印记基因XIST和H19 DNA甲基化水平与猪克隆胚胎发育效率关联分析

目的 体细胞核移植(Somatic cell nuclear transfer，SCNT)在农业、生物医学等领域应用广泛，但是克隆效率太低制约了该技术的应用和推广。本研究的目的在于探究印记基因XIST和H19 DNA甲基化水平与克隆效率的联系。方法 利用同一头猪源耳朵成纤维细胞培养获得14个细胞克隆团，分别作为供体细胞进行SCNT试验，统计比较以各克隆团为供体细胞生产克隆胚胎的囊胚率以及各囊胚的XIST和H19基因调控区的DNA甲基化水平，对XIST和H19基因差异甲基化区域DNA甲基化水平与克隆胚胎的囊胚率进行相关性分析。结果 以1号克隆团为供体细胞得到囊胚的XIST基因DNA甲基化水平最高且囊胚率最低，分别为65.04%、8.6%，以14号克隆团为供体细胞得到XIST基因囊胚的DNA甲基化水平最低但囊胚率最高，分别为16.68%、38.2%；相关性分析表明XIST基因的DNA甲基化水平与囊胚率之间存在高度负相关(|r|=0.8125>0.8)。H19基因A侧甲基化平均水平最高和最低分别为5.12%和0.61%，相关性分析表明H19基因A侧DNA甲基化水平与囊胚率间只有极弱的相关(|r|=0.1647<0.3)；H19基因G侧DNA甲基化水平最高和最低分别为90.92%和72.69%，相关性分析表明H19基因G侧DNA甲基化水平与囊胚率间只有低度相关(0.3<|r|=0.3098<0.5)。结论 XIST基因DNA甲基化水平越低，则克隆团囊胚率越高。研究结果对提高猪SCNT胚胎发育效率，改善现有的猪SCNT技术体系提供了基础。

Correlation between DNA methylation levels of imprinted genes XIST and H19 and developmental efficiency of porcine cloned embryo

Objective Somatic cell nuclear transfer (SCNT) is widely used in agriculture, biomedicine and other fields, but the low cloning efficiency restricts the application and promotion of this technology. The purpose of this study was to explore the relationship between the DNA methylation levels of imprinted genes (XIST, H19) and cloning efficiency.Method Fourteen cell clone groups were obtained by culturing the same pig-derived ear fibroblasts, and were used as the donor cells for SCNT experiments. We statistically compared the blastocyst rates and DNA methylation levels of XIST and H19 gene regulatory regions for each sac of cloned embryos produced with each clone group as donor cells. We analyzed the correlation between the DNA methylation levels of differentially methylated regions for XIST and H19 genes and the blastocyst rates of cloned embryos.Result The DNA methylation level of XIST gene of blastocysts obtained from clone group 1 as donor cells was the highest (65.04%) and the blastocyst rate was the lowest (8.6%). The DNA methylation level of XIST gene of blastocysts obtained from clone group 14 as donor cells was the lowest (16.68%) and the blastocyst rate was the highest (38.2%). The correlation analysis showed that there was a strong negative correlation between the XIST gene methylation level and the blastocyst rate (|r|=0.8125>0.8). The highest and lowest DNA methylation levels of H19 gene A side were 5.12% and 0.61% respectively. Correlation analysis showed that there was only a very weak correlation between the DNA methylation level of H19 gene A side and blastocyst rate (|r|=0.1647< 0.3). The highest and lowest DNA methylation levels on the G side of H19 gene were 90.92% and 72.69% respectively. Correlation analysis showed that there was only a low correlation between the DNA methylation level on the G side of H19 gene and the blastocyst rate (0.3<|r|=0.3098<0.5).Conclusion The lower the DNA methylation level of XIST gene, the higher the blastocyst rate of the clone group. The research results provide a basis for improving the efficiency of pig SCNT embryonic development and improving the existing pig SCNT technology system.