Generating Recombinant CFTR Plasmids

The plasmid pcDNA5.FRT-wtCFTR containing the coding sequence for the WT CFTR gene was kindly provided by the Cystic Fibrosis Foundation Therapeutics (CFFT) Lab. The plasmid contains the CFTR gene coding sequence in the pCDNA5 FRT/TO plasmid backbone (Life Technologies). The CFTR coding sequence with a carboxyl-terminal T7 tag from pcDNA5.FRT-wtCFTR was cloned into the multiple cloning site of the pCW57-GFP-2A-MCS plasmid, resulting in the plasmid pGFP-P2A-CFTR-WT-T7. The pCW57-GFP-2A-MCS plasmid was a gift from Adam Karpf, Eppley Institute for Cancer Research, University of Nebraska Medical Center, Omaha, NE (Addgene plasmid No. 71783; n2t.net/addgene:71783; RRID: Addgene_71783). pGFP-P2A-CFTR-WT-T7 was mutagenized to delete the coding sequence in CFTR exon 23, resulting in pGFP-P2A-CFTR-Δex23-T7. pGFP-P2A-CFTR-WT-T7 was mutagenized to introduce a PTC at amino acid position 1282 (W1282X) to generate the pGFP-P2A-CFTR-W1282X-T7 plasmid. Plasmid constructions were conducted by GenScript. The plasmids pGFP-P2A-CFTR-WT-T7, pGFP-P2A-CFTR-W1282X-T7, and pGFP-P2A-CFTR-Δex23-T7 encode recombinant GFP-P2A-CFTR proteins, which are Turbo GFP and T7-tagged CFTR polypeptides linked by a self-cleaving 2A peptide (31 (link)).