调控DNA修复途径影响绵羊胚胎成纤维细胞中基因打靶频率的研究

In many mammalian cell types, non-homologous end-joining (NHEJ), homologous recombination (HR), and microhomology mediated end-joining (MMEJ) are the three main pathways involved in the repair of genomic double strand breaks (DSBs). NHEJ events far outnumber those that depend on MMEJ or HR. Repair via NHEJ pathway is a competing reaction for target integration of a donor construct, eventually leading to mutation of the engineered endonuclease target site without integration of the donor construct. .DNA Ligase 4 (Lig4) is responsible for sealing of double-strand breaks (DSBs) during NHEJ. In ovine embryo fibroblasts, the translation of the factor Lig4 is transiently depleted by siRNA, or Lig4-mediated joining by interfering with its DNA binding is blocked by 5,6-Bis(benzylideneamino)-2-mercaptopyrimidin-4-ol (SCR7) which is a new anti cancer molecule. In order to establish effective programs to selectively inhibit NHEJ inside the ovine embryo fibroblasts, an extrachromosomal assay system is used to evaluate the effect of siRNA or SCR7 on NHEJ. Transcription activator-like effector nucleases (TALENs) are used to target and break the β-casein gene in the ovine genome using ovine embryo fibroblasts. Meanwhile, NHEJ are inhibited by effective siRNA or SCR7. Transient depletion of the lig4 gene expression via RNA interference or blocking of Lig4-DNA binding function via SCR7 prior to induction of the DNA break substantially increase the proportion of cells harbouring the desired integrations, such as reporter gene or chicken infectious bursal disease virus VP2 antigen gene. The frequencies of targeted integration are identified by PCR. Exogenous gene expression results are determined by reverse transcription PCR and Western after transcription activator-like effector transcription factors (TALE-TFs) activate β- casein gene promoter and initiate the transcription of precise integration gene . The research will provide the basis for constructing sheep mammary gland bioreactor research.

在多数哺乳动物细胞中，非同源末端连接（NHEJ）修复基因组双链断裂（DSBs）的频率远高于微同源末端连接（MMEJ）和同源重组（HR）的频率。NHEJ途径造成人工核酸酶作用靶点产生突变，竞争抑制基因打靶事件。.NHEJ中的DNA连接酶4（Lig4）负责密封DSBs。在绵羊胚胎成纤维细胞中，用siRNA瞬间抑制Lig4的mRNA翻译或SCR7阻碍Lig4蛋白因子与DNA结合，应用染色体外检测系统，确定有效抑制NHEJ的方案。应用类转录激活因子效应物核酸酶靶向断裂细胞基因组的β-酪蛋白基因位点，抑制NHEJ，促使细胞采用MMEJ或HR修复DSBs，提高报告基因或鸡法氏囊病毒VP2抗原基因的精确整合效率，PCR检测基因打靶效率。人工转录因子（TALE-TF）激活β-酪蛋白基因启动子，反转录PCR和Western确定精确整合的外源基因表达结果，为构建绵羊乳腺生物反应器的研究提供基础。

哺乳动物细胞中，非同源末端连接（NHEJ）和同源重组（HR）是修复基因组双链断裂（DSBs）的两个主要途径。Lig4基因是NHEJ途径修复双链断裂末端的重要因子。. PCR扩增获得绵羊Lig4基因的CDS序列（KT180155），确定了绵羊Lig4的mRNA和氨基酸序列。. 针对绵羊Lig4的mRNA序列，设计合成4对siRNA，筛选出有效抑制Lig4基因表达的2个siRNA。应用质粒重连法检测HR修复效率，HR质粒和siRNA共转染绵羊胚胎成纤维细胞，经72h培养及流式细胞仪检测，与对照组细胞比较，结果表明HR质粒重连效率提高3～4倍。. SCR7是Lig4的特异性抑制剂。绵羊成纤维细胞在375μM的SCR7培养液中，出现细胞致死现象。SCR7达50μM，能阻碍绵羊胚胎成纤维细胞的NHEJ修复途径，提高HR修复效率。. 采用CRISPaint成功实现绵羊ACTG1基因羧基端标记，获得了单克隆细胞,为绵羊基因组定点导入外源大片段基因提供基础。. 为了获得突变或基因整合的单克隆细胞，利用分裂期细胞贴壁较弱，建立了人工手动筛选单克隆方法。. 调控DSBs途径的功能因子，对研究哺乳动物细胞DSBs修复机理具有重要意义。同时需要注意，原代哺乳动物细胞转染Cas9质粒效率仍然很低，提高转染物质导入细胞核的效率是哺乳动物细胞基因编辑的一大挑战。. 项目正在完善绵羊体细胞克隆程序，力争获得奶用绵羊乳腺生物反应器模型。