LONG-TERM EXPRESSION OF A T-CELL RECEPTOR BETA-CHAIN GENE IN MICE RECONSTITUTED WITH RETROVIRUS-INFECTED HEMATOPOIETIC STEM-CELLS

To determine the feasibility of retrovirus-mediated gene transfer into stem cells for studying T-cell development, we constructed a high-titer retrovirus vector containing the neomycin phosphotransferase (neo) gene and a murine T-cell receptor (TCR) β-chain gene with the Vβ6 variable segment. The TCR gene was placed under the control of the human β-actin promoter and enhancer. Bone marrow cells pretreated with 5-fluorouracil were infected by coculturing with ψ-2 virus-producing cells in the presence of recombinant interleukins 1, 2, 4, and 6 as well as interleukin 3 from WEHI-3 conditioned medium. The infected cells were transplanted into irradiated mice, and expression of the exogenous Vβ6 was examined with a Vβ6-specific monoclonal antibody, RNase protection, and polymerase chain reaction amplification. Three of seven mice expressed the retroviral TCR gene on the surface of a significant proportion of mature T cells 5-6 months after transplantation. In mice analyzed month after transplantation, up to 30% of mature T cells expressed Vβ6 TCRs, an increase of at least 20% above the level of endogenous Vβ6 expression. DNA analysis revealed that pluripotent bematopoietic stem cells were infected by the retroviral vector in a long-term reconstituted mouse that showed increased Vβ6 expression.