Journal: International Journal of Molecular Sciences

Article Title: Pre-Treatment with Dacarbazine Sensitizes B16 Melanoma to CAR T Cell Therapy in Syngeneic Mouse Model

doi: 10.3390/ijms27010189

Figure Lengend Snippet: Design of human/murine hybrid epidermal growth factor receptor (EGFR) and EGFR murine chimeric antigen receptor (CAR). ( A ) Schematic of the human/murine hybrid EGFR with cetuximab-binding substitutions (G442S, R467K, M491I, N492S, K497N) and its engagement by a cetuximab-derived single-chain variable fragment (scFv) on mCAR T cells. ( B ) The retroviral vector encoding the EGFR-specific murine CAR and green fluorescent protein (GFP); I—domain I; II—domain II; III—domain III; IV—domain IV.

Article Snippet: The synthetic CAR gene was cloned by PCR amplification with primers containing restriction sites, restriction digest, and ligation (restriction enzymes and ligase from New England Biolabs, Ipswich, MA, USA) into the retroviral vector pMSCV (a gift from Dr. Reya, Addgene, #20672) right before IRES followed by the reporter gene of GFP.

Techniques: Binding Assay, Derivative Assay, Retroviral, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Pre-Treatment with Dacarbazine Sensitizes B16 Melanoma to CAR T Cell Therapy in Syngeneic Mouse Model

doi: 10.3390/ijms27010189

Figure Lengend Snippet: Generation of murine EGFR CAR T cells. ( A ) Workflow for EGFR mCAR T cell production, including T cell isolation, activation, retroviral transduction, and subsequent in vitro and in vivo experiments created with BioRender ( https://www.biorender.com/ (accessed on 1 September 2025)). ( B ) Representative flow cytometry histogram showing the transduction efficiency of murine T cells with the EGFR-targeted murine CAR construct. ( C ) Summary of independent transduction preparations: percentage of GFP + cells in each preparation. ( D ) Expansion ( left ) and viability ( right ) of EGFR mCAR T cells compared to non-transduced T cells over 14 days of culture. * p < 0.05 and *** p < 0.001.

Article Snippet: The synthetic CAR gene was cloned by PCR amplification with primers containing restriction sites, restriction digest, and ligation (restriction enzymes and ligase from New England Biolabs, Ipswich, MA, USA) into the retroviral vector pMSCV (a gift from Dr. Reya, Addgene, #20672) right before IRES followed by the reporter gene of GFP.

Techniques: Cell Isolation, Activation Assay, Retroviral, Transduction, In Vitro, In Vivo, Flow Cytometry, Construct