Abstract
Neutrophils and related phagocytic leukocytes are notoriously difficult to transfect, making the introduction of proteins into these cells for biological studies problematic. We describe here two methods that have been successfully used to introduce proteins into intact primary human neutrophils while maintaining normal functional responses. The first utilizes a lipid-based reagent that transports proteins into intact neutrophils. This method is quick, easy, and is capable of transducing greater than 90% of the neutrophils in the population being studied. The second method involves the addition of a sequence derived from the HIV TAT protein to the protein to be introduced into the neutrophil. This requires both molecular biology to generate the initial construct as well as special procedures for protein isolation and renaturation. However, it also results in highly effective functional protein delivery into human neutrophils.
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References
Bokoch, G. M. (2005) Regulation of innate immunity by Rho GTPases. Trends Cell Biol. 15, 163–171.
Joloit, A. and Prochiantz, A. (2004) Transduction peptides: from technology to physiology. Nat. Cell Biol. 6, 189–196.
Schwarze, S. R., Hruska, K. A., and Dowdy, S. F. (2000) Protein transduction: unrestricted delivery into all cells? Trends Cell Biol. 10, 290–295.
Becker-Hapak, M., McAllister, S. S., and Dowdy, S. F. (2001) TAT-mediated protein transduction into mammalian cells. Methods 24, 247–256.
Han, H., Fuortes, M., and Nathan, C. (2003) Critical role of the carboxyl terminus of proline-rich tyrosine kinase (Pyk2) in the activation of human neutrophils by tumor necrosis factor: separation of signals for the respiratory burst and degranulation. J. Exp. Med. 197, 63–75.
Richard, J. P., Melikov, K., Vives, E., et al. (2003) Cell-penetrating peptides-A reevaluation of the mechanism of cellular uptake. J. Biol. Chem. 278, 585–590.
Gardiner, E. M., Pestonjamasp, K. N., Bohl, B. P., Chamberlin, C., Hahn, K. M., and Bokoch, G. M. (2002) Spatial and temporal analysis of Rac activation during live neutrophil chemotaxis. Current Biol. 12, 2029–2034.
Zhao, T. and Bokoch, G. M. (2005) Critical role of proline-rich tyrosine kinase 2 in reversion of the adhesion-mediated suppression of reactive oxygen species generation by human neutrophils. J. Immunol. 174, 8049–8055.
Zhao, T., Benard, V., Bohl, B. P., and Bokoch, G. M. (2003) The molecular basis for adhesion-mediated suppression of reactive oxygen species generation by human neutrophils. J. Clin. Invest. 112, 1732–1740.
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© 2007 Humana Press Inc., Totowa, NJ
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Zhao, T., Bokoch, G.M. (2007). Transduction of Proteins into Intact Neutrophils. In: Quinn, M.T., DeLeo, F.R., Bokoch, G.M. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology™, vol 412. Humana Press. https://doi.org/10.1007/978-1-59745-467-4_9
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DOI: https://doi.org/10.1007/978-1-59745-467-4_9
Publisher Name: Humana Press
Print ISBN: 978-1-58829-788-4
Online ISBN: 978-1-59745-467-4
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