DNA Transposons for Modification of Human Primary T Lymphocytes

  • Xin Huang
  • Andrew Wilber
  • R. Scott McIvor
  • Xianzheng Zhou
Part of the Methods In Molecular Biology™ book series (MIMB, volume 506)


Genetic modification of peripheral blood T lymphocytes (PBL) or hematopoietic stem cells (HSC) has been shown to be promising in the treatment of cancer (Nat Rev Cancer 3:35–45, 2003), transplant complications (Curr Opin Hematol 5:478–482, 1998), viral infections (Science 285:546–551, 1999), and immunodeficiencies (Nat Rev Immunol 2:615–621, 2002). There are also significant implications for the study of T cell biology (J Exp Med 191:2031–2037, 2000). Currently, there are three types of vectors that are commonly used for introducing genes into human primary T cells: oncoretroviral vectors, lentiviral vectors, and naked DNA. Oncoretroviral vectors transduce and integrate only in dividing cells. However, it has been shown that extended ex vivo culture, required by oncoretroviral-mediated gene transfer, may alter the biologic properties of T cells (Nat Med 4:775–780, 1998; Int Immunol 9:1073– 1083, 1997; Hum Gene Ther 11:1151–1164, 2001; Blood 15:1165–1173, 2002; Proc Natl Acad Sci U S A, 1994). HIV-1-derived lentiviral vectors have been shown to transduce a variety of slowly dividing or nondividing cells, including unstimulated T lymphocytes (Blood 96:1309–1316, 2000; Gene Ther 7:596–604, 2000; Blood 101:2167–2174, 2002; Hum Gene Ther 14:1089–1105, 2003). However, achieving effective gene transfer and expression using lentivirus vectors can be complex, and there is at least a perceived risk associated with clinical application of a vector based on a human pathogen (i.e., HIV-1). Recently it has been found that oncoretroviral and lentiviral vectors show a preference for integration into regulatory sequences and active genes, respectively (Cell 110:521–529, 2002; Science 300:1749–1751, 2003). Additionally, insertional mutagenesis has become a serious concern, after several patients treated with an oncoretroviral vector for X-linked SCID developed a leukemia-like syndrome associated with activation of the LMO2 oncogene (Science 302:415–419, 2003). Naked DNA-based genetic engineering of human T lymphocytes also requires T cells to be activated prior to gene transfer (Mol Ther 1:49–55, 2000; Blood 101:1637–1644, 2003; Blood 107:2643–2652, 2006). In addition, random integration by electroporation is of low efficiency. We have recently reported that the Sleeping Beauty transposon system can efficiently mediate stable transgene expression in human primary T cells without prior T cell activation (Blood 107:483–491, 2006). This chapter describes methodology for the introduction of SB transposons into human T cell cultures with subsequent integration and stable long-term expression at noticeably high efficiency for a nonviral gene transfer system.

Key words

Transposons Sleeping Beauty transposon Human T lymphocytes Nonviral DNA Gene transfer 



This work was supported by a Grant-in-Aid of Research, Artistry and Scholarship from the Graduate School, University of Minnesota, the Minnesota Medical Foundation, the Children's Cancer Research Fund, the Alliance for Cancer Gene Therapy Young Investigator Award, the G&P Foundation for Cancer Research, the Sidney Kimmel Foundation for Cancer Research Kimmel Scholar Award, and the National Blood Foundation. X.Z. is the recipient of an American Society of Hematology Junior Faculty Scholar Award.


  1. 1.
    Ivics, Z., Hackett, P. B., Plasterk, R. H., Izsvak, Z. (1997) Molecular reconstruction of Sleeping Beauty, a Tc1-like transposon from fish, and its transposition in human cells. Cell 91, 501 –510CrossRefGoogle Scholar
  2. 2.
    Izsvak, Z., Ivics, Z. (2004) Sleeping Beauty transposon: biology and applications for molecular therapy. Mol Ther 9, 147 –156CrossRefPubMedGoogle Scholar
  3. 3.
    Geurts, A. M., Yang, Y., Clark, K. J., et al. (2003) Gene transfer into genomes of human cells by the Sleeping Beauty transposon system. Mol Ther 8, 108 –117CrossRefPubMedGoogle Scholar
  4. 4.
    Yant, S. R., Wu, X., Huang, Y., et al. (2005) High-resolution genome-wide mapping of transposon integration in mammals. Mol Cell Biol 25, 2085 –2094CrossRefPubMedGoogle Scholar
  5. 5.
    Yant, S. R., Meuse, L., Chiu, W., Ivics, Z., Izsvak, Z., Kay, M. A. (2000) Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system. Nat Genet 25, 35 –41CrossRefPubMedGoogle Scholar
  6. 6.
    Belur, L. B., Frandsen, J. L., Dupuy, A. J., et al. (2003) Gene insertion and long-term expression in lung mediated by the Sleeping Beauty transposon system. Mol Ther 8, 501 –507CrossRefPubMedGoogle Scholar
  7. 7.
    Luo, G., Ivics, Z., Izsvak, Z., Bradley, A. (1998) Chromosomal transposition of a Tc1/mariner-like element in mouse embryonic stem cells. Proc Natl Acad Sci USA 95, 10769 –10773CrossRefPubMedGoogle Scholar
  8. 8.
    Dupuy, A. J., Clark, K., Carlson, C. M., et al. (2002) Mammalian germ-line transgenesis by transposition. Proc Natl Acad Sci USA 99, 4495 –4499CrossRefPubMedGoogle Scholar
  9. 9.
    Collier, L. S., Carlson, C. M., Ravimohan, S., Dupuy, A. J., Largaespada, D. A. (2005) Cancer gene discovery in solid tumours using transposon-based somatic mutagenesis in the mouse. Nature 436, 272 –276CrossRefPubMedGoogle Scholar
  10. 10.
    Dupuy, A. J., Akagi, K., Largaespada, D. A., Copeland, N. G., Jenkins, N. A. (2005) Mammalian mutagenesis using a highly mobile somatic Sleeping Beauty transposon system. Nature 436, 221 –226CrossRefPubMedGoogle Scholar
  11. 11.
    Keng, V. W., Yae, K., Hayakawa, T., Mizuno, S., Uno, Y., Yusa, K., Kokubu, C., Kinoshita, T., Akagi, K., Jenkins, N. A., Copeland, N. G., Horie, K., Takeda, J. (2005) Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system. Nat Methods 2, 763 –769CrossRefPubMedGoogle Scholar
  12. 12.
    Kitada, K., Ishishita, S., Tosaka, K., Takahashi, R., Ueda, M., Keng, V. W., Horie, K., Takeda, J. (2007) Transposon-tagged mutagenesis in the rat. Nat Methods 4, 131 –133CrossRefPubMedGoogle Scholar
  13. 13.
    Huang, X., Wilber, A. C., Bao, L., Tuong, D., Tolar, J., Orchard, P., et al., (2006) Stable gene transfer and expression in human primary T cells by the Sleeping Beauty transposon system. Blood 107, 483 –491CrossRefPubMedGoogle Scholar
  14. 14.
    Akagi, Y., Isaka, Y., Akagi, A., et al. (1999). Transcriptional activation of a hybrid promoter composed of cytomegalovirus enhancer and beta-actin/beta-globin gene in glomeru-lar epithelial cells in vivo. Kidney Int 1999 51, 1265 –1269CrossRefGoogle Scholar
  15. 15.
    Amendola, M., Venneri, M. A., Biffi, A., Vigna, E., Naldini, L. (2004) Coordinate dual-gene transgenesis by lentiviral vectors carrying synthetic bidirectional promoters. Nat Biotechnol 23, 108 –116CrossRefPubMedGoogle Scholar
  16. 16.
    Levine, B. L., Bernstein, W. B., Aronson, N. E., et al. (2002) Adoptive transfer of cos-timulated CD4+ T cells induces expansion of peripheral T cells and decreased CCR5 expression in HIV infection. Nat Med 8, 47 –53CrossRefPubMedGoogle Scholar
  17. 17.
    Riddell, S. R., Greenberg, P. D. (1990) The use of anti-CD3 and anti-CD28 monoclonal antibodies to clone and expand human antigen-specific T cells. J Immunol Methods 128, 189 –201CrossRefPubMedGoogle Scholar
  18. 18.
    Sambrook, J., Russell, D. W. (2001) Molecular cloning: a laboratory manual. 3rd Edition. New York, NY : Cold Spring Harbor Laboratory PressGoogle Scholar
  19. 19.
    Hirt, B. (1967) Selective extraction of poly-oma DNA from infected mouse cell cultures. J Mol Biol 26, 365 –369CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press 2009

Authors and Affiliations

  • Xin Huang
    • 1
  • Andrew Wilber
    • 2
  • R. Scott McIvor
    • 3
  • Xianzheng Zhou
    • 1
  1. 1.The Division of Blood and Marrow Transplantation, Department of PediatricsThe Cancer Center, University of MinnesotaMinneapolisUSA
  2. 2.Department of Genetics, Cell Biology and DevelopmentUniversity of MinnesotaMinneapolisUSA
  3. 3.The Cancer Center, Department of GeneticsCell Biology and Development, University of MinnesotaMinneapolisUSA

Personalised recommendations