TALENs和CRISPR／Cas9诱导绵羊成纤维细胞基因组定点双链断裂提高Rad51基因表达量

The Improvement of Rad51 Expression by TALENs and CRISPR/Cas9 Induced Targeting Double-Srtand Breaks in Ovis Fibroblasts

为检测不同限制性人工核酸内切酶对绵羊成纤维细胞Rad51基因m RNA转录和蛋白表达的影响,本实验将有效切割MSTN(myostatin)基因位点的TALENs和CRISPR/Cas9人工核酸酶电转染到体外培养的绵羊成纤维细胞中诱导产生DNA双链定点断裂,与正常和电转染p Max绿色荧光蛋白报告基因质粒的绵羊成纤维细胞进行对照,采用实时荧光定量PCR和Western Blot方法对不同处理的绵羊成纤维细胞Rad51基因m RNA转录和蛋白质的表达情况进行研究。结果表明:TALENs和CRISPA/Cas9人工核酸酶诱导产生的MSTN基因组DNA双链定点断裂能提高绵羊成纤维细胞Rad51基因表达量。

To detect the effects of different kinds of artificial restriction enzymes on ovis fibroblast Rad51 mRNA transcription and Rad51 protein expression level.The TALENs or CRISPR/Cas9 artificial nuclease,which could cut MSTN （myostatin） gene,was transfected into ovis fibroblasts in vitro culture by electroporation,and the fixed-point double-stranded DNA rupture was induced. Normal and ovis fibroblasts transfected pMax GFP plasmid as positive control.Ovis fibrohlast Rad51 mRNA and protein expression was studied by real-time fluorescence quantitative PCR and Western Blot respectively.The results showed that the TALENs or CRISPR/Cas9 artificial nuclease could induce fixed-point double-stranded DNA rupture,and could improve the Rad51 gene expression in ovis fibroblasts.