研究非洲爪蟾卵提取物中纺锤体组装和染色体凝聚的方法。

Methods for studying spindle assembly and chromosome condensation in Xenopus egg extracts.

作者信息

Maresca Thomas J, Heald Rebecca

机构信息

Department of Molecular and Cell Biology, University of California, Berkeley, USA.

出版信息

Methods Mol Biol. 2006;322:459-74. doi: 10.1007/978-1-59745-000-3_33.

DOI:10.1007/978-1-59745-000-3_33

PMID:16739744

Abstract

Methods are presented for preparing cytoplasmic extracts from Xenopus laevis eggs and their utilization to reconstitute and monitor events of the cell cycle in vitro. Addition of sperm nuclei to crude extracts and "cycling" of the reaction through interphase and back into metaphase promotes formation of bipolar spindles capable of segregating their duplicated chromosomes. Reactions can be "spun down" onto cover slips for immunofluorescence analysis. High-speed extracts support mitotic chromosome condensation, which can be observed live by fluorescence time-lapse video microscopy. Because of the biochemically accessible nature of the egg extract system, a wide array of biochemical techniques can be combined with spindle and chromosome assembly reactions to evaluate the roles of specific proteins in these processes.

摘要

本文介绍了从非洲爪蟾卵中制备细胞质提取物的方法，以及利用这些提取物在体外重建和监测细胞周期事件的方法。将精子细胞核添加到粗提取物中，并使反应通过间期再回到中期的“循环”，可促进能够分离其复制染色体的双极纺锤体的形成。反应可以“沉淀”到盖玻片上进行免疫荧光分析。高速提取物支持有丝分裂染色体凝聚，这可以通过荧光延时视频显微镜实时观察到。由于卵提取物系统具有生物化学可及性，因此可以将多种生化技术与纺锤体和染色体组装反应相结合，以评估特定蛋白质在这些过程中的作用。