克隆牛重编程中核质互作分子机理的探讨

Domesticated animals cloned by somatic cell nuclear transfer (SCNT) generally have poor developmental competency, with many abnormalities occurring in the course of development which may due to incomplete reprogramming of the nuclear genome and abnormal expression of genes important for the regulation of growth and development. The interaction between the karyoplast and cytoplast plays an important role in the efficiency of SCNT, but the underlying mechanism remains unclear.In cattle, oocytes with various mitochondrial DNA (mtDNA) haplotypes usually have different ATP content and can further affect the efficiency of in vitro production of embryos. As mitochondrial DNA (mtDNA) comes nearly exclusively from the recipient oocyte during SCNT and is regulated by genes in the donor nucleus, it is a perfect model to investigate the interaction between donor nuclei and host oocytes in SCNT. We investigated whether the reprogramming and in vitro development potential of cloned bovine embryos produced by SCNT would be influenced by mtDNA haplotype compatibility between the oocytes and donor cells. Embryos from homotype A-A or B-B showed significantly higher developmental ability at blastocyst stages than the heterotype A-B or B-A combinations. Post-implantation development ability were also higher in the homotype SCNT groups than in the heterotype groups. Moreover, the autologous cloned embryos, for which the donor cells and oocytes come from the same individual have even higher developmental potential. To investigate the molecular mechanism, we established a method of extraction and amplification of trace amount of RNA from single cloned blastocyst, followed by microarray analysis. The gene expression patterns of the various types of reconstructed embryos and differentially expressed candidate genes could be screened for the researches on karyoplast and cytoplast interaction and reprogramming during the pre-impalantation developmental stage of cloned embryos. Further, the specific siRNA for these candidate would be designed and screened for next knockdown experiment in mice embryos. Bioinformatics analysis and network constrcuction of the expression profile data may elucidate the potential functional role of the differentially expressed genes in karyoplast and cytoplast interaction and reprogramming in SCNT embryos.

动物克隆中的核-质相互作用对体细胞重编程起决定性作用，但具体机制不明。我们将从卵母细胞、供体细胞和核质互作多方面考察重编程的分子机理。本课题以活体取卵获得的遗传背景清晰且经线粒体DNA分型的牛卵母细胞为核受体进行核移植，以克隆效率有差异的同体、线粒体同型异体和异型克隆胚胎为模型，体外受精胚作为对照，研究不同核-质组合克隆胚胎的重编程情况。结合已建立的单胚胎极微量RNA（pg级）抽提技术和RNA线性扩增技术，采用牛基因组表达谱芯片研究同体、同型和异型克隆胚胎表达模式，通过生物信息学手段筛选差异表达基因，重点分析在重编程和核质互作中起重要作用的基因，设计特异的siRNA，在胚胎水平验证差异基因功能，揭示重编程的分子机制，构建克隆动物着床前胚胎发育过程中核质互作和表观遗传调控网络图，阐述两者之间的关系。

为了阐明克隆重编程中重编程的关键分子机制，本课题以活体取卵(OPU)获得的遗传背景清晰的牛卵母细胞，利用PCR-RFLP技术对线粒体DNA进行分型，并与不同线粒体分型的体细胞组合进行核移植，在此基础上开展以下研究：比较不同线粒体单倍型卵母细胞与供核细胞不同组合在着床前和着床后的克隆效率；对发育过程中的关键因子如Oct4、Nanog和Sox2等在同型和异型克隆胚胎中的DNA甲基化状态进行分析。为了阐明核移植的分子机制，本课题进一步建立了单胚胎pg级RNA分析技术平台，建立同体、同型和异型克隆不同时期的胚胎cDNA文库，并利用包含23,000多个牛转录本的Affymetrix芯片对单胚胎的基因表达谱进行分析，通过生物信息学分析得到了基因表达谱聚类结果，初步筛选出4个差异表达基因，并利用设计其RNAi序列，利用小鼠胚胎进行干扰实验，初步验证候选基因的功能，系统地探讨表观遗传修饰对克隆胚胎发育的作用机理。胚胎干细胞来自囊胚的内细胞团，早期胚胎发育的研究也将为胚胎干细胞的分子机制提供理论基础。