下调组蛋白修饰H3K9me3的水平对猪克隆胚胎基因表达模式及发育效率的影响

The pig cloning technique has valuable applications in swine industry and biomedical field. However, currently cloned pig embryos only have a full-term developmental efficiency at about 1%. Such a low efficiency limits the application of pig cloning technique. Recent studies on mouse discovered that nuclear donor cells-derived histone modification H3K9me3 is a major barrier for cloned embryo development. Over-expression of H3K9me3 demethylases Kdm4a/b/d in cloned mouse embryos significantly activated expression of genes modified by H3K9me3 through down-regulation of H3K9me3 level, and significantly enhanced the full-term developmental rate of cloned mouse embryos by 8-16 folds. Recently, a study showed that cloned pig embryos have a significantly higher H3K9me3 level than fertilization-generated embryos, suggesting that H3K9me3 also is an epigenetic modification that impairs the developmental ability of cloned pig embryos. The purpose of this project is to investigate the effects of decrease of H3K9me3 level on gene expression patterns and developmental rate of cloned pig embryos by over-expression of Kdm4a/b/c/d in nuclear donor cells. The present study will determine the roles of H3K9me3 in regulation of gene expression patterns and developmental ability of cloned pig embryos, and develop a new method that can increase the success rate of pig cloning, therefore promote the research and applications of pig cloning technique.

猪克隆技术在养猪业及生物医药业都有重要应用价值，但目前猪克隆胚胎发育出生的效率只有约1%。这限制了猪克隆技术的应用。最近在小鼠的研究中发现，核供体细胞来源的组蛋白修饰H3K9me3是克隆胚胎发育的一种主要障碍，在克隆胚胎中过表达H3K9me3去甲基化酶Kdm4a/b/d可显著下调H3K9me3水平并激活其结合的基因表达，从而将克隆胚胎发育出生的效率提高8-16倍。最新的研究显示猪克隆胚胎的H3K9me3水平显著高于受精胚胎。这暗示H3K9me3也是抑制猪克隆胚胎发育的一种表观遗传修饰。本项目将通过在核供体细胞中过表达猪Kdm4a/b/c/d来下调猪克隆胚胎的H3K9me3水平，研究其对猪克隆胚胎基因表达及发育效率的影响。本项目将揭示H3K9me3对猪克隆胚胎发育的调控作用，并探索建立一种基于过表达H3K9me3去甲基化酶的可显著提高猪克隆技术效率的新方法，为促进该技术的研究及应用提供基础。

猪体细胞克隆技术可用于复制扩繁优秀种猪和制备各种用途的基因修饰猪，所以它在养猪业及生物医药业都有重要应用价值。但目前猪克隆胚胎发育出生的效率只有约1%。这限制了猪克隆技术的应用。最近在小鼠的研究中发现，通过过表达组蛋白H3K9me3去甲基化酶KDM4家族基因，敲除XIST基因或干扰其表达，以及用组蛋白去乙酰化酶抑制剂TSA处理等方式可矫正小鼠克隆胚胎的基因表达模式并将其发育出生的效率提高约6-10倍。本项目通过注射猪KDM4A家族基因表达载体，注射抗XIST的siRNA和shRNA表达载体，注射抗RLIM(XIST转录激活因子)的siRNA，注射抗组蛋白去乙酰化酶HDAC2和SIRT1的siRNA等方式矫正了猪克隆胚胎的基因表达模式并显著提高了其发育效率。本项目建立了多种可显著提高猪克隆胚胎发育效率的新方法，为促进猪克隆技术的应用和发展提供了基础。