Abstract
The generation of chimeric antigen receptor (CAR) T cells requires the transfer of the CAR gene into primary T cells. Among various gene transfer strategies, gammaretroviral vectors have been widely used to generate CAR T cells for both preclinical and clinical settings. Here we describe the detailed method of generating CAR T cells utilizing gammaretroviral vectors. This approach consists of two parallel parts: (1) production of the gammaretroviral particles and (2) gammaretroviral transduction of activated T cells. The gammaretroviral particles are produced by co-transfecting the gammaretroviral vector with packaging plasmids into 293T cells. The manufactured viral particles then efficiently infect activated T cells where the CAR transgene is integrated into host genomic DNA, resulting in stable expression of the CAR molecule on the surface of T cells.