Design and test of Pol II-driven miRNA-expression constructs. (A) A schematic illustration of the construct design. From top to bottom: an example of miRNA against the E1k subunit (the bold, underlined sequence complements the E1k mRNA); construct pCAG-RFP-miR that constitutively expresses RFP and a miRNA, which is placed in the 3'-UTR of the RFP gene; construct pCAG-RFP-miRint that constitutively expresses RFP and a miRNA that is placed in an intron located in the 3'-UTR of the RFP gene; and structure of the artificial intron (SS = splicing site, BrP = branch point with the sequence TCCTGACCATTCAT, PPT = poly pyrimidine track with the sequence CCTCTTTCTTTTTCCT, and ESE = exon splicing enhancer with the sequence TTGTGGAAGAAAT). (B) Detection of intron-splicing in RNA extracted from NF1 cells that were transfected with pCAG-RFP-miR-E1-1int. The RT-PCR product was resolved on a 2% agarose gel (lane 1). MW (molecular weight) lane shows the 100 bp DNA ladder (New England Biolab). The PCR product was sequenced and the spliced exon junction sequence was ...TAATTAATTAACAG/GCTTGT..., which matched exactly as predicted from the correctly spliced junction. (C) Northern blots detecting E1k and E2k mRNAs. The RNA was extracted from cells 48 hours after transfection with the constructs pCAG-RFP-miR or pCAG-RFP-miRint. Ethidium bromide staining is shown as loading controls. (D) Northern blots detecting the pre-miR-E2-4 and miR-E2-4. The constructs pCAG-RFP-miR and pCAG-RFP-miRint were used in the transfection. The mark on the right side of the pre-miR-E2-4 gel indicates the position of a 60 nt single stranded DNA. The mark on the right side of the miR-E2-4 gel indicates the position of a 22 nt single stranded DNA.