睾丸内注射pEGFP-N1体内转染精子并在山羊早期胚胎成功表达

目的探讨睾丸内注射法pEGFP-N1在精细胞的整合和在早期胚胎中表达.方法选择4头本地山羊,双侧睾丸注射不同剂量质粒DNA pEGFP-N1,注射后PCR和Southern杂交检测pEGEP-N1在精子中的整合.结果pEGFP-N1整合到精子基因组中,转染效率最高发生在注射后第40天,转染阳性率最高为81%;绿色荧光蛋白在精子及其体外受精的部分胚胎中表达,胚胎阳性率最高的达66.7%.结论通过睾丸内注射pEGFP-N1能整合进入精子基因组,并能通过体外受精在山羊早期胚胎中表达;睾丸内注射法可能是一种可行、简单并利于推广的制备转基因山羊的方法.

Transfection of pEGFP - N1 Gene into Goat Spermatozoa in vivo and Its Expression in Early Embryos after Transfection

Objective Intratesticular injection of pEGFP-N 1 was used to investigate its integration in spermatozoa and expression in early embryos . Methods\ Four local male goats were selected and different doses of pEGFP N 1 plasmid DNA were injected into the bilateral testes. The integration of pEGFP N 1 in the sperms was determined by PCR and Southern blotting. Results pEGFP N 1 was integrated into spermatozoa, and the transfection rate peaked 81% at 40 d after intratesticular injection. Expression of green fluorescent protein was found in spermatozoa and early goat embryos with a maximum positive rate of 66 7%. Conclusion\ Intratesticularly injected pEGFP N 1 can be integrated into spermatozoa and then green fluorescent protein can be detected in early embryos produced by in vitro fertilization. Intratesticular injection may be a feasible and simple approach to produce transgenic goats.