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目的构建稳定携带Oct4、Klf4、Sox2、c-myc(以下简称为OKSM)和增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)基因的小鼠肿瘤细胞株。方法将载体pLentG-KOSM用NotI、XbaI线性化,并进行聚合酶链式反应(polymerase chain reaction,PCR),以鉴定其结构。按Invitrogen公司推荐的标准程序进行慢病毒包装和确认慢病毒是否成功生产;携带OKSM和EGFP基因的慢病毒感染小鼠肝癌细胞(Hep1-6)、小鼠乳腺癌细胞(4T1)和小鼠肺癌细胞(LLC),以建立相应病毒感染体系。结果 pLentG-KOSM确实含有OKSM4种基因,按标准程序生产的携带OKSM和EGFP基因的慢病毒上清高效率感染Hep1-6、4T1及LLC,成功建立稳定携带OKSM和EGFP基因的小鼠肿瘤细胞株。结论成功构建携带OKSM和EGFP基因的Hep1-6、4T1、LLC细胞株,为相关的后续研究打下了良好基础。
Objective To construct a mouse tumor cell line stably carrying Oct4, Klf4, Sox2, c-myc (hereinafter referred to as OKSM) and enhanced green fluorescent protein (EGFP) gene. Methods The vector pLentG-KOSM was linearized with NotI and XbaI and subjected to polymerase chain reaction (PCR) to identify its structure. Lentivirus packaging was performed according to the standard procedure recommended by Invitrogen and whether the lentivirus was successfully produced; Lentivirus-infected mouse hepatoma cells (Hep 1-6), mouse mammary carcinoma cells (4T1) and mouse lung cancer cells carrying OKSM and EGFP genes Cells (LLC) to establish the corresponding viral infection system. Results pLentG-KOSM did contain OKSM4 gene. Hep1-6, 4T1 and LLC were efficiently transfected with the lentivirus supernatants carrying OKSM and EGFP gene by standard procedure. Mouse tumor cell lines stably carrying OKSM and EGFP gene were successfully established. Conclusion The successful construction of Hep1-6, 4T1 and LLC cell lines carrying OKSM and EGFP genes laid a good foundation for the follow-up research.