Abstract
Argonautes (Agos) are core effectors of RNA silencing. In several nonmammalian organisms, multiple Agos are known to exhibit specialized functions for distinct RNA silencing pathway. Mammals have four closely related Agos. To examine the functions of mammalian Agos in the microRNA silencing pathway, we generated mouse embryonic stem (ES) cells that are nullizygous for all Agos. This chapter describes a variety of techniques including BAC recombineering, gene targeting, and inducible Cre-loxP recombination, used to generate inducible Ago knock-out ES cells. The Ago-deficient ES cells provide an important tool for the study of mammalian RNA silencing.
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References
Hammond, S.M., Boettcher, S., Caudy, A.A., Kobayashi, R. and Hannon, G.J. (2001) Argonaute2, a link between genetic and biochemical analyses of RNAi. Science, 293, 1146–1150.
Okamura, K., Ishizuka, A., Siomi, H. and Siomi, M.C. (2004) Distinct roles for Argonaute proteins in small RNA-directed RNA cleavage pathways. Genes Dev, 18, 1655–1666.
Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G. and Tuschl, T. (2004) Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell, 15, 185–197.
Liu, J., Carmell, M.A., Rivas, F.V., Marsden, C.G., Thomson, J.M., Song, J.J., Hammond, S.M., Joshua-Tor, L. and Hannon, G.J. (2004) Argonaute2 is the catalytic engine of mammalian RNAi. Science, 305, 1437–1441.
Ramirez-Solis, R., Liu, P. and Bradley, A. (1995) Chromosome engineering in mice. Nature, 378, 720–724.
Su, H., Trombly, M.I., Chen, J. and Wang, X. (2009) Essential and overlapping functions for mammalian Argonautes in microRNA silencing. Genes Dev, 23, 304–317.
Hasty, P., Crist, M., Grompe, M. and Brad-ley, A. (1994) Efficiency of insertion versus replacement vector targeting varies at different chromosomal loci. Mol Cell Biol, 14, 8385–8390.
Zhang, Y., Buchholz, F., Muyrers, J.P. and Stewart, A.F. (1998) A new logic for DNA engineering using recombination in Escherichia coli. Nat Genet, 20, 123–128.
Muyrers, J.P., Zhang, Y., Testa, G. and Stewart, A.F. (1999) Rapid modification of bacterial artificial chromosomes by ET-recombination. Nucleic Acids Res, 27, 1555–1557.
Matsuda, T. and Cepko, C.L. (2007) Controlled expression of transgenes introduced by in vivo electroporation. Proc Natl Acad Sci USA, 104, 1027–1032.
Acknowledgments
The authors would like to thank A. Bradley and G. Guo for providing NM5 ES cells. Funding was provided by National Institute of Health (5R21GM079528), Illinois Department of Public Health (to X.W.).
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Su, H., Wang, X. (2011). Generation of an Inducible Mouse ES Cell Lines Deficient for Argonaute Proteins. In: Hobman, T., Duchaine, T. (eds) Argonaute Proteins. Methods in Molecular Biology, vol 725. Humana Press. https://doi.org/10.1007/978-1-61779-046-1_19
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DOI: https://doi.org/10.1007/978-1-61779-046-1_19
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