Nuclear remodeling assay in Xenopus egg extract.
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.
Gonda
Koichi
K
Department of Medicine, Stem Cell Institute, University of Minnesota, Minneapolis, USA.
Kikyo
Nobuaki
N
D000818:Animals; D002466:Cell Nucleolus / Q000378:metabolism; D002467:Cell Nucleus / Q000235:genetics* / Q000502:physiology; D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D008856:Microscopy, Fluorescence; D010063:Ovum / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000502:physiology*; D014157:Transcription Factors / Q000502:physiology*; D014461:Ultracentrifugation; D014981:Xenopus / Q000235:genetics* / Q000378:metabolism; D029867:Xenopus Proteins / Q000378:metabolism / Q000502:physiology*
D016601:RNA-Binding Proteins; D014157:Transcription Factors; D029867:Xenopus Proteins; C075593:YBX2 protein, Xenopus