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目的 :通过 PK D1基因打靶载体的胚胎干细胞转染、药物筛选以及分子鉴定 ,获得发生同源重组的胚胎干细胞克隆 ,为产生 PK D 1基因缺陷小鼠品系准备条件。 方法 :体外培养小鼠胚胎干细胞 ,用电穿孔的方法进行 PK D1基因打靶载体的转移 ,并用 G4 18进行筛选培养 ,得到阳性克隆后 ,进一步扩大培养 ,抽提胚胎干细胞的基因组 DNA ,采用 DNA印迹法进行分子鉴定。结果 :经 G4 18筛选培养得到 192个阳性克隆 ,分子鉴定获得 2个发生同源重组的胚胎干细胞克隆。结论 :鉴定得到的 2个同源重组胚胎干细胞克隆 ,为进一步建立 PK D1基因缺陷小鼠奠定了基础
OBJECTIVE: To clone embryonic stem cells (PKCs) derived from PKD1-targeting vector and obtain the homologously recombined embryonic stem cell clones in order to generate the strain of PKD1-deficient mice. Methods: Murine embryonic stem cells were cultured in vitro. The PK D1 gene targeting vector was transfected by electroporation and then screened by G418. After positive clones were obtained, the clones were further expanded and the genomic DNA of embryonic stem cells was extracted. Law for molecular identification. Results: 192 positive clones were screened by G418 screening, and 2 clones of embryonic stem cells were obtained by homologous recombination. Conclusion: Two homologous recombined ES cell clones were identified, which laid the foundation for the further establishment of PKD1-deficient mice