构建稳定表达RFP及嘌呤霉素抗性的K562细胞株

Establishment of K562 Cell Line with Stable Expression of RFP and Puromycine Resistance

目的构建稳定表达红色荧光蛋白（red fluorescent protein，RFP）和嘌呤霉素（puromycin）抗性的K562．PM．RFP细胞株，便于慢性粒细胞性白血病研究中K562细胞的观察和筛选。方法采用PCR法获得RFP片段，将其插入到慢病毒pGC-FU-3FLAG-IRES—Puromycin载体中获得pGC—PM—RFP重组质粒，经脂质体转染到293T细胞中获得慢病毒LV—PM—RFP，有限稀释法检测慢病毒在293T细胞中的转染效率，用包装获得的慢病毒感染K562细胞，经嘌呤霉素筛选获得RFP阳性的K562-PM—RFP细胞株。结果PCR及测序结果证实目的基因RFP正确克隆至慢病毒质粒中，经慢病毒LV—PM-RFP感染的K562细胞能在嘌呤霉素抗性培养基中存活，并稳定表达RFP。结论成功构建了慢病毒重组质粒pGC—PM-RFP，并获得了携带RFP及嘌呤霉素抗性基因的K562-PM—RFP细胞株。

Objective To construct a K562-PM-RFP cell line with stable expression of red fluorescent protein （RFP） and puromycine resistance, for the study of chronic myelogenous leukemia. Methods RFP gene was amplified by PCR, and then inserted into pGC-FU-3FLAG- IRES-Pu- romycin lentiviral vector. The recombinant lentiviral pGC-PM-RFP plasmid was transfected into 293T cells by lipofectamine 2000. Transfection efficiency of LV-PM-RFP in 293T cells was detected by limiting dilution assay. The stable K562-PM-RFP cell line containing RFP was selected by puromy- cine. Results Using plasmid PCR and sequencing, the RFP target gene was confirmed to be cor- rectly cloned to the lentiviral plasmid. The K562 cells which were transfected with recombinant lenti- virus LV-PM-RFP survived from puromycine selection and stably expressed RFP. Conclusion The recombinant lentivirual plasmid pGC-PM-RFP was successfully constructed and the K562-PM-RFP stable cells which continuously express RFP and resist to puromycine were established.