Production and Validation of Lentiviral Vectors

The lentiviral expression vector pLV-hTERT-IRES-hygro [19 (link)] and FUW-M2rtTA [20 (link)] were obtained from Addgene (#85140 and #20342, respectively). The lentiviral vector TetO-HHFG (Additional file 1: Fig. S1A) was described in detail previously ([9 (link)]. Lentivirus was generated in 293 T cells by cotransfection of pHIV vector with pPAX2 and pMD2.G in 10:7.5:5 ratio. Lentivirus was collected and concentrated using Lenti-X concentrator following manufacturer´s instructions (Takara). We routinely performed two analysis to confirm lentivirus production and concentration. First, we used Lenti-X GoStrix (Takara) to confirm a titer above 5 × 10⁵ infectious units per ml after concentration. Second, we always produced in parallel a control lentiviral vector expressing GFP (pHIV-eGFP-FOXA3) and infect human fibroblasts. GFP-positivity should be higher than 75% by flow cytometer.