Pcdna5 frt expression vector

FRT cells were provided by M. Welsh (University of Iowa, Iowa City, Iowa, USA). Cells were modified by our laboratory to express WT or mutant CFTR on an isogenic background using the Flp-in system (Thermo Fisher). Briefly, CFTR cDNAs (WT, F508del, P67L) were cloned into NheI and XhoI sites of the pcDNA5/FRT expression vector according to the manufacturer’s instructions (Thermo Scientific). Cells were cotransfected with plasmids containing CFTR and a flp recombinase construct (pOG44) and cultured in media with hygromycin (100 µg/ml) to select site-specific recombinants. Hygromycin-resistant clonal lines were isolated and screened for loss of β-galactosidase. Clonal cells were found to have CFTR mRNA levels that varied less than by 50% compared with cells expressing WT CFTR, in contrast with more variable levels of mRNA in FRT lines tested previously (25 (link)). Stability of expression in the new FRT models was verified to at least passage 31. All new lines were validated by Western blot and I_sc measurements with and without lumacaftor or ivacaftor. The P67L biogenesis defect and responses to lumacaftor were also shown following transient transfection in human embryonic kidney (HEK) 293 cells (ATCC, catalog CRL-3216; E.J. Sorscher, unpublished observations).

The full-length hCB1R gene (1.4 kbp) was amplified from pRC/CMV CB1 construct as template, using Pfu DNA polymerase (Stratagene) under the following thermocycling conditions: 95 °C for 1 min, followed by 30 cycles of 95 °C for 30 s and 68 °C for 2 min, followed by an extension time of 5 min at 68 °C in an Eppendorf Mastercycler (Westbury, NY). NheI and BamHI restriction sites were incorporated into forward 5′-CGCTAGCATG-AAGTCGATCCTAGATGGCCT-3′ and reverse 5′-TATGGATCC-TCACAGAGCCTCGGCAGACGTG-3′ primers, respectively. The PCR product was purified using a MinElute PCR kit (Qiagen) and was digested using BamHI and NheI restriction enzymes. The same restriction enzymes were used for digestion of pcDNA5/FRT expression vector (Invitrogen). The vector and PCR fragment were purified using a MinElute kit and ligated at room temperature for 1 h. The ligated products were then transformed into One Shot Top10 competent E. coli cells following the vendor’s protocol (Invitrogen). Plasmid preparations were cultured in Luria broth containing 0.1 mg/mL ampicillin. Recombinant plasmid DNA was isolated using a pure link kit (Invitrogen), and DNA insertion was confirmed by sequencing (University of Connecticut Biotechnology Center, Storrs, CT).

A HeLa FRT clone selected for a single FRT integration site was a generous gift from Dr. Woodring Wright and Dr. Jerry Shay (UT Southwestern Medical Center at Dallas, TX). Human sequences were inserted into the pcDNA5/FRT expression vector (Invitrogen) using a variety of restriction sites. The sequences of the minigenes are available at https://github.com/jbkinney/15_splicing. For stable integration, 0.4 μg of minigene plasmid DNA and 3.6 μg of pOG44 (Invitrogen) were co-transfected into 10⁶ HeLa FRT cells using Lipofectamine 2000 (Life Technologies). After 48 hours, transfected cells were selected with 200 μg/ml hygromycin B (Invitrogen). For transient transfection, 1 μg of minigene plasmid was transfected into HeLa cells. Cells were collected after 48 hours and RNA analyzed by radioactive RT-PCR.