应用Southern 印迹杂交法检测转移基因在宿主细胞中的整合

摘要

目的　应用Southern 印迹杂交法（Southern blot）证实转移基因整合至宿主细胞基因组DNA。方法　酚氯仿法提取基因转导的PA317/AIM及K562/AIM细胞基因组DNA，以聚合酶链反应（PCR）扩增转移基因片段；随机引物法标记32P-DNA探针，与经BamHI酶切、琼脂糖凝胶电泳、转移至尼龙膜的基因组DNA进行Southern杂交反应，检测转移基因的整合。结果　PCR反应和Southern blot分析证实转移基因存在于受体细胞基因组DNA中。结论　Southern 印迹杂交法证实转移基因稳定整合至逆转录病毒生产细胞PA317/AIM及靶细胞K562/AIM中。%Objective　To confirm the integration of transduced genes in the genomic DNA of host cells by Southern blot analysis. Methods　The genomic DNA of transduced PA317/AIM and K562/AIM cells were extracted by phenol-chloroform method. The sequences of foreign genes were detected by polymerase chain reaction (PCR). The BamHI-digested DNA resolved by electrophoresis gel was transferred to nylon member and hybridized with the random primer labeled 32　P-DNA probe. The integration of transduced genes is thus determined by so-called Southern blot. Results　The transduced genes were integrated into the genomic DNA of target cells tested by PCR and Southern Blot.Conclusion　The integration of transduced genes in retroviral producing cells PA317/AIM and target cells K562/AIM can be confirmed by Southern blot analysis.