFP胚胎阳性率显著高于线性质粒组(99.3%与76.6%);DNaseⅠ消化组GFP胚胎阳性率极显著低于不消化组(25.4%与99.3%)。结果表明,用环形质粒DNA转染牦牛精子不会影响其受精及早期胚胎发育能力,但DNaseⅠ消化洗涤显著降低GFP胚胎阳性率。
The objective of this study was to evaluate the effect of yak sperm-mediated exogenous DNA on yak sperm fertilizability,and subsequent embryonic development and expression of green fluorescent protein（GFP） in yak hybrid embryos.Yak sperms were transfected with plasmid pAcGFP1-hLF,and then fertilized bovine oocytes in vitro,which were observed under a fluorescent microscope.The results showed that cleavage rates for bovine oocytes inseminated with yak sperms transfected with circular plasmid,linearized plasmid pAcGFP1-hLF and non-transfected were 56.4%,54.8% and 61.0%,respectively.The subsequent blastocyst rate was significantly lower（P〈0.05） for those transfected with linearized plasmids than circular plasmids（17.7% vs.35.7%）.The cleavage rates for those inseminated with the cirlular plasmid transfected sperms after digested with DNaseⅠ,without digestion and control were 44.8%,55.2 %and 56.3%,respectively.The subsequent blastocyst rates were 33.3%,33.3% and 30.2%,respectively.The percentage of GFP positive embryos was greater（P〈0.05） for those produced from circular plasmids than linearized plasmids transfection without DNaseⅠdigestion（99.3% vs.76.6%）,but the percentage of GFP positive embryos was lower after digested with DNaseⅠcompared with non-digestion（25.4% vs.99.3%）.It was concluded that yak sperms transfected with circular plasmid DNA does not reduce their fertilizability and subsequent embryonic development,but DNaseⅠdigestion of the transfected sperms can significantly decrease GFP expression in the subsequent embryos.
出处
《生物技术通报》
CAS
CSCD
北大核心
2010年第8期168-173,共6页
Biotechnology Bulletin
基金
四川省学术和技术带头人培养基金资助项目
关键词
牦牛
精子介导基因转移
胚胎体外生产
绿色荧光蛋白
Yak Sperm-mediated gene transfer In vitro embryo production Green fluorescent protein
