追踪细胞周期蛋白B1-绿色荧光蛋白传感器以描绘有丝分裂与有丝分裂旁路的模式。

Tracking the Cyclin B1-GFP Sensor to Profile the Pattern of Mitosis Versus Mitotic Bypass.

作者信息

Griesdoorn Victoria, Brown M Rowan, Wiltshire Marie, Smith Paul J, Errington Rachel J

机构信息

Institute of Cancer and Genetics, School of Medicine, Cardiff University, Tenovus Building, Heath Park, Cardiff, CF14 4XN, UK.

出版信息

Methods Mol Biol. 2016;1342:279-85. doi: 10.1007/978-1-4939-2957-3_17.

DOI:10.1007/978-1-4939-2957-3_17

PMID:26254931

Abstract

This chapter provides a method for quantitative single cell analysis to track the transition of single cells from G2, indicated by high cyclin B1 levels, to G1 polyploidy phase (G1(p)), indicated by low cyclin B1 levels, in a 4n population. The cell tracking methodology described provides a fluorescence fingerprint suitable for deriving G2/M or G2/G1p transitions. Notably, during late G2 the absolute cyclin B1-eGFP reporter levels obtained were high and the switch-off point identifiable, with destruction rates of a similar order across all cell cycle routing avenues. The three principle parameters extracted were defined as (1) G2-to-G1(p) transition duration (tGFP(off)); (2) rate of sensor destruction (kGFP(off)), and (3) peak sensor expression (GFP(peak)).

摘要

本章提供了一种定量单细胞分析方法，用于追踪4n群体中单个细胞从细胞周期蛋白B1水平高所指示的G2期向细胞周期蛋白B1水平低所指示的G1多倍体期（G1(p)）的转变。所描述的细胞追踪方法提供了一个适用于推导G2/M或G2/G1p转变的荧光指纹图谱。值得注意的是，在G2晚期，所获得的细胞周期蛋白B1-eGFP报告基因的绝对水平很高，且关闭点可识别，在所有细胞周期途径中的降解速率处于相似水平。提取的三个主要参数定义为：(1) G2到G1(p)的转变持续时间（tGFP(off)）；(2) 传感器降解速率（kGFP(off)），以及(3) 传感器表达峰值（GFP(peak)）。