【摘要】 类转录激活因子效应物(Transcription activator-like effector,TALE)已经成为基因组编辑和基因转录调控的有效工具,在多个物种的基因组中实现特定位点的删除、插入和突变。针对绵羊成纤维细胞生长因子5(Fibroblast growth factor 5,FGF5)基因起始密码子ATG位点,设计构建TALENs(Transcription activator-like effector nucleases,TALENs)。通过比较分析正常培养和基因组编辑处理的绵羊成纤维细胞,电转TALENs组合,Surveyor突变检测,筛选获得1对有效的TALENs。有限稀释细胞传代培养,PCR扩增FGF5基因片段,经PAGE检测筛选发生突变的细胞,测序确认FGF5基因ATG位点产生缺失突变细胞。获得具有编辑活性的TALENs,为该基因的定点编辑奠定基础。Surveyor检测和测序结果表明,在绵羊FGF5基因ATG起始密码子上游104碱基位点,存在1个G/C单核苷酸多态。更多还原

【Abstract】 Transcriptional activator-like effector(TALE)technologies were established over the last decade as useful tools for genomic editing and transcriptional modulation.Moreover,TALE nucleases(TALENs)technology can be used to bring about targeted gene deletion,insertion and mutagenesis in a siries of species genome.TALENs were designed to target the ATG initiation codon site of ovine FGF5 gene.According to the results of comparing normal culture and genome editing ovine fibroblasts,the activities of TALEN pairs were tested and screened by Surveyor nuclease after electric transfection TALEN plasmid pairs into ovine fibroblasts.After ovine fibroblast clones were cultured by limiting dilution,the screening of mutant cells was identified by PAGE electrophoresis after PCR amplification.The deletion mutations were introduced into ovine FGF5 gene of fibroblasts and identified by DNA sequencing.Mutated cells missing the ATG initiation codon of ovine FGF5 were obtained by valid TALENs.The acquisition of a pair of valid TALENs provides the basis for targeted disruption ovine FGF5 gene.A point mutation was identified at 104 base site of the ATG initiation codon upstream of ovine FGF5 gene by Surveyor mutation detection and sequence analysis.更多还原