Adoptive immunotherapy using chimeric antigen receptor-expressing T (CART) cells has attracted attention as an efficacious strategy for cancer treatment. To prove the efficacy and safety of CART cell therapy, the elucidation of immunological mechanisms underlying it in mice is required. Although a retroviral vector (Rv) is mainly used for the introduction of CAR to murine T cells, gene transduction efficiency is generally less than 50%. The low transduction efficiency causes poor precision in the functional analysis of CART cells. We attempted to improve the Rv gene transduction protocol to more efficiently generate functional CART cells by optimizing the period of pre-cultivation and antibody stimulation. In the improved protocol, gene transduction efficiency to murine T cells was more than 90%. In addition, almost all of the prepared murine T cells expressed CAR after puromycin selection. These CAR-T cells had antigen -specific cytotoxic activity and secreted multiple cytokines by antigen stimulation. We believe that our optimized gene transduction protocol for murine T cells contributes to the advancement of T cell biology and development of immunotherapy using genetically engineered T cells. (C) 2016 Elsevier Inc. All rights reserved.