猪孤雌发育中印迹基因H19/IGF2表达和甲基化模式的分析

Parthenogenesis is a form of reproduction without being fertilized by a spermatozoon. It is a process that an oocyte is able to develop into an embryo alone. Imprinted genes of H19/IGF2 play an important role for parthenogenetic embryo development. The previously studies of our group have demonstrated that the abnormal expression and methylation status of imprinted genes may be responsible for developmental failure in porcine parthenogenesis fetus. In the present study, parthenogenetic fetal fibroblasts were treated by small molecule drugs, such as testosterone. The results suggested that the expression and methylation patterns of H19 were altered after testosterone treated. To confirm the effect of H19/IGF2 expression, DNMTs and histone acetylation were detected. In addition, the system of CRISPR/Cas9 were used to deletion in H19 DMR for down-regulated the expression of H19 in PA cells. Here, to verify if altered expression and methylation patterns of imprinted gene H19/IGF2 was enhance to parthenogenetic development, the PA cells which was treated by small molecule drugs as nuclear donors using somatic cell nuclear transfer and PA reconstructed embryos which using CRISPR/Cas9 were transferred to recipient sows. The purpose of this study is to explicit the expression and methylation status of H19/IGF2 in parthenogenetic development, and provides useful information for future study of birthing the full-term development parthenote pig.

孤雌生殖是指不需要精子，仅靠卵子自身就可以发育成为个体。印迹基因H19/IGF2对于孤雌发育具有重要的调控作用。课题组之前的结果证明孤雌胎儿发育失败与印迹基因异常表达和甲基化模式有关。本项目采用睾丸酮等小分子药物对孤雌细胞进行了处理，前期结果证明其可以改变H19的表达以及甲基化状态。同时检测甲基转移酶DNMTs和组蛋白乙酰化对H19/IGF2表达的影响。拟采用CRISPR/Cas9系统对H19的DMR区域进行了靶向敲除，抑制H19在孤雌细胞中的表达，探索其调控机制。通过体细胞核移植技术和胚胎移植技术，将小分子药物处理后的孤雌体细胞核供体和CRISPR/Cas9介导的重组胚胎分别移植入代孕母猪体内，验证印迹基因H19/IGF2的表达和甲基化状态的改变是否可以延长孤雌发育。本研究将明确印迹基因H19/IGF2的表达和甲基化状态在猪孤雌发育中的作用，为构建发育到期的孤雌猪提供理论依据。

孤雌生殖是指不需要精子，仅靠卵子自身就可以发育成为个体。印迹基因H19/IGF2对于孤雌发育具有重要的调控作用。本课题在孤雌细胞中分析印迹基因H19/IGF2的表达和甲基化模式，结果显示，H19/IGF2存在异常的表达和甲基化状态。进一步验证印迹基因NNAT，NAP1L5和MKRN3在孤雌胎儿中的表达和甲基化模式，结果显示，这些印迹基因存在紊乱的表达和甲基化模式。本课题还利用孤雌胎儿胎盘，分析了FLT1的甲基化模式。结果显示，孤雌胎儿胎盘发育失败与FLT1甲基化状态异常有关。采用维生素C（Vc）和二甲基乙二酰氨基乙酸（DMOG）对孤雌胚胎进行了处理，结果显示，Vc提高了小鼠孤雌囊胚率；而DMOG抑制了小鼠孤雌囊胚率。而且，验证了DNA甲基化关键酶TETs蛋白在孤雌胚胎发育过程中的表达模式。同时，本课题分析了N6-腺苷酸甲基化（m6A）在孤雌胚胎发育过程中的表达水平。结果显示，孤雌胚胎发育中存在异常的m6A修饰。本课题通过分析孤雌发育过程中印迹基因的表达和甲基化模式，确认了其在孤雌发育过程中的重要作用，为构建发育到期的孤雌猪提供理论依据。