Open AccessLong-term expression of a T-cell receptor beta-chain gene in mice reconstituted with retrovirus-infected hematopoietic stem cells.
Author(s) -
Joonsoo Kang,
Joan E. Wither,
Nobumichi Hozumi
Publication year - 1990
Publication title -
proceedings of the national academy of sciences of the united states of america
Language(s) - English
Resource type - Journals
eISSN - 1091-6490
pISSN - 0027-8424
DOI - 10.1073/pnas.87.24.9803
Subject(s) - biology , microbiology and biotechnology , stem cell , retrovirus , stem cell factor , haematopoiesis , t cell receptor , viral vector , interleukin 3 , transplantation , genetic enhancement , t cell , virology , il 2 receptor , gene , virus , recombinant dna , immunology , immune system , medicine , biochemistry , surgery
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.