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) 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.
Kang
J
J
Division of Molecular Immunology and Neurobiology, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Canada.
Wither
J
J
Hozumi
N
N
D000199:Actins / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001854:Bone Marrow Cells; D002460:Cell Line; D004742:Enhancer Elements, Genetic; D005260:Female; D015870:Gene Expression; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000276:immunology*; D019868:Kanamycin Kinase; D046911:Macromolecular Substances; D051379:Mice; D008810:Mice, Inbred C57BL; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000235:genetics*; D015345:Oligonucleotide Probes; D010770:Phosphotransferases / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011948:Receptors, Antigen, T-Cell / Q000235:genetics*; D012091:Repetitive Sequences, Nucleic Acid; D014162:Transfection
D000199:Actins; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D011948:Receptors, Antigen, T-Cell; D010770:Phosphotransferases; D019868:Kanamycin Kinase