Lentiviral shRNA Knockdown of Apoptosis Regulators

Target sequences for Bak, Bax, and Bim (23 (link)) were selected and were synthesized by the Mayo Clinic Molecular Biology Core Facility. The targeting sequence for Bid was AAGAAGACATCATCCGGAATA. The targeting sequence was CCCATTCACTACAGGTGAA for Bak (26 (link)), was GCTCTGAGCAGATCATGAA for Bax (26 (link)), and was GATCCTCCCCGAATACCTC for apoptosis-inducing factor (AIF; ref. 27 (link)). After annealing, the template forms a double-stranded DNA flanked by EcoRI and BamHI sites and can be ligated into the lentiviral shRNA cloning and expression vector pSIH-H1 (System Biosciences). Insertion at the intended site was confirmed by DNA sequencing. For pseudovirus production, 2 µg endotoxin-free lentivector expression construct was mixed with lentivector packaging plasmid mix (System Biosciences) and diluted in serum reduction Opti-MEM containing Plus reagent (Invitrogen). After incubation at room temperature for 15 min, the Lipofectamine reagent containing Opti-MEM was added dropwise into the above DNA/Plus complex and incubated for another 15 min. Lentivirus producer cell line 293T was transfected with the DNA/Lipofectamine/Plus complex in Opti-MEM overnight in the 5% CO₂ incubator at 37°C. The next day, the medium was replaced with fresh DMEM containing 2% heat-activated fetal bovine serum and the incubation at 37°C was continued. At 48 h post-transfection, the supernatants were collected, clarified, and filtered through Millex-HV 0.45 µm polyvinylidene difluoride filters (Millipore). The supernatants were then concentrated by adding 10% (final concentration) of PEG-8000 (Sigma), incubated at 4°C overnight for no less than 12 h, and centrifuged at 1,500 × g for 10 min at 4°C. The pseudovirus pellet was resuspended in a small volume of Opti-MEM and stored at −80 °C. For transduction of the lentivector expression construct (packaged in pseudo-type viral particles) into target cells, appropriate amounts of virus in Opti-MEM were directly added to target cells grown in Opti-MEM containing 8 µg/mL polybrene (Sigma) and incubated overnight at 37°C. Target gene knockdown was then tested 72 h post-transduction.