Oct4基因在绵羊早期胚胎表达模型的建立及相关作用机制分析

Oct4 gene plays an important role in the development of pre-implantation embryos. To the best of our knowledge, the heterogeneities of Oct4-expression in ovine pre-implantation embryos have not been clarified; the existence of Oct4-expression in ovine trophectoderm has not been decided yet. Firstly, In this study, we aim to establish ovine sOct4ΔPE-EGFP embryos by applying CRISPR editing to insert EGFP gene into the termination codon of ovine Oct4 gene. Secondly, by tracking the EGFP expression of the sOct4ΔPE-EGFP embryos, the expression pattern of Oct4 gene in the different blastomeres of ovine embryos will be illustrated. Thirdly, sOct4ΔPE-EGFP embryos will be co-cultured with small molecular inhibitor, which activate or inhibit Oct4 gene by regulating Wnt signaling to clarify the function of Oct4 on the formation of inner cell mass and trophectoderm. Moreover, the regulators, the co-operators and the target genes of Oct4 gene will be examined to clarify the involved molecular mechanism in pre-implantation ovine embryos.

Oct4基因在绵羊早期胚胎发育中起到了重要的调控作用。迄今为止，在绵羊早期胚胎不同卵裂球中Oct4基因表达的异质性和规律性还不清楚，对绵羊胚胎滋养层细胞中是否还存在Oct4基因表达尚有争议。首先，本项目拟利用CRISPR基因编辑技术和受精卵显微注射技术靶向绵羊胚胎Oct4终止密码子区域插入报告基因EGFP，构建绵羊sOct4ΔPE-EGFP胚胎模型。其次，应用sOct4ΔPE-EGFP胚胎模型，示踪分析Oct4基因在绵羊胚胎中的差异表达变化规律。第三，应用小分子抑制剂和绵羊胚胎共培养，通过调控Wnt信号通路激活或抑制Oct4基因的表达，分析其对内细胞团和滋养层细胞发育形成的影响。最后，通过分析Oct4基因的上游调控因子，协作因子和靶基因，阐明Oct4基因在绵羊早期胚胎发育中的作用机制。

Oct4基因和Cdx2基因是附植前胚胎发育的重要调控基因，并最终调控了胎儿和胎盘的发育，也在胚胎妊娠识别和附植时调控了干扰素（interferon tau， IFNT） 的表达。本项目研究结果表明：绵羊胚胎16细胞期胚胎开始表达Oct4 蛋白；绵羊囊胚阶段分化的滋养层细胞中仍表达多能基因OCT4。利用CRISPR/CAS9技术和受精卵显微注射技术构建了绵羊OCT4-EGFP报告基因胚胎和OCT4移码突变的基因编辑胚胎，结果表明：OCT4敲减显著影响了绵羊桑胚阶段的胚胎发育。利用Cas9蛋白，小分子化合物Scr7，ssDNA模板优化了绵羊精准编辑技术方法，提高了绵羊基因精准编辑的效率，OCT4移码突变的编辑效率达到51.4%。对OCT4基因编辑胚胎进行单胚胎转录组测序，结果表明：OCT4基因表达敲减后，影响绵羊胚胎早期三胚层相关功能基因的表达。

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