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Retrovirus packaging and transduction

RD Rong Du
DS Delaney K. Sullivan
NA Nancy G. Azizian
YL Yuanhui Liu
YL Yulin Li
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Retroviral vectors for the expression of fluorescent markers were combined with pCL-10A1 retrovirus packaging vector at 1:1 ratio and transfected into HEK293FT cells using lipofectamine 3000 transfection reagent (Invitrogen). Supernatants containing the viral particles were collected at 48 h post-transfection and passed through a sterile 0.45 μm syringe filter (VWR). Wild type, SYVN1 KO1, and SEL1L KO1 cells were infected by viral supernatant supplemented with 6 μg/ml polybrene in 6-well plate by centrifugation at 1000 g for 90 min. Infected cells were selected by growing in DMEM medium containing 3 μg/ml puromycin for 2 days.

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