小鼠Pkhd1条件性基因打靶载体的构建

Construction of targeting vector for conditional knockout of murine Pkhdl

目的构建小鼠Pkhd1条件性基因打靶载体,为建立Pkhd1条件性敲除小鼠模型奠定基础。方法以正常小鼠(129xl/SvJ)基因组DNA为模板,扩增小鼠包括第6号外显子的Pkhd1基因部分序列,通过引入LoxP和Neo基因等步骤,建立条件性敲除Pkhd1第6号外显子的条件性基因打靶载体。结果经多个限制性核酸内切酶酶切鉴定和测序证实,构建的小鼠Pkhd1基因条件性打靶载体符合设计要求。结论成功构建了小鼠Pkhd1条件性基因打靶载体,为建立Pkhd1基因条件性敲除小鼠打下了基础。

Objective To construct the targeting vector for conditional knockout for polycystic kidney and hepatic disease 1 gene （Pkhdl） .Methods A 9.3 kb Pkhdl genomie DNA frasment containing exon 6, obtained from routine genomie DNA（ 129xl/SvJ ）by PCR, was inserted into T vector（ pCB2. 1-TOPO）whieh was used as the homologous ann. Two LoxP was inserted into intron 5 and 6 for conditional knockout of exon 6 of Pkhdl. Resnlts The correct structure of the targeting vector was confu-mcd by restriction enzyme digestion and sequencing analysis. Conclusion A targeting vector for conditional knockout of routine Pkhdl has been successfully constructed.The construction of targeting vector paved the way for conditional knockout mouse strain generated by targeted mutation of Pkhdl.