体细胞克隆牛Dlk1/Dio3印记区域ncRNA和miRNA重编程研究

In somatic cell nuclear transfer (SCNT), the incomplete epigenetic reprogramming of donor nuclei has been implicated as a primary reason for the low efficiency of SCNT and the development abnormality of cloned animals. Genomic imprinting refers to the differential expression of the two alleles that is dependent on their parental origin, which has allele-specific epigenetic modification. Many of symptoms defects observed in SCNT animal are similar to experimentally induced imprinting disruptions (biallelic expression of imprinted genes) in mice and naturally occurring imprinting diseases in humans.Genomic imprinting is a fine area to study epigenetic reprogramming of donor nuclei in SCNT. The Dlk1/Dio3 imprinted domain play an important role in embryo development and organogenesis.Activation of the imprinted Dlk1-Dio3 region correlates with pluripotency levels of mouse stem cells. In our previous studies,aberrant expression of Gtl2, severe loss of methylation of IG-DMR and hypermethylation of the Dlk1 5' promoter region has been found in organs of died cloned bovine, which indicatid the important role of normal reprogramming in DLK1/DIO3 imprinted domain in SCNT animal. The ncRNA gene and miRNA in DLK1/DIO3 imprinted domain in cattle and epigenetic reprogramming in cloned cattle of the domain remain an underdeveloped area of research, largely because of the lack of information on sequence and polymorphisms of Dlk1/Dio3 imprinted region. The aims of this study are to identify the useful SNP in bovine Dlk1/Dio3 imprinted region, to establish the expression patterns of ncRNA gene and miRNA of bovine Dlk1/Dio3 region and to analysis the epigenetic reprogramming status of ncRNA gene and miRNA in cloned cattle. These results may increase our understanding on nuclear reprogramming events, and may provide a new way for improving cloned efficiency.

供体核的不完全重编程导致发育重要基因异常表达是造成克隆效率低下和克隆动物发育异常的主要原因。印记基因异常表达的遗传病人和印记基因定点敲除的小鼠中，存在着和体细胞克隆动物类似的表型异常。Dlk1/Dio3印记区域在胚胎发育过程中起非常重要的作用，尤其是该区域母系表达的ncRNA基因和miRNA与干细胞发育直接相关。在前期工作中我们发现此区域的Gtl2基因、IG DMR区以及Dlk1启动子区在死亡克隆动物中重编程异常，说明Dlk1/Dio3印记区域的正常重编程在决定克隆动物的命运中起重要作用。然而由于缺少基因序列以及多态性信息，牛Dlk1/Dio3区域的ncRNA基因和miRNA还没有被系统研究。该项目拟在前期工作的基础上，建立正常牛中该区域ncRNA基因和miRNA时空表达规律，分析克隆牛中ncRNA基因和miRNA的重编程，寻找克隆牛发育异常的重要原因，为提高体细胞克隆的效率提供理论依据。

供体核的不完全重编程导致发育重要基因异常表达是造成克隆效率低下和克隆动物发育异常的主要原因。Dlk1-Dio3印记区域在胚胎发育过程中起非常重要的作用，尤其是该区域母系表达的 ncRNA 基因和 miRNA 与干细胞发育直接相关。本研究中，我们以牛的Dlk1-Dio3印记区域中的ncRNA和miRNA为研究对象，克隆得到了牛Dlk1-Dio3印记区域中位于IG-DMR、具有增强子特征的非编码RNA基因MICO1和MICO1OS；分析了MICO1和MICO1OS基因在自然繁殖牛和体细胞克隆牛中的印记状态，发现MICO1和MICO1OS在克隆牛多个组织中表达异常，并利用亚硫酸氢盐测序法对6个候选调控区的甲基化进行了分析；得到了牛Dlk1-Dio3印记区域中位于Meg8基因内元的3个长非编码RNA基因，分析了其在自然繁殖牛和体细胞克隆牛中的印记状态；克隆得到了牛Dlk1-Dio3印记区域中位于Meg8基因和Meg9基因之间的3个长非编码RNA基因，分析了其在自然繁殖牛和体细胞克隆牛中的印记状态；对Dlk1-Dio3印记区域8个miRNA在自然繁殖牛和体细胞克隆牛中的印记状态进行了分析。以上研究结果将为揭示牛Dlk1-Dio3印记区域在体细胞克隆牛中表观遗传重编程机制，寻找克隆牛发育异常的重要原因，为提高体细胞克隆效率提供新的思路。在本项目的资助下，我们发表论文17篇，其中9篇被SCI收录，培养硕士研究生5名。