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. 1990 Dec;87(24):9803–9807. doi: 10.1073/pnas.87.24.9803

Long-term expression of a T-cell receptor beta-chain gene in mice reconstituted with retrovirus-infected hematopoietic stem cells.

J Kang 1, J Wither 1, N Hozumi 1
PMCID: PMC55262  PMID: 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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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bassin R. H., Tuttle N., Fischinger P. J. Rapid cell culture assay technic for murine leukaemia viruses. Nature. 1971 Feb 19;229(5286):564–566. doi: 10.1038/229564b0. [DOI] [PubMed] [Google Scholar]
  2. Bender M. A., Gelinas R. E., Miller A. D. A majority of mice show long-term expression of a human beta-globin gene after retrovirus transfer into hematopoietic stem cells. Mol Cell Biol. 1989 Apr;9(4):1426–1434. doi: 10.1128/mcb.9.4.1426. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bodine D. M., Karlsson S., Nienhuis A. W. Combination of interleukins 3 and 6 preserves stem cell function in culture and enhances retrovirus-mediated gene transfer into hematopoietic stem cells. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8897–8901. doi: 10.1073/pnas.86.22.8897. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bordignon C., Yu S. F., Smith C. A., Hantzopoulos P., Ungers G. E., Keever C. A., O'Reilly R. J., Gilboa E. Retroviral vector-mediated high-efficiency expression of adenosine deaminase (ADA) in hematopoietic long-term cultures of ADA-deficient marrow cells. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6748–6752. doi: 10.1073/pnas.86.17.6748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bowtell D. D., Johnson G. R., Kelso A., Cory S. Expression of genes transferred to haemopoietic stem cells by recombinant retroviruses. Mol Biol Med. 1987 Aug;4(4):229–250. [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Davis M. M., Bjorkman P. J. T-cell antigen receptor genes and T-cell recognition. Nature. 1988 Aug 4;334(6181):395–402. doi: 10.1038/334395a0. [DOI] [PubMed] [Google Scholar]
  8. Dick J. E., Magli M. C., Huszar D., Phillips R. A., Bernstein A. Introduction of a selectable gene into primitive stem cells capable of long-term reconstitution of the hemopoietic system of W/Wv mice. Cell. 1985 Aug;42(1):71–79. doi: 10.1016/s0092-8674(85)80102-1. [DOI] [PubMed] [Google Scholar]
  9. Hantzopoulos P. A., Sullenger B. A., Ungers G., Gilboa E. Improved gene expression upon transfer of the adenosine deaminase minigene outside the transcriptional unit of a retroviral vector. Proc Natl Acad Sci U S A. 1989 May;86(10):3519–3523. doi: 10.1073/pnas.86.10.3519. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kawamoto T., Makino K., Niwa H., Sugiyama H., Kimura S., Amemura M., Nakata A., Kakunaga T. Identification of the human beta-actin enhancer and its binding factor. Mol Cell Biol. 1988 Jan;8(1):267–272. doi: 10.1128/mcb.8.1.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kisielow P., Blüthmann H., Staerz U. D., Steinmetz M., von Boehmer H. Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature. 1988 Jun 23;333(6175):742–746. doi: 10.1038/333742a0. [DOI] [PubMed] [Google Scholar]
  12. Koike K., Nakahata T., Takagi M., Kobayashi T., Ishiguro A., Tsuji K., Naganuma K., Okano A., Akiyama Y., Akabane T. Synergism of BSF-2/interleukin 6 and interleukin 3 on development of multipotential hemopoietic progenitors in serum-free culture. J Exp Med. 1988 Sep 1;168(3):879–890. doi: 10.1084/jem.168.3.879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lemischka I. R., Raulet D. H., Mulligan R. C. Developmental potential and dynamic behavior of hematopoietic stem cells. Cell. 1986 Jun 20;45(6):917–927. doi: 10.1016/0092-8674(86)90566-0. [DOI] [PubMed] [Google Scholar]
  14. Lim B., Apperley J. F., Orkin S. H., Williams D. A. Long-term expression of human adenosine deaminase in mice transplanted with retrovirus-infected hematopoietic stem cells. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8892–8896. doi: 10.1073/pnas.86.22.8892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lo D., Sprent J. Identity of cells that imprint H-2-restricted T-cell specificity in the thymus. Nature. 1986 Feb 20;319(6055):672–675. doi: 10.1038/319672a0. [DOI] [PubMed] [Google Scholar]
  16. Mann R., Mulligan R. C., Baltimore D. Construction of a retrovirus packaging mutant and its use to produce helper-free defective retrovirus. Cell. 1983 May;33(1):153–159. doi: 10.1016/0092-8674(83)90344-6. [DOI] [PubMed] [Google Scholar]
  17. Miltenyi S., Müller W., Weichel W., Radbruch A. High gradient magnetic cell separation with MACS. Cytometry. 1990;11(2):231–238. doi: 10.1002/cyto.990110203. [DOI] [PubMed] [Google Scholar]
  18. Myers R. M., Larin Z., Maniatis T. Detection of single base substitutions by ribonuclease cleavage at mismatches in RNA:DNA duplexes. Science. 1985 Dec 13;230(4731):1242–1246. doi: 10.1126/science.4071043. [DOI] [PubMed] [Google Scholar]
  19. Robertson E., Bradley A., Kuehn M., Evans M. Germ-line transmission of genes introduced into cultured pluripotential cells by retroviral vector. Nature. 1986 Oct 2;323(6087):445–448. doi: 10.1038/323445a0. [DOI] [PubMed] [Google Scholar]
  20. Snodgrass R., Keller G. Clonal fluctuation within the haematopoietic system of mice reconstituted with retrovirus-infected stem cells. EMBO J. 1987 Dec 20;6(13):3955–3960. doi: 10.1002/j.1460-2075.1987.tb02737.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Spinella D. G., Hansen T. H., Walsh W. D., Behlke M. A., Tillinghast J. P., Chou H. S., Whiteley P. J., Kapp J. A., Pierce C. W., Shevach E. M. Receptor diversity of insulin-specific T cell lines from C57BL (H-2b) mice. J Immunol. 1987 Jun 1;138(11):3991–3995. [PubMed] [Google Scholar]
  22. T-cell repertoire. Immunol Rev. 1988 Jan;101:1–215. [PubMed] [Google Scholar]
  23. Uchida N., Cone R. D., Freeman G. J., Mulligan R. C., Cantor H. High efficiency gene transfer into murine T cell clones using a retroviral vector. J Immunol. 1986 Mar 1;136(5):1876–1879. [PubMed] [Google Scholar]
  24. Williams D. A., Lemischka I. R., Nathan D. G., Mulligan R. C. Introduction of new genetic material into pluripotent haematopoietic stem cells of the mouse. Nature. 1984 Aug 9;310(5977):476–480. doi: 10.1038/310476a0. [DOI] [PubMed] [Google Scholar]
  25. Wilson J. M., Danos O., Grossman M., Raulet D. H., Mulligan R. C. Expression of human adenosine deaminase in mice reconstituted with retrovirus-transduced hematopoietic stem cells. Proc Natl Acad Sci U S A. 1990 Jan;87(1):439–443. doi: 10.1073/pnas.87.1.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Wilson J. M., Johnston D. E., Jefferson D. M., Mulligan R. C. Correction of the genetic defect in hepatocytes from the Watanabe heritable hyperlipidemic rabbit. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4421–4425. doi: 10.1073/pnas.85.12.4421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Zwiebel J. A., Freeman S. M., Kantoff P. W., Cornetta K., Ryan U. S., Anderson W. F. High-level recombinant gene expression in rabbit endothelial cells transduced by retroviral vectors. Science. 1989 Jan 13;243(4888):220–222. doi: 10.1126/science.2911735. [DOI] [PubMed] [Google Scholar]

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