小鼠T279基因敲除胚胎干细胞的构建
Construction of T279 gene knockout ES cells

目的:利用打靶载体技术构建小鼠T279基因敲除胚胎干细胞模型。方法:订购含小鼠T279基因的129 BAC质粒和PL452,PCR扩增Retrieving同源臂及loxp-neo-loxp片段,利用PL253质粒进行目的片段的连接,并在EL350大肠杆菌中进行同源重组,构建T279打靶载体,将其电转染小鼠胚胎干细胞,利用Southern blot方法鉴定同源重组的阳性克隆。结果:经限制性内切酶酶切鉴定及DNA测序鉴定,T279打靶载体构建成功。Southern blot结果显示成功筛选到同源重组的敲除T279基因的胚胎干细胞克隆。结论:T279基因敲除打靶载体构建成功并筛选到了同源重组阳性的胚胎干细胞克隆。
Aim: To construct T279 gene knockout embryonic stem(ES)cells using gene knockout targeting technique.Methods: The 129 BAC plasmid which contain T279 gene was ordered,and Retrieving homologous arms and loxp-neo-loxp fragment were amplified by PCR method. PL253 plasmid was used for ligation and homologous recombination between different fragments, and the 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: The correct structure of the targeting vector has been 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: T279 gene knockout targeting vector has been successfully constructed and homologous recombination-positive ES cell clones are obtained