Figure 5

Design and test of a Cre-inducible miRNA-expression construct with fluorescent markers. (A) The basic construct of pCAG-EGFP/RFP-miRNAint is composed of the CAG promoter, a loxP site, the EGFP gene with 3 consecutive SV40 poly A signals (to ensure the cessation of the Pol II synthesis), a second loxP site, the RFP gene, a 3' UTR carrying the pre-miRNA hairpin, and a poly A signal. Cre mediates the excision of EGFP and the 3 poly A signals, allowing the CAG promoter to synthesize RFP and miRNA. (B) Top: after transient transfection with pCAG-EGFP/RFP-miRNAint and without Cre, EGFP was expressed and RFP was not. Bottom: cotransfection with Cre diminished EGFP expression and induced the RFP expression. (C) Northern blot of RNA extracted from cells transfected with pCAG-EGFP/RFP-miRNAint carrying miRNA against E1k or E2k but without Cre (lane 1). Lanes 2–6 are standard dilution of the RNA extracted from cells transfected with the scrambled miRNA construct. The relative RNA loadings are indicated at the bottom. One hundred represents 6 μg RNA for E1k blot (top) and 20 μg RNA for E2k blot (bottom). β-actin was detected as a loading control. (D) Northern blot of RNA extracted from cells cotransfected with pCAG-EGFP/RFP-miRNAint carrying miRNA against E1k or E2k and CMV-Cre (lane 7). Lane 1 is RNA from cells transfected with constitutively expressing pCAG-RFP-miRNAint constructs, which serve as positive knockdown control. Lanes 2–6 are standard dilution of the RNA extracted from cells transfected with the scrambled miRNA construct. The relative RNA loadings are indicated at the bottom. One hundred represent 6 μg RNA for E1k blot (top) and 20 μg RNA for E2k blot (bottom). β-actin was detected as a loading control. (E) Confirmation of protein knockdown by measurement of KGDHC activity. Three days after transfection of constructs CAG-RFP-miR-E1-1int, -miR-E2-4int or -miR-Scrint, NF-1 cells were lysed and assayed for KGDHC activity as described in Methods.