Vector titration by flow cytometry: 1 × 105 HEK293T cells were plated into each well of a 6-well plate and transduced with a range of volumes of concentrated lentivirus. At 72 hr after transduction, cells were trypsinized and EGFP-positive cells were quantified using a BD Cyan flow cytometer or BD FACSArray Bioanalyzer 3 days after transduction.
HEK293T cells were not used for qPCR titration due to their reported abnormal karyotype.56 (link) Briefly, 1 × 105 HT1080 cells were plated into each well of a 6-well plate and transduced with a range of volumes of concentrated lentivirus. At 72 hr after transduction, genomic DNA was extracted and the provirus titer calculated by qPCR, as described previously.57 (link) The viral capsid number was determined using a p24 ELISA kit (Clontech - 632200). The capsid number was determined according to the kit manufacturer’s calculations, where 1 ng p24 is equivalent to 1.25 × 107 lentiviral particles (lp). The vector RNA genome titer was determined using a qRT-PCR RNA titration kit containing pre-designed primers and standards (Clontech - 631235). For all titer comparisons, LTR1 and third generation vectors were produced side-by-side to account for variations between production batches.
HEK293T cells were not used for qPCR titration due to their reported abnormal karyotype.56 (link) Briefly, 1 × 105 HT1080 cells were plated into each well of a 6-well plate and transduced with a range of volumes of concentrated lentivirus. At 72 hr after transduction, genomic DNA was extracted and the provirus titer calculated by qPCR, as described previously.57 (link) The viral capsid number was determined using a p24 ELISA kit (Clontech - 632200). The capsid number was determined according to the kit manufacturer’s calculations, where 1 ng p24 is equivalent to 1.25 × 107 lentiviral particles (lp). The vector RNA genome titer was determined using a qRT-PCR RNA titration kit containing pre-designed primers and standards (Clontech - 631235). For all titer comparisons, LTR1 and third generation vectors were produced side-by-side to account for variations between production batches.
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