Genjet dna transfection reagent

Mouse Bpnt2 cDNA was cloned into the pBABE-puro (Addgene, #1764) retroviral vector using BamHI and SalI restriction sites. The plasmid was subsequently mutagenized using traditional site-directed mutagenesis methods to generate D108A, T181P, and D175N mutants, and mutagenesis was verified by Sanger sequencing. Retroviral vectors were each cotransfected with VSV.G (Addgene, #14888) and gag/pol (Addgene, #14887) vectors into HEK 293T cells using GenJet DNA transfection reagent (SignaGen). Two milliliter of the viral supernatant was harvested 48 h after transfection, filtered to remove cells, and added directly to separate Bpnt2-KO MEF cultures containing 8 μg/ml polybrene (Invitrogen). Approximately 24 h after viral transduction, 3 μg/ml puromycin was added to kill nontransduced cells. The cells were incubated in puromycin media for 3 days before being passaged into media without puromycin. Efficacy of transduction was confirmed by measuring Bpnt2 mRNA and protein expression.

Transient transfection with plasmids was performed using GenJet DNA transfection reagent (SignaGen Laboratories, SL100489) according to the manufacturer’s instruction. Lentiviral transduction was performed as before for stable expression of the mitophagy reporter mito-mCE, shRNAs, and sgRNAs [45 (link)]. In brief, to produce infectious lentiviruses, 293T cells were co-transfected with a lentiviral transfer vector together with the packaging plasmid psPAX2 (a gift from Didier Trono, Addgene plasmid #12260) and the vesicular stomatitis virus G protein expression plasmid pVGV-G at a ratio of 5:4:1 for 2 days. Transduction units (TUs) of 60x concentrated lentiviruses were determined in 293T cells in the presence of appropriate antibiotics to select transduced cells. Target cells were transduced with lentiviruses at a single dose of 10 TUs in the presence of 10 μg/ml polybrene for 6 h. Stably transduced cells were selected for 2–3 weeks in the presence of appropriate antibiotics.