Human embryonic kidney 293FT cells (Invitrogen) were transfected with lentiviral vectors (pLKO-puro-shRNA, pGIPZ, and pHAGE) and packaging vectors (pMD.2G and psPAX2) by using Lipofectamine 3000 (Invitrogen) with a standard protocol. Briefly, culture supernatants were collected at 48 hours after transfection and ultracentrifuged at 25,000 rpm at 4°C for 2 hours to precipitate the virus. Viral pellets were dissolved in PBS and kept frozen at −80°C until use. Virus titers were estimated by using a qPCR-based method to detect long terminal repeat (LTR) sequences as described previously (53). The following primers and probe were used: LTR F, 5′-TGTGTGCCCGTCTGTTGTGT-3′; LTR R, 5′-GAGTCCTGCGTCGAGAGAGC-3′; and LTR probe, 5′-CAGTGGCGCCCGAACAGGGA-3′.

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