*4.4. Production of Lentiviral Particles*

To generate lentiviral particles, three vectors were used including transfer vector (LL3.7 or pCDH or pLV-hTERT-IRES-hygro), packaging plasmid (psPAX2 from Addgene, Watertown, MA, USA, Plasmid # 12260), and envelope plasmid (pMD2.G from Addgene, Watertown, MA, USA, Plasmid # 12259). The 293FT cells (Invitrogen, Carlsbad, CA, USA) were cultured in dulbecco's modified eagle medium (DMEM) with high-glucose supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1x penicillin-streptomycin-amphotericin B (PSA) solution, at 37 ◦C and 5% CO2. Then, 8.82 µg transfer vector DNA, 6.66 µg psPAX2 packaging plasmid DNA, and 2.70 µg pMD2.G envelope plasmid DNA was mixed with 180 µL of polyfect transfection reagent (Qiagen Inc., Germantown, MD, USA) and the final volume was brought up to 855 µL using DMEM high-glucose media without any supplements. The plasmids-polyfect mixture was incubated at room temperature for 10 min and then gently mixed in the media on 70-80 % confluent 293FT cells. Cells were incubated for 4–6 h at 37 ◦C and 5% CO2. After incubation time, the transfection media was replaced by fresh DMEM high-glucose media supplemented with 10% FBS and 1x PSA solution. After 72 h, the medium containing lentiviral particles was collected and ultra-centrifuged over a 20% sucrose cushion at 25,000 RPM for 2 h at 4 ◦C. LL3.7 vector-based lentiviral particles were resuspended in chemically defined medium-2 (CDM-2), whereas pCDH or pLV-hTERT-IRES-hygro vector-based lentiviral particles were resuspended in 1x PBS, aliquoted, and stored at −80 ◦C.

To infect the cells by pCDH or pLV-hTERT-IRES-hygro based-lentiviral particle, the median tissue culture infectious dose (TCID50) of lentiviral particles was calculated. The frozen viral aliquot was resuspended in 0.5-1 mL of appropriate media with 8 µg/mL polybrene. The target cells were incubated with lentiviral particles at multiplicity of infection (MOI) of10 for 24 h at 37 ◦C and 5% CO2. After 72 h of culture, cells were selected with appropriate selection antibiotic. The LL3.7 vector-based lentiviral particles were tittered by infecting human embryonic kidney (HEK) cells and counting green fluorescent protein (GFP)-positive cells [61].
