小鼠MPI基因的打靶载体的构建和筛选

Construction of a Mouse Novel Gene Targeting Vector and Recombination Screening

目的 构建小鼠MPI基因的基因打靶载体转染ES细胞 ,构建用于同源重组筛选的对照载体。方法根据计算机分析小鼠MPI基因的基因组序列 ,构建用于同源重组载体的长臂和短臂并且转染小鼠ES细胞 ,经抗性筛选后得到阳性克隆 ,抽提基因组DNA后用PCR的方法进行重组子的初步筛选。结果 成功构建了MPI基因的基因打靶载体并且摸索了用PCR的方法进行重组细胞初步筛选的方法。结论 这个载体的构建为MPI基因功能的研究打下了基础 ,同时用PCR方法进行初步筛选大大减少了Southern杂交的工作量 ;利用实验小鼠来研究印迹基因是非常有效的方法 ,它不仅能了解印迹基因在小鼠生长发育过程中的功能 ,而且进而有助于研究人的相应印迹区。

ObjectiveTo investigate the function of a novel mouse imprinting gene which was cloned by DD -PCR and RACE, a knockout strategy was applied to target this gene, so in this study the targeting vector was constructed and the recombinant cell clones were primarily screened by PCR. Methods Two large fragments flanking the coding region of this gene were cloned into the pKO Scrambler NTKV -1906 vector after the genomic sequence of this gene was analyzed by computer, and then the linearized replacement vector was transfected into the mouse ES cells and drug -resistant cell clones were picked out for screening by a PCR -based strategy. Results The targeting vector and a control vector were constructed successfully and three recombinants were obtained by PCR screening for further use. Conclusion This targeting vector and the recombinants provided a support for identification of the function of the mouse novel imprinting gene . At the same time, a PCR -based screening was established and successfully applied to obtain three positive clones for further use, which would reduce the work of screening in the knockout process.