Objective: To construct an acute myeloid leukemia cell line stably expressing CD123-CLL1 so as to provide an "in vitro" model for studying the role of CD123 and CLL-1 in leukemia and the treatment targeting CD123 and CLL-1.
Methods: The recombinant plasmid of lentivirus was constructed by synthesizing CD123 and CLL-1 sequences and PCR homologous recombination. The lentivirus vector was packaged by three-plasmid packaging system. After collecting the supernatant of lentivirus, the virus titer was determined by quantitative PCR. K562 leukemia cells were collected and transtected with virus supernatant. Leukemia cell line stably expressing the target gene were screened by purinomycin. The expression levels of CD123 and CLL-1 were detected by RT-PCR and flow cytometry.
Results: The lentiviral vector was successfully constructed, and identified by agarose gel electrophoresis and gene sequencing, then the virus titer of the supernatant was up to 5.81×108 after quantitative PCR assay. The K562 leukemia cell line obtained positive expression cells after being infected by puromycin. The high expression of CD123 and CLL-1 was confirmed by RT-PCR, while the significantly high expression of CD123 and CLL-1 was confirmed by flow cytometry.
Conclusion: Lentiviral vector expressing CD123-CLL1 has been successfully constructed, and K562 leukemia cell line stably expressing CD123 and CLL-1 has been successfully obtained.
题目: 稳定表达CD123-CLL1双基因白血病细胞株的构建及鉴定.
目的: 构建稳定表达CD123-CLL1的慢病毒载体及急性髓系白血病细胞株，为研究CD123、CLL-1在白血病中的作用及以CD123和CLL-1为靶点的治疗方法提供一种体外模型.
方法: 通过合成CD123及CLL-1序列及PCR同源重组的方式构建慢病毒重组质粒，通过3质粒包装系统对慢病毒质粒载体进行包装，收集慢病毒上清液后采用定量PCR法测定病毒滴度。收集白血病K562细胞株，使用病毒上清液对其进行感染，通过嘌呤霉素筛选以获得稳定表达目的基因的白血病细胞系，采用RT-PCR和流式细胞术检测CD123和CLL-1的表达水平.
结果: 结果：经琼脂糖凝胶电泳及基因测序鉴定，成功构建了慢病毒载体，且经过定量PCR法检测显示，收集后的病毒上清液病毒滴度高达5.81×108 IU/ml。 白血病K562细胞株经过感染及通过嘌呤霉素分选后获得阳性表达细胞系，RT-PCR法证实CD123和CLL-1的mRNA高表达，流式细胞术证实了CD123及CLL-1的表达明显高于对照细胞.
结论: 成功构建了表达CD123-CLL-1的慢病毒载体，同时成功获得了能够稳定表达CD123及CLL-1的K562白血病细胞株.