Peg3, Cdkn1c和 Gtl2基因在克隆绵羊和自然分娩绵羊中的DNA甲基化水平检测

尽管研究证明很多克隆动物存在DNA甲基化异常的情况，却很少有研究比较克隆绵羊与自然分娩绵羊之间的甲基化情况，可能是由于克隆绵羊的获得、绵羊基因组、绵羊基因组印记等因素的限制．本研究中，为了证明克隆绵羊重编程的状况，克隆了Peg3基因的差异甲基化区域(differential methylated region，DMR)，并且分析了Peg3、Cdkn1c、Gtl2在克隆绵羊和自然分娩绵羊不同组织中的甲基化水平．研究发现，在克隆绵羊和自然分娩绵羊中Peg3呈现为超甲基化水平，在克隆绵羊的肾脏和肺脏中DNA甲基化水平为95.45%、81.18%，相对于正常分娩的绵羊组织中的98.18%、87.27%无显著性差异，而Cdkn1c在两组实验动物中的肾脏和肺脏中表现为非甲基化水平，分别为0%、0.53%、0.53%和0.53%，Gtl2则是低甲基化水平，并且克隆绵羊与正常分娩绵羊之间的DNA甲基化水平无显著性差异(r² = 0.77)．这些结果表明，Peg3、Cdkn1c、Gtl2三个印记基因在克隆绵羊和自然分娩绵羊组织中呈现类似甲基化水平，无显著性差异．

Discovery of DNA Methylation Status of Peg3, Cdkn1c and Gtl2 in Cloned and Natural Lambs

Current research has determined that many cloned animals have heterogeneous DNA methylation profiles. However, few studies have compared the methylation profiles of both naturally produced lambs and cloned lambs created using somatic cell nuclear transfer. The paucity of research in this area is because of insufficient resources to study limited cloned offspring, the ovine genome, and ovine genomic imprinting. In this study, to show the degree of reprogramming in cloned lambs, we cloned the putative differentially methylated regions (DMRs) of Peg3 from sheep and analyzed the DNA methylation patterns in CpG islands and DMRs of the putative imprinted genes Peg3, Cdkn1c and Gtl2 in cloned lambs. We have provided evidence that Peg3 was highly methylated. The degree of methylation was 95.45% in the kidney and 88.18% in the lung of a natural sheep and 98.18% in the kidney and 87.27% in the lung for one cloned sheep. The bisulphite sequencing results for Cdkn1c show complete non-methylation (0%, 0.53%, 0.53%, 0.53%) in all samples. In addition, Gtl2 was hypomethylated in all lambs, from a linear correlation analysis, there were some differences in the quantitative values from both groups (correlation r² = 0.77). These data show that the DNA methylation status of the three imprinted genes was similar in cloned and natural lambs.