Abstract
A major goal in cell biology is to understand the molecular mechanisms of the biological process under study, which requires functional information about the roles of individual proteins in the cell. For many non-genetic model organisms researchers have relied on the use of inhibitory reagents, such as antibodies that can be microinjected into cells. More recently, the advent of RNA-mediated interference (RNAi) has allowed scientists to knockdown individual proteins and to examine the consequences of the knockdown. In this chapter we present a comparison between microinjection of inhibitory reagents and RNAi for the analysis of protein function in mammalian tissue culture cells, providing both a description of the techniques as well as a discussion of the benefits and drawbacks of each approach. In addition, we present a strategy to employ RNAi for organisms without a sequenced genome. While the focus of our research is on the organization of the mitotic spindle during cell division and thus the examples utilized are from that system, the approaches described here should be readily applicable to multiple experimental models.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Scholey, J. M. (1998). Functions of motor proteins in echinoderm embryos: an argument in support of antibody inhibition experiments. Cell Motil. Cytoskel. 39, 257–260.
Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E. & Mello, C. C. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811.
Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K. & Tuschl, T. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature 411, 494–498.
Khodjakov, A., Copenagle, L., Gordon, M. B., Compton, D. A. & Kapoor, T. M. (2003). Minus-end capture of preformed kinetochore fibers contributes to spindle morphogenesis. J. Cell. Biol. 160, 671–683.
Khodjakov, A. & Rieder, C. L. (2006). Imaging the division process in living tissue culture cells. Methods 38, 2–16.
Stout, J. R., Rizk, R. S., Kline, S. L. & Walczak, C. E. (2006). Deciphering protein function during mitosis in PtK cells using RNAi. BMC Cell Biol. 7, 26.
Mayer, T. U., Kapoor, T. M., Haggarty, S. J., King, R. W., Schreiber, S. L. & Mitchison, T. J. (1999). Small molecule inhibitor of mitotic spindle bipolarity identified in a phenotype-based screen. Science 286, 971–974.
Weil, D., Garcon, L., Harper, M., Dumenil, D., Dautry, F. & Kress, M. (2002). Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells. Biotechniques 33, 1244–1248.
Freshney, R. I. (2005). Culture of Animal Cells: A Manual of Basic Techniques (Freshney, R. I., ed.), Wiley-Liss, New Jersey.
Phelan, M. C. (2003). Basic techniques for mammalian cell tissue culture. In Current Protocols in Cell Biology (Bonifacino, J. S., Dasso, M., Harford, J. B., Lippincott-Schwartz, J. & Yamada, K. M., eds.). Wiley-Liss, New Jersey.
Spector, D. L., Goldman, R. D. & Leinwand, L. A. (1998). Visualization of Organelles, Proteins, and Gene Expression. Cells: A Laboratory Manual, 3. 3 vols, Cold Spring Harbor Laboratory Press, New York.
Harlow, E. & Lane, D. (1988). Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Harborth, J., Elbashir, S. M., Bechert, K., Tuschl, T. & Weber, K. (2001). Identification of essential genes in cultured mammalian cells using small interfering RNAs. J Cell Sci. 114, 4557–4565.
Caplen, N. J., Parrish, S., Imani, F., Fire, A. & Morgan, R. A. (2001). Specific inhibition of gene expression by small double-stranded RNAs in invertebrate and vertebrate systems. Proc Natl Acad Sci U S A 98, 9742–9747.
Liu, S. T., Rattner, J. B., Jablonski, S. A. & Yen, T. J. (2006). Mapping the assembly pathways that specify formation of the trilaminar kinetochore plates in human cells. J Cell Biol. 175, 41–53.
Acknowledgments
The authors would like to thank Susan Kline for early instruction in microinjection and suggestions on transfections of PtK2 cells. The authors would also like to thank Chantal LeBlanc for editing of the manuscript. Work in the Walczak lab is supported by NIH R01GM059618, an ACS Scholar award RSG CSM-106128, and in part by the Indiana METACyt Initiative of Indiana University, funded in part through a major grant from the Lilly Endowment, Inc. Rania Rizk is supported by a predoctoral fellowship from the American Heart Association.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Stout, J.R., Rizk, R.S., Walczak, C.E. (2009). Protein Inhibition by Microinjection and RNA-Mediated Interference in Tissue Culture Cells: Complementary Approaches to Study Protein Function. In: Carroll, D. (eds) Microinjection. Methods in Molecular Biology, vol 518. Humana Press. https://doi.org/10.1007/978-1-59745-202-1_7
Download citation
DOI: https://doi.org/10.1007/978-1-59745-202-1_7
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-58829-884-3
Online ISBN: 978-1-59745-202-1
eBook Packages: Springer Protocols