猪体细胞核移植胚胎发育过程中染色质开放与关键组蛋白修饰的动态变化及调控机制研究

猪体细胞核移植（克隆）效率低阻碍该技术在异种器官移植和人类疾病模型构建上的应用，在鼠等动物上的研究表明体细胞基因组上存在重编程障碍区域，我们前期试验证实在猪体细胞基因组上也存在特异的重编程障碍区域，但该区域的调控机制还不清楚，因此本研究拟开展：1）利用低量细胞DNase-seq和ChIP-seq技术研究猪早期克隆胚胎发育过程中的染色质开放性和关键组蛋白修饰的动态变化；2）通过比较猪体内受精和克隆胚胎的基因表达，确定猪体细胞基因组上重编程障碍区域及该区域上所富集的转录因子和关键组蛋白修饰；3）调控猪核移植胚胎或体细胞基因组上重编程障碍区域所富集的转录因子或表观遗传修饰来确定对重编程效率的影响；4）结合基因编辑技术研究该调控对基因敲除/敲入猪制备的影响。该项目的研究将为揭示猪体细胞核移植的重编程机制提供依据，同时可提高猪的克隆效率，促进克隆猪技术在生物医学领域上的应用。

The low efficiency of porcine somatic cell nuclear transfer (cloning) hinders the application of this technique in the construction of xenotransplantation and human disease models, and studies in mouse and other animals show that there are reprogramming resistant regions in the somatic genome, and our prelimary studies have confirmed that there are specific reprogramming resistant regions in the porcien somatic genome; however, the related regulation mechanism in these regions is not clear, so our study is proposed: 1) Using low input DNase-seq and ChIP-seq techniques to study the dynamic changes of chromatin accessibility and key histone modification during the development of early cloned embryos in pigs; 2) Comparing the gene expression of fertilization and cloned embryos in pigs and determining reprogramming resistant regions in porcine somatic genome and the transcription factors and key histone modification enriched in thess regions; 3) Regulating key transcription factors or epigenetic modifications enriched in the reprogramming resistant regions of cloned embryos or somatic cells to determine the effect on the efficiency of reprogramming; 4) Confirming the effect of this regulation on the producing gene knockout/tapping pigs by pig cloning and gene editing techniques.This project will provide a basis for revealing the reprogramming mechanism of porcine somatic cell nuclear transfer, improve the cloning efficiency of pigs and promote the application of cloned pig technology in biomedical fields.