绵羊成纤维细胞中TALE-TFs激活β酪蛋白基因启动子研究

Activation of β-casein Gene Promoter By Using Engineered TALE-TFs in Ovine Fibroblasts

针对绵羊β-酪蛋白基因启动子序列设计4个人工转录因子TALE-TFs。电转染TALE-TFs进入绵羊成纤维细胞中，通过实时荧光定量和Western blotting检测β-酪蛋白基因表达，筛选获得激活效果较好的TALE-TF1。将TALE-TF1和β-酪蛋白基因启动子、Red报告基因质粒电转染进入绵羊成纤维细胞，通过荧光显微镜直接观察报告基因Red表达。利用TALE人工转录因子，在绵羊成纤维细胞中能够激活β-酪蛋白基因启动子表达框，为替代乳腺上皮细胞表达验证系统提供了一种检测途径。

Four TALE-TFs targeting promoter of ovine β-casein gene were designed and TALE-TFs of eukaryotic expression plasmid were constructed. These TALE-TFs plasmids were nucleofected into sheep embryo fibroblasts respectively. Compared with the control groups, TALE-TF1 was identified to effectively activate the expression of sheep β-casein gene through qRT-PCR and Western blotting. TALE-TF1 plasmid and expression cassette with ？-casein gene promoter and Red fluorescent protein reporter gene were co-nucleofected into ovine fibroblasts through Amaxa Nucleofector. ？-casein gene promoter can be activated by artificial TALE-TFs in ovine fibroblasts. TALE-TFs were adopted to provide a new approach for detecting the expression result of ？-casein gene promoter-interesting gene expression cassettes in ovine fibroblasts.