title: Analysis of nuclear reconstitution, nuclear envelope assembly, and nuclear pore assembly using Xenopus in vitro assays.
abstract: The large and complex eukaryotic nucleus is the arbiter of DNA replication, RNA transcription, splicing, and ribosome assembly. With the advent of in vitro nuclear reconstitution extracts derived from Xenopus eggs in the 1980s, it became possible to assemble multiple nuclei in vitro around added DNA or chromatin substrates. Such reconstituted nuclei contain a nuclear lamina, double nuclear membranes, nuclear pores, and are competent for DNA replication and nuclear import. In vitro nuclear reconstitution has allowed the assembly of "wild-type" and "biochemically mutant" nuclei in which the impact of individual components can be assessed. Here, we describe protocols for preparation of the nuclear reconstitution extract, nuclear reconstitution in vitro, assessment of nuclear membrane integrity, and a more specialized assay for nuclear pore assembly into preformed pore-free nuclear intermediates.
authors.affiliation: Cell and Developmental Biology, University of California, San Diego, California, USA.
authors.forename: Cyril
authors.initials: C
authors.lastname: Bernis
authors.affiliation: Cell and Developmental Biology, University of California, San Diego, California, USA.
authors.forename: Douglass J
authors.initials: DJ
authors.lastname: Forbes
mesh_terms: D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism*; D002843:Chromatin / Q000235:genetics; D004247:DNA / Q000235:genetics; D004261:DNA Replication; D022022:Nuclear Pore / Q000378:metabolism*; D009687:Nuclear Proteins; D010063:Ovum / Q000166:cytology*; D014982:Xenopus laevis
chemical_list: D002843:Chromatin; D009687:Nuclear Proteins; D004247:DNA
keywords: 70kDa rhodamine dextran; BAPTA; Membrane integrity; Nuclear envelope; Nuclear membrane assembly; Nuclear pore complex; Nuclear pore complex assembly; Nuclear pore-free intermediates; Nuclear reconstitution assay; Vesicle–vesicle fusion; Xenopus egg extract; mAb414