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  • Title: Long-term expression of a T-cell receptor beta-chain gene in mice reconstituted with retrovirus-infected hematopoietic stem cells.
    Author: Kang J, Wither J, Hozumi N.
    Journal: Proc Natl Acad Sci U S A; 1990 Dec; 87(24):9803-7. PubMed ID: 2175916.
    Abstract:
    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) beta-chain gene with the V beta 6 variable segment. The TCR gene was placed under the control of the human beta-actin promoter and enhancer. Bone marrow cells pretreated with 5-fluorouracil were infected by coculturing with psi-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 V beta 6 gene was examined with a V beta 6-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 less than 1 month after transplantation, up to 30% of mature T cells expressed V beta 6 TCRs, an increase of at least 20% above the level of endogenous V beta 6 expression. DNA analysis revealed that pluripotent hematopoietic stem cells were infected by the retroviral vector in a long-term reconstituted mouse that showed increased V beta 6 expression.
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