单胚胎RT-qPCR对猪体细胞核移植囊胚全能性的分析

为了检测猪体细胞核移植囊胚的质量和全能性基因表达量,采用体细胞核移植技术制备克隆胚胎并于体外培养5 d后获得囊胚,对照组为从人工授精5 d后的长白母猪体内获取的体内囊胚。在高倍镜下检测两组囊胚的形态,用Hoechest 33342染色细胞核DNA,记录囊胚细胞总数;建立单胚胎cDNA的制备方法,并用qPCR检测囊胚中全能性基因(Oct4、Nanog、Sox2)的表达量。结果表明,与体内囊胚相比,猪体细胞核移植囊胚质量较差,细胞数目显著降低(分别为110±10.3和54±12.6);并且全能性基因表达量显著下降(P<0.05)。由此可见,全能性基因表达量偏低是影响猪体细胞核移植囊胚发育能力的因素之一。

The analysis of totipotency in pig somatic cell nuclear transfer blastocysts using single-embryo RT-qPRC

In order to investigate the quality and expression level of pluripotency genes of somatic cell nuclear transfer blastocysts in pigs,the reconstructed embryos were produced by somatic cell nuclear transfer and blastocysts were harvested after cultured in vitro for 5 days.The control group was in vivo blastocysts obtained from Landrace sows after being artificial inseminated for 5 days.The morphology of blastocysts in the two groups was investigated using high power microscope,and the total number of cells in blastocysts was counted through Hoechest 33342 staining.cDNA from single embryo was generated and the expression level of pluripotency genes(Oct4,Nanog,Sox2)was detected by qPCR.The results showed that the quality of somatic cell nuclear transfer blastocyst was poor,the number of cells(110 ±10.3 and 54 ±12.6,respectively) and the expression level of pluripotency genes were also decreased significantly(P < 0.05),compared with in vivo blastocysts.It was concluded that the lower expression level of pluripotency genes affected the developmental competence of somatic cell nuclear transfer blastocysts.