四环素调控的WT1基因表达系统在U937中的建立

目的：在白血病细胞系U937中建立四环素诱导的WT1基因表达系统。方法：运用脂质体将质粒pRevTet-On、pRevTRE-Luc和pRevTRE-WT1分别转染逆转录病毒的包装细胞PT67以产生逆转录病毒。将逆转录病毒RevTet-On感染U937细胞，经G418筛选，产生抗性细胞，用有限稀释法将U937/Tet-On抗性细胞单克隆化。14天后，克隆逐步形成，在显微镜下标记并移出。再将逆转录病毒RevTRE-Luc瞬时感染这些克隆，培养基中加入强力酶素(Dox)2mg/L或不加Dox，应用荧光素酶报告基因分析试剂盒检测荧光素酶的活性。挑选一株低背景和高诱导倍数的U937/Tet-On克隆，最后将逆转录病毒RevTRE-WT1感染这株克隆。结果：在U937中建立四环素调控的WT1基因表达系统。结论：建立逆转录病毒整合的U937细胞株，可用四环素及其衍生物精确调控WT1基因的表达，为研究WT1在白血病细胞系的功能提供一种有力的实验手段。

Establishment of Expression of WT1 in U937 Cell Line Controlled by the RevTet - On Regulatory System

Objectve:To establish a tetracycline-controlled inducible gene expression system in U937 cell line.Methods:To produce recombinant retrovirus, the package cell line PT67 was transfected with vectors pTet-On, pTRE-Luc and pTRE-WT1respectively by using liposome transfection reagent. U937 cell line was infected with recombinant retrovirus RevTet-On. The infected cells were selected in growth medium containing G418. G418-resistant cells were then subcloned with limited dilution. Fourteen days later, all individual G418-resistant clones were screened by transient infection with retrovirus TRE-Luc for clones with low background and high induction of luciferase in response to doxycycline. A U937 cell subline with high levels of induction and high gene expression levels was obtained. Then the cell line was infected with retrovirus TRE-WT1. Results:A U937 cell line expressing WT1 regulated by the RevTet-On regulatory system was established. Conclusion:The RevTet-On regulatory system could modulate the expression of WT1 by tetracycline and its derivatives, which provides a useful model for research on the function of WT1 in leukemic cells.