使用生物传感器测量小鼠卵母细胞中分离酶对减数分裂黏连蛋白亚基 Rec8 切割效率的方案。

Protocol to measure cleavage efficiency of the meiotic cohesin subunit Rec8 by separase in mouse oocytes using a biosensor.

机构信息

CIRB, Collège de France, UMR7241/U1050, 75005 Paris, France.

Institut de Biologie Paris Seine, Sorbonne Université, 7 quai St. Bernard, 75252 Paris, France; CNRS UMR7622 Developmental Biology Lab, Sorbonne Université, 7 quai St. Bernard, 75252 Paris, France.

出版信息

STAR Protoc. 2022 Dec 16;3(4):101714. doi: 10.1016/j.xpro.2022.101714. Epub 2022 Sep 22.

DOI:10.1016/j.xpro.2022.101714

PMID:36149797

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9508607/

Abstract

Here, we describe a biosensor to assess meiotic cohesin subunit Rec8 cleavage in mouse oocytes. We detail oocyte collection and microinjection of the mRNA expressing the biosensor. The biosensor is targeted to chromosomes and consists of two fluorophores flanking a Rec8 fragment containing separase cleavage sites. Cleavage leads to dissociation of one fluorophore from chromosomes, and the efficiency can be estimated by live imaging. We detail the use of this biosensor in mouse oocytes with or without Aurora B/C inhibitor. For complete details on the use and execution of this protocol, please refer to Nikalayevich et al. (2022).

摘要

在这里，我们描述了一种用于评估小鼠卵母细胞中减数分裂黏连蛋白亚基 Rec8 切割的生物传感器。我们详细介绍了卵母细胞的收集和表达生物传感器的 mRNA 的显微注射。该生物传感器靶向染色体，由两个荧光团组成，两侧是含有分离酶切割位点的 Rec8 片段。切割导致一个荧光团从染色体上解离，其效率可以通过活细胞成像来估计。我们详细介绍了该生物传感器在有或没有 Aurora B/C 抑制剂的小鼠卵母细胞中的应用。有关该方案使用和执行的完整详细信息，请参阅 Nikalayevich 等人（2022 年）。