Abstract
Xenopus egg extract is an ideal material to identify nuclear remodeling activities important for nuclear cloning. Since the protocol for egg extract preparation was established more than 20 yr ago, egg extract has been widely used as a source for the purification of factors associated with a number of physiological activities. The eggs are large, easily obtained in large quantities, and are abundant in a variety of bioactive proteins. Many aspects of somatic nuclear remodeling observed in nuclear cloning are recapitulated in somatic nuclei incubated in egg extract. Our in vitro nuclear remodeling assay has proven effective for the purification of two novel nuclear remodeling activities, ISWI, a key player in the dissociation of TATA binding protein from chromatin, and FRGY2a and FRGY2b, two proteins capable of nucleolar disassembly. Here we outline our protocol of egg extract preparation and in vitro nuclear remodeling assay, as well as the purification method for FRGY2a and FRGY2b. Our in vitro nuclear remodeling assay in combination with egg extract will serve as a powerful tool with which to biochemically uncover molecular events involved in nuclear cloning.
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References
Gurdon, J. B., Laskey, R. A., De Robertis, E. M., and Partington, G. A. (1979) Reprogramming of transplanted nuclei in amphibia. Int. Rev. Cytol. Suppl. 161–178.
Hochedlinger, K. and Jaenisch, R. (2002) Nuclear transplantation: lessons from frogs and mice. Curr Opin Cell Biol 14, 741–748.
Rideout, W. M., 3rd, Eggan, K., and Jaenisch, R. (2001) Nuclear cloning and epigenetic reprogramming of the genome. Science 293, 1093–1098.
Solter, D. (2000) Mammalian cloning: advances and limitations. Nat Rev Genet 1, 199–207.
Kikyo, N. and Wolffe, A. P. (2000) Reprogramming nuclei: insights from cloning, nuclear transfer and heterokaryons. J Cell Sci 113, 11–20.
Lohka, M. J. and Masui, Y. (1983) Formation in vitro of sperm pronuclei and mitotic chromosomes induced by amphibian ooplasmic components. Science 220, 719–721.
Blow, J. J. and Laskey, R. A. (1986) Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs. Cell 47, 577–587.
Murray, A. W. and Kirschner, M. W. (1989) Cyclin synthesis drives the early embryonic cell cycle. Nature 339, 275–280.
Laskey, R. A., Honda, B. M., Mills, A. D., and Finch, J. T. (1978) Nucleosomes are assembled by an acidic protein which binds histones and transfers them to DNA. Nature 275, 416–420.
Newmeyer, D. D., Farschon, D. M., and Reed, J. C. (1994) Cell-free apoptosis in Xenopus egg extracts: inhibition by Bcl-2 and requirement for an organelle fraction enriched in mitochondria. Cell 79, 353–364.
Gorlich, D., Prehn, S., Laskey, R. A., and Hartmann, E. (1994) Isolation of a protein that is essential for the first step of nuclear protein import. Cell 79, 767–778.
Wade, P. A. and Kikyo, N. (2002) Chromatin remodeling in nuclear cloning. Eur. J. Biochem. 269, 2284–2287.
Kikyo, N., Wade, P. A., Guschin, D., Ge, H., and Wolffe, A. P. (2000) Active remodeling of somatic nuclei in egg cytoplasm by the nucleosomal ATPase ISWI. Science 289, 2360–2362.
Gonda, K., Fowler, J., Katoku-Kikyo, N., Haroldson, J., Wudel, J., and Kikyo, N. (2003) Reversible disassembly of somatic nucleoli by the germ cell proteins FRGY2a and FRGY2b. Nat. Cell Biol. 5, 205–210.
Smythe, C. and Newport, J. W. (1991) Systems for the study of nuclear assembly, DNA replication, and nuclear breakdown in Xenopus laevis egg extracts. Methods Cell Biol. 35, 449–468.
Gurdon, J. B. (1976) Injected nuclei in frog oocytes: fate, enlargement, and chromatin dispersal. J. Embryol. Exp. Morphol. 36, 523–540.
Adam, S. A., Sterne-Marr, R., and Gerace, L. (1992) Nuclear protein import using digitonin-permeabilized cells. Methods Enzymol. 219, 97–110.
Madine, M. A., and Coverley, D. (1997) Xenopus replication assays. Methods Enzymol. 283, 535–549.
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© 2006 Humana Press Inc.
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Gonda, K., Kikyo, N. (2006). Nuclear Remodeling Assay in Xenopus Egg Extract. In: Verma, P.J., Trounson, A.O. (eds) Nuclear Transfer Protocols. Methods in Molecular Biology™, vol 348. Humana Press. https://doi.org/10.1007/978-1-59745-154-3_17
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DOI: https://doi.org/10.1007/978-1-59745-154-3_17
Publisher Name: Humana Press
Print ISBN: 978-1-58829-280-3
Online ISBN: 978-1-59745-154-3
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