Retrovirus production, retroviral infection, and stable cell line selection

Retroviral human CPT1A WT and G710E constructs were kindly provided by Taro Hitosugi from Mayo Clinic. These plasmids were pLHCX-hygro- Gateway destination vector-based and were constructed as previously described144. Each of the pLHCX vector plasmid (RV), CPT1A WT (RW), and CPT1A G710E (RM) plasmid was co-transfected with packaging plasmids (EcoPac, pAmphopac, pVSVG) into HEK293T (Sigma-Aldrich) cells using lipofectamine 2000. Retrovirus was harvested 48 h after transfection, filtered w/ 0.45 μm filter, and 8 μg/ml final concentration of polybrene was added. Retroviral infection of the PEO1 WT (A3) and PEO1-CPT1A KO clones (B85), PEO4 WT (C5) and PEO4-CPT1A KO clones (C6) was conducted with freshly harvested retrovirus. Infected cell lines were selected in 25 μg/ml hygromycin for 3 weeks to obtain stable cell lines. Stable expression of the CPT1A WT and G710E mutant protein was verified by western blot of cell lysates as described below.