Bernis Cyril, Forbes Douglass J
Cell and Developmental Biology, University of California, San Diego, California, USA.
Methods Cell Biol. 2014;122:165-91. doi: 10.1016/B978-0-12-417160-2.00008-4.
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.
大型且复杂的真核细胞核是DNA复制、RNA转录、剪接和核糖体组装的仲裁者。随着20世纪80年代从非洲爪蟾卵中提取的体外细胞核重建提取物的出现，围绕添加的DNA或染色质底物在体外组装多个细胞核成为可能。这种重建的细胞核包含核纤层、双层核膜、核孔，并且具备DNA复制和核输入的能力。体外细胞核重建使得能够组装“野生型”和“生化突变型”细胞核，从而可以评估单个组件的影响。在这里，我们描述了制备细胞核重建提取物、体外细胞核重建、评估核膜完整性以及一种更专门的将核孔组装到预先形成的无孔核中间体的检测方法的方案。
