Model to block promoter-associated nascent antisense lncRNA. (A) Schematic of the CMV-EGFP-SV40pA vector for Cre-induced poly(A) inversion. (B) Strategy for generating PA clones and testing the role of nascent antisense RNAs in TGA. (C) Quantitative RT-PCR for eGFP mRNA 4 d after treatment with irrelevant shRNA (ctrl-shRNA), shRNA(0)S, shRNA(0)AS, shRNA(-4)S, and shRNA(-4)AS in clone PA-5. The shRNA(0)AS and shRNA-(-4)AS are designed to target the promoter-associated antisense lncRNA, while shRNA(0)S and shRNA-(-4)S target in the sense direction. (D) PCR genotyping to confirm that the poly(A) signal is inverted in clone PA-5-6, in contrast to clone PA-5-1 and the parent clone PA-5. (E) Strand-specific RT-PCR to detect promoter-associated antisense RNAs in clones PA-5, PA5-1, and PA5-6. Clone PA5-6 contains an inverted SV40-poly(A) sequence.
