Quantitative Assessment of Transduced CD8 T Cells

Genomic DNA was isolated from transduced CD8 T cells with the iPrep Purification Instrument (Thermo fisher scientific) and qPCR analysis was performed using ABI Taqman technology, with a modified version of the previously described assay designed to detect the integrated CD4-zeta sequence in genomic DNA (gDNA) [22 (link)]. To determine copy number per unit DNA, a standard curve was generated consisting of 5 to 10⁶ plasmid copies spiked into 200 ng nontransduced control gDNA. The plasmid copy number in the standard curve was verified using digital qPCR with the same CD4-z primer/probe set, and performed on a QuantStudio 3D digital PCR instrument (Life Technologies). Each data-point was evaluated in triplicate with a positive Ct value and % CV less than 0.95% for all quantifiable values. To control for the quantity of interrogated DNA, a parallel amplification reaction was performed using 10 ng gDNA and a primer/probe set specific for a non-transcribed genomic sequence upstream of the CDKN1A (p21) gene as previously described [72 (link)]. These amplification reactions generated a correction factor to adjust for calculated versus actual DNA input. Copies of transgene per cell were calculated according to the formula: [Average copies of transgene(from qPCR)x gDNA input Correction Factor/Input gDNA(ng)]x 0.0063 ng gDNA/cell.