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. 2017 Aug 31;10:273. doi: 10.3389/fnmol.2017.00273

Figure 4.

Figure 4

Design and characterization FLEX-Cre system. (A,B) Schematic representation of the FLEX vector which encodes a double-Floxed, inverted shRNA and GFP. In the presence of Cre, a FLip-EXcision (FLEX) switch occurs, leading to a U6 promoter-driven expression of the gene-specific shRNA, and a CMV promoter- driven expression of GFP. (C) The FLEX-shFyn plasmid was incubated with (+ Cre) or without (− Cre) Cre recombinase in vitro and infection was evaluated by staining cells with anti-GFP antibodies (right panels). (D,E) Characterization of Cre-driven Fyn knockdown in HEK293T cells. (D,E) FLEX-shFyn or FLEX-SCR plasmids were pretreated with (+ Cre) or without Cre recombinase then co-transfected with Fyn plasmid as indicated. mRNA expression (D) and protein levels (E) were analyzed by RT-PCR and western blot analysis, respectively. 1. pUSE-empty plasmid, 2. pUSE-Fyn, 3. pUSE-Fyn + pLVX-FLEX-SCR, 4. pUSE-Fyn + pLVX-FLEX-SCR + Cre, 5. pUSE-Fyn + pLVX-FLEX-shFyn, 6. pUSE-Fyn + pLVX-FLEX-shFyn + Cre. Two-tailed t-test. ***p < 0.001; **p < 0.01. (D) n = 3, (E) n = 4. (F) Verification of Cre-dependent pLVX-FLEX-shFyn infection in vivo. The DMS of Drd1-Cre-Ai14 mice was infected with Ltv-FLEX-shFyn (2 × 107 pg/ml). Colocalization of infected neurons (GFP, green, left panels) with TdTomato (D1R neurons, red, right panels) was determined 3 weeks later. Merged image, right panel. Left panel, 5X; middle and right panels, 20X.