【摘要】 采用牛体外成熟卵母细胞和冷冻解冻的精子为材料,以pEGFP-N1为模式基因,探讨DNA浓度、精子质膜破损方法、注射台温度对牛精子胞质内注射(ICSI)介导转基因效果的影响。结果表明:线性pEGFPN1质粒DNA浓度为5μg/mL、10μg/mL两组的早期胚胎发育率显著高于50μg/mL组(19.8%,16.7%VS 7.9%,p<0.05)。以冻融、TritonX100、超声波断尾三种方法破损精子质膜,冻融组的囊胚发育率(24.7%)最高,且冻融组的早期胚胎基因表达率极显著高于超声断尾组(41%VS 20.5%,p<0.01);当分别在25℃、38℃的注射台进行显微注射时,两组之间胚胎的囊胚率无显著差异(p>0.05),但二者之间胚胎的基因表达率差异显著(46.83%VS 28.57%,p<0.05)。以上结果表明:(1)牛精子转染外源GFP基因的浓度不宜过高,转染高浓度的DNA会影响胚胎发育;(2)精子质膜是阻碍外源DNA与牛精子相结合的主要因素,将精子冻融处理可有效破损其质膜,利于精子与外源DNA的结合,从而提高ICSI介导转基因效率;(3)25℃和38℃热台温度对牛ICSI胚胎的早期发育无影响,但25℃热台温度可提高牛ICSI介导转基因的效果。更多还原

【Abstract】 Cattle oocytes in vitro maturation(IVM) and sperm freezing and thawing were used as the materials, and pEGFP-N1 as a model gene to explore the effects of DNA concentrations, sperm plasma membrane damage method and injection temperature on the efficiency of the production of intracytoplasmic sperm injection(ICSI)-mediated cattle transgenic embryos. The results showed that: When linear pEGFP-N1 plasmid DNA concentration was 5 μg/mL and 10 μg/mL respectively, early embryonic development was significantly higher than that in 50 μg/mL group(19.8%, 16.7% vs. 7.9%, 1.4%, p<0.05). For sperm plasma membrane damage treatment by using freezing and thawing, TritonX100 and ultrasonic method, the blastocyst rate(24.7%) was the highest in freezing and thawing group, and the gene expression rate of early embryonic in freezing-thawing group was significantly higher than that in ultrasound tail group(41% vs.20.5%, p<0.01); when microinjection were at 25 degrees and 38 degrees, the blastocyst between the two groups showed no significant difference(p>0.05), but embryonic gene expression significantly different(46.83% vs. 28.57%, p<0.05). These results suggested that: Firstly, cattle sperm exogenous GFP gene transfection concentration could not be too high, and high concentration of DNA trasfection impeded embryonic development. Secondly, the sperm plasma membrane was the main factor that hindered the combination of exogenous DNA with cattle sperm, sperm freezing and thawing treatment could effectively damage the sperm plasma membrane, which was conducive to combine exogenous DNA with sperm, and therefore improve the transgenic efficiency. Thirdly, there was no effects of the microinjection platform temperatures(25 degrees and 38 degrees) on the early development of the cattle ICSI embryos, but there was higher transgenic efficiency in cattle when the platform temperature of microinjection was 25 degrees.更多还原