小鼠ADAM10条件性基因打靶载体的构建与鉴定

Construction and characterization of the targeting vector of conditional knockout of mouse ADAM10 gene

目的构建小鼠ADAM10条件性基因打靶载体,为建立ADAM10条件性敲除小鼠模型奠定基础。方法以正常小鼠(129/Svj)基因组DNA为模板,采用长片段PCR方法,扩增小鼠ADAM10基因第2外显子及其侧翼序列。通过引入LoxP位点和TK基因等步骤,获得ADAM10条件性基因打靶载体。结果经限制性内切酶酶切鉴定和测序证实,构建的小鼠ADAM10基因条件性打靶载体符合设计要求。结沦成功构建了小鼠ADAM10条件性基因打靶载体,为建立ADAM10条件性基因敲除小鼠打下了基础。

Objective： To construct the targeting vector of conditional knockout of ADAM10 gene for the generation of ADAM10 - deficient mouse. Methods： 6286bp and 6007bp genomic DNA fragments containing exon 2 of ADAM10 gene from 129/Svj mouse strain were amplified by PCR and inserted into T vector （pMD - 18T）, respectively, which were used as the homologous arms. Two LoxP sites were inserted into intron 1 and 2 for conditional knockout of exon 2 of ADAM10 gene. Results ： The correct structure of the targeting vector was confirmed by restriction endonuclease digestion and sequencing analysis. Conclusion： A targeting vector for conditional knockout of ADAM10 gene has been successfully constructed, which lay a foundation for generating the conditional ADAM10 - deleted mouse.