基础医学与临床 ›› 2012, Vol. 32 ›› Issue (2): 149-153.

• 研究论文 • 上一篇    下一篇

用慢病毒载体介导的RNAi感染CB3细胞后抑制FLI-1的表达

李岩1,宋伟1,李薇1,王雪梅1,王冠军1,崔久嵬2   

  1. 1. 吉林大学白求恩第一医院
    2. 吉林大学第一医院
  • 收稿日期:2011-10-13 修回日期:2011-12-08 出版日期:2012-02-05 发布日期:2012-01-12
  • 通讯作者: 崔久嵬 E-mail:cuijiuwei1@yahoo.com

Suppress the expression of Fli-1 through lentivirus-based vectors mediated RNAi

  • Received:2011-10-13 Revised:2011-12-08 Online:2012-02-05 Published:2012-01-12

摘要: 【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。

关键词: Fli-1, RNA干扰, 慢病毒载体

Abstract: 【Abstract】 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 ) and provide powerful technical support for follow-up experiments. Methods Small interfering RNA that is directed 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 were 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). Conclusions The lentivirus RNAi vectors toward FLI-1 could be used to infect the CB3 cell line. After delivering this sort of vectors into the CB3 cells, the experession of FLI-1 was inhibited dramatically. Current research has provided a stable infection tool for CB3 cell line and this will facilitate the follow-up experiments.

Key words: Fli-1, RNA interference, lentivirus-based vectors

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