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目的:建立体内稳定表达HER2多表位基因的小鼠黑色素瘤B16细胞系。方法:利用分子克隆技术构建真核表达载体pcDNA3-GFP-HER2,阳离子脂质体介导法将其稳定转染入B16细胞,G418克隆筛选及体内传代后,流式细胞术(FCM)分选出GFP-HER2表达阳性细胞群,对该群细胞进行无G418条件下的单克隆筛选并进行体内稳定性检测。结果:经限制性内切酶鉴定及序列分析,pcDNA3-GFP-HER2构建正确;最终建立的表达HER2基因B16细胞系体内传代后GFP-HER2阳性率高于90%。结论:成功建立了体内稳定表达HER2多表位基因的小鼠黑色素瘤细胞系,为其他体内稳定表达外源基因的转染细胞的建立提供了借鉴。
OBJECTIVE: To establish a mouse melanoma B16 cell line stably expressing the HER2 polytope gene in vivo. Methods: The eukaryotic expression vector pcDNA3-GFP-HER2 was constructed by molecular cloning technique. The recombinant plasmid was transfected into B16 cells by cationic liposome-mediated method. After G418 clones were screened and passaged, the cells were sorted by flow cytometry (FCM) A positive population of GFP-HER2 expressing cells was obtained and the cells were subjected to monoclonal screening without G418 and tested for in vivo stability. Results: The pcDNA3-GFP-HER2 was constructed correctly by restriction enzyme analysis and sequence analysis. The positive rate of GFP-HER2 was higher than 90% in the B16 cell line expressing HER2 gene. CONCLUSION: The mouse melanoma cell line stably expressing HER2 polytope gene in vivo has been successfully established and provided a reference for the establishment of transfected cells stably expressing foreign genes in vivo.