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Virologica Sinica, 21 (6) : 604-608, 2006
Research Article
Inhibition of Dicer Gene and Its Influence on the Function of shRNA
1.The Key Laboratory of Molecular Biology of Infectious Diseases of Ministry of Education, The Institute for Viral Hepatitis, Chongqing University of Medical Sciences, Chongqing 400016, China
2.Molecular Oncology Laboratory, The University of Chicago Medical Center, IL 60637,USA
In order to determine the effect of silencing Dicer on shRNA-mediated RNA interference, we constructed two shRNA expression vectors that targeted the RNAse III domain of the Dicer gene. The shRNA vectors were transfected into 2215 cells, colon cancer TC cells and Green Fluorescent Protein (GFP)-tagged HepG2 A9 cells using lipofectamineTM 2000. The level of Dicer mRNA was assessed by RT-PCR. After the knocking-down of the Dicer gene was confirmed in the GFP-tagged HepG2 A9 cells, another shRNA expression vector that targeted the GFP gene was transfected in these cells. The suppression of GFP expression was evaluated by RT-PCR and fluorescence microscopy. We found that anti-Dicer shRNA expression vectors efficiently inhibited the expression of Dicer in the tested cells lines. Moveover, when the Dicer expression was silenced in GFP-tagged HepG2 A9 cells, the GFP-targeting shRNA expression vector was less effective in silencing GFP expression. Thus, our results indicated that the expression of Dicer could be inhibited by anti-Dicer shRNA expression vectors, and that the expression silencing activity of shRNA expression vectors is dependent on the presence of Dicer enzyme.
  Published online: 20 Nov 2006
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