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. 2013 Feb 28;8(2):e53072. doi: 10.1371/journal.pone.0053072

Figure 3. Effectiveness and tissue-specificity of AFP-Cre/LoxP-shRNA system for HCC tissue-specific RNAi.

Figure 3

(A) Construction of the AFP-Cre/LoxP-shRNA system which consists of AFP-Cre and LoxP-shRNA vectors. (B) Schematic illustration of the AFP-Cre/LoxP-shRNA system. When the AFP-Cre and LoxP-shRNA vectors co-infect target cells, the AFP promoter drives the downstream Cre recombinase gene expression which subsequently cuts the LoxP-CMV-eGFP-LoxP inside the U6 promoter and activates it. The activated U6 promoter then drives the downstream shRNA expression. The LoxP-shRNA harbors a CMV-eGFP indicator which can indicate the RNAi expression (GFP fluorescence will diminish if the AFP promter initiates the RNAi expression). (C, D) CMV-eGFP indicator and gene silencing analysis by Q-PCR and western blot showed that the AFP-Cre/LoxP-shRNA system targeting Beclin 1 (AFP-Cre/LoxP-shRNA-Beclin1) could efficiently silence target gene (Beclin 1) in a HCC-specific manner. The GFP fluorescence diminished in HCC cells (HCCLM3, HepG2, and Hep3B) but not in non-HCC cells (L-02, Hela, and SW1116) after infection with the AFP-Cre/LoxP-shRNA-Beclin1. Q-PCR and western blot analysis were exemplified by HCC cell line HCCLM3 and non-HCC cell line L-02.