用慢病毒载体介导的RNAi感染CB3细胞后抑制FLI-1的表达

 【摘要】目的 观察慢病毒载体介导的RNA干扰(RNA interference, RNAi)方法对小鼠红白血病细胞（CB3）中FLI-1（Friend leukemia integration-1,FLI-1）基因表达的抑制作用，以为后续实验提供有力的技术支持。方法 设计制备针对目的基因FLI-1的小干扰RNA(small interfering RNA, siRNA),构建制备目的基因的siRNA慢病毒载体，PCR阳性菌落进行测序鉴定，对慢病毒包装与滴度测定后感染CB3细胞,观察绿色荧光蛋白(green fluorescent protein，GFP)表达情况。RT-PCR和Western blot检测病毒感染CB3细胞后FLI-1的表达。结果 成功构建了针对FLI-1基因的siRNA慢病毒载体,并包装成慢病毒颗粒，病毒滴度为1.5E+9TU/ml，并可将其高效感染CB3细胞，病毒感染后FLI-1在转录水平和翻译水平的表达量显著低于对照组和未感染病毒组（P<0.001）。结论 成功构建了针对FLI-1基因的siRNA慢病毒载体，并可有效抑制CB3细胞中FLI-1基因的表达,为后续实验提供稳定的感染细胞载体。

Suppression of FLI-1 expression through lentivirus-based vectors mediated RNAi

Objective To observe the inhibition of lentiviral vector-mediated RNA interference on the expression of Friend leukemia integration-1 gene in mouse erythroleukemia cell line(CB3).Methods Small interfering RNA towards FLI-1 gene was designed and RNAi mediated with lentivirus-based vectors was constructed.Those colonies with positive PCR result were sequenced.After the packaging and the titer assaying,the lentivirus was used to infect the CB3 cells.Then,the expression of GFP was observed,and the inhibition of the expression of FLI-1 was analyzed via both RT-PCR and Western blot approaches.Results The lentivirus RNAi vectors with target gene were successfully constructed and the titer of concentrated virus was 1.5E+9TU/ml.Compared to the negative and non-infected controls,the expression of FLI-1 gene was significantly inhibited after the CB3 cells were infected by lentivirus(P<0.001).Conclusion The lentivirus RNAi vectors toward FLI-1 may be used to infect the CB3 cell line.After delivering this sort of vectors into the CB3 cells,the experession of FLI-1 is significantly inhibited.