Abstract
Integrins play critical roles in adhesion and migration of T cells during an immune response and inflammation. It is of great importance to understand the molecular pathways that regulate integrin function in T cells. Lentiviral vector-based gene transfer method has emerged in the past decade as an efficient means of transferring genes into both resting and activated hard-to-transfect cells, including T cells to knockdown gene expression. Therefore, this technology could be utilized effectively to study different aspects of integrin function or even to perform genome-wide RNAi screens to look globally for regulators of integrin function in T cells. In this chapter, we provide the simplest protocol to infect activated CD4+ human T cells with high efficiency.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Brown, N. H. (2000) Cell-cell adhesion via the ECM: Integrin genetics in fly and worm. Matrix Biol. 19, 191–201.
Cram, E. J., Shang, H., and Schwarzbauer, J. E. (2006) A systematic RNA interference screen reveals a cell migration gene network in C. elegans. J. Cell Sci. 119, 4811–4818.
Wang, X., Bo, J., Bridges, T., Dugan, K. D., Pan, T. C., Chodosh, L. A., and Montell, D. J. (2006) Analysis of cell migration using whole-genome expression profiling of migratory cells in the Drosophila ovary. Dev. Cell 10, 483–495.
Cram, E. J., Clark, S. G., and Schwarzbauer, J. E. (2003) Talin loss-of-function uncovers roles in cell contractility and migration in C. elegans. J. Cell Sci. 116, 3871–3878.
Hemler, M. E. (1990) VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu. Rev. Immunol. 8, 365–400.
Luo, B. H., Carman, C. V., and Springer, T. A. (2007) Structural basis of integrin regulation and signaling. Annu. Rev. Immunol. 25, 619–647.
Springer, T. A. (1990) Adhesion receptors of the immune system. Nature 346, 425–434.
Askari, J. A., Buckley, P. A., Mould, A. P., and Humphries, M. J. (2009) Linking integrin conformation to function. J. Cell Sci. 122, 165–170.
Stephens, L. E., Sutherland, A. E., Klimanskaya, I. V., Andrieux, A., Meneses, J., Pedersen, R. A., and Damsky, C. H. (1995) Deletion of beta 1 integrins in mice results in inner cell mass failure and peri-implantation lethality. Genes. Dev. 9, 1883–1895.
Yang, J. T., Rayburn, H., and Hynes, R. O. (1995) Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development. Development 121, 549–560.
Radtke, F., Wilson, A., Mancini, S. J., and MacDonald, H. R. (2004) Notch regulation of lymphocyte development and function. Nat. Immunol. 5, 247–253.
Fischer, K. D., Zmuldzinas, A., Gardner, S., Barbacid, M., Bernstein, A., and Guidos, C. (1995) Defective T-cell receptor signalling and positive selection of Vav-deficient CD4+ CD8+ thymocytes. Nature 374, 474–477.
Etzioni, A. (2009) Genetic etiologies of leukocyte adhesion defects. Curr. Opin. Immunol. 21, 481–486.
Svensson, L., Howarth, K., McDowall, A., Patzak, I., Evans, R., Ussar, S., Moser, M., Metin, A., Fried, M., Tomlinson, I., and Hogg, N. (2009) Leukocyte adhesion deficiency-III is caused by mutations in KINDLIN3 affecting integrin activation. Nat. Med. 15, 306–312.
Manevich-Mendelson, E., Feigelson, S. W., Pasvolsky, R., Aker, M., Grabovsky, V., Shulman, Z., Kilic, S. S., Rosenthal-Allieri, M. A., Ben-Dor, S., Mory, A., Bernard, A., Moser, M., Etzioni, A., and Alon, R. (2009) Loss of Kindlin-3 in LAD-III eliminates LFA-1 but not VLA-4 adhesiveness developed under shear flow conditions. Blood 114, 2344–2353.
Rubinson, D. A., Dillon, C. P., Kwiatkowski, A. V., Sievers, C., Yang, L., Kopinja, J., Rooney, D. L., Zhang, M., Ihrig, M. M., McManus, M. T., Gertler, F. B., Scott, M. L., and Van Parijs, L. (2003) A lentivirus-based system to functionally silence genes in primary mammalian cells, stem cells and transgenic mice by RNA interference. Nat. Genet. 33, 401–406.
Zhao, Y., Zheng, Z., Cohen, C. J., Gattinoni, L., Palmer, D. C., Restifo, N. P., Rosenberg, S. A., and Morgan, R. A. (2006) High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation. Mol. Ther. 13, 151–159.
Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. H., Verma, I. M., and Trono, D. (1996) In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science 272, 263–267.
Lois, C., Hong, E. J., Pease, S., Brown, E. J., and Baltimore, D. (2002) Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors. Science 295, 868–872.
Baker, R. G., Hsu, C. J., Lee, D., Jordan, M. S., Maltzman, J. S., Hammer, D. A., Baumgart, T., and Koretzky, G. A. (2009) The adapter protein SLP-76 mediates “outside-in” integrin signaling and function in T cells. Mol. Cell. Biol. 29, 5578–5589.
Gong, H., Shen, B., Flevaris, P., Chow, C., Lam, S. C., Voyno-Yasenetskaya, T. A., Kozasa, T., and Du, X. G protein subunit Galpha13 binds to integrin alphaIIbbeta3 and mediates integrin “outside-in” signaling. Science 327, 340–343.
Primo, L., di Blasio, L., Roca, C., Droetto, S., Piva, R., Schaffhausen, B., and Bussolino, F. (2007) Essential role of PDK1 in regulating endothelial cell migration. J. Cell Biol. 176, 1035–1047.
Jani, K., and Schock, F. (2007) Zasp is required for the assembly of functional integrin adhesion sites. J. Cell Biol. 179, 1583–1597.
Lafuente, E. M., van Puijenbroek, A. A., Krause, M., Carman, C. V., Freeman, G. J., Berezovskaya, A., Constantine, E., Springer, T. A., Gertler, F. B., and Boussiotis, V. A. (2004) RIAM, an Ena/ VASP and Profilin ligand, interacts with Rap1-GTP and mediates Rap1-induced adhesion. Dev. Cell 7, 585–595.
Zhang, Z., Rehmann, H., Price, L. S., Riedl, J., and Bos, J. L. (2005) AF6 negatively regulates Rap1-induced cell adhesion. J. Biol. Chem. 280, 33200–33205.
Verhoeyen, E., Costa, C., and Cosset, F. L. (2009) Lentiviral vector gene transfer into human T cells. Methods Mol. Biol. 506, 97–114.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2011 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Banerjee, D., Shimaoka, M. (2011). Lentiviral Gene Transfer Method to Study Integrin Function in T Lymphocytes. In: Shimaoka, M. (eds) Integrin and Cell Adhesion Molecules. Methods in Molecular Biology, vol 757. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-166-6_4
Download citation
DOI: https://doi.org/10.1007/978-1-61779-166-6_4
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-165-9
Online ISBN: 978-1-61779-166-6
eBook Packages: Springer Protocols