兔早期胚胎PCR性别鉴定体系的建立

根据兔SRY基因序列设计两对引物作为兔雄性特异性引物,根据兔APP基因序列设计1对引物作为内标引物,分别建立了兔早期胚胎性别鉴定的双重PCR和巢式PCR反应体系,在不同浓度的基因组DNA和兔早期胚胎上进行性别鉴定应用,同时,对兔SRY巢式PCR引物特异性进行了分析。结果表明,多重PCR扩增兔基因组DNA可以准确判定其性别,扩增灵敏度为100pg基因组DNA;多重PCR鉴定24枚兔32细胞桑椹胚性别,只能对整胚成功鉴定。巢式PCR,公兔基因组DNA扩增出282bp的SRY基因片段,母兔没有扩增产物,扩增灵敏度为10pg;对24枚兔32细胞桑椹胚性别鉴定结果表明,巢式PCR可以对少至4个胚胎细胞进行准确鉴定,同一胚胎结果符合率为100%(24/24)。SRY引物只对兔雄性基因组DNA特异,而其他动物(人、牛、绵羊、小鼠)雄性DNA及兔的冲卵液,均无PCR产物。

Establishment of PCR sex identification system of rabbit early embryos

In this study, two pairs of primers were designed according to SRY gene of rabbit as male specific primers, and a pairs of primers were designed according to the APP gene as control primers. Multiplex PCR and nest PCR system were established, chosen and optimized to sex rabbit embryos. Then, the specific of the SRY primers were detected.The genomic DNA from blood of 24 male rabbits and 9 female rabbits were used to multiplex PCR and nest PCR. The result shows that the sensitivity of the multiplex PCR is 100pg genomic DNA. The products of nest PCR were detected using electrophoresis, the result shows that the sensitivity is 10pg genomic DNA. The sensitivity of multiplex PCR is obviously lower than nest PCR, but the accuracy of two kinds of PCR are both 100%. The result of sexing 46 embryos shows that the multiplex PCR can sex embryos within at least 32 cells. The nest PCR can sex the embryos about 4 cells and the accuracy was 100%. The primers of rabbit SRY gene designed in this study are very specific in embryo sexing.