小鼠TSC21基因敲除打靶载体的构建和胚胎干细胞同源重组克隆的鉴定
Construction of mouse TSC21 gene knockout targeting vector and identification of homologous recombination clone of embryonic stem cells

目的:构建小鼠TSC21基因敲除打靶载体,电转胚胎干细胞(ES细胞)并筛选同源重组阳性克隆。方法:订购含TSC21基因的129 BAC克隆,PCR扩增2对同源臂,利用PL253、PL452质粒进行目的片段的连接和同源重组,构建打靶载体并电转ES细胞,利用Southern blot方法鉴定同源重组阳性ES细胞克隆。结果:构建了TSC21基因敲除打靶载体,经过限制性内切酶鉴定及DNA测序鉴定,TSC21基因敲除打靶载体构建成功。Southern blot结果显示成功筛选到打靶序列同源重组阳性ES细胞克隆。结论:TSC21基因敲除打靶载体构建成功并筛选到同源重组阳性ES细胞克隆。
Aim: To construct mouse TSC21 gene knockout targeting vector, transform the targeting vector into embryonic stem(ES)cells using electroporate method,and screen homologous recombination occurred ES cells.Methods: 129 BAC clones which contain TSC21 gene was ordered, two pairs of homologous arms were amplified by PCR method. PL253, PL452 plasmid were used for ligation and homologous recombination between different fragments.Targeting vector was constructed and transformed into ES cells by electroporate method.Southern blot was used to identify homologous recombination occurred ES cell clones.Results: TSC21 gene knockout targeting vector was constructed. The correct structure of the targeting vector was confirmed by the results of restriction endonuclease enzyme digestion and DNA sequencing analysis. Southern blot result showed that homologous recombination-positive ES cell clones existed.Conclusion: TSC21 gene knockout targeting vector has been successfully constructed and homologous recombination-positive ES cell clones are obtained