Marchant Jonathan S
Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455.
Cold Spring Harb Protoc. 2018 Apr 2;2018(4):pdb.prot096990. doi: 10.1101/pdb.prot096990.
The oocyte is a specialized single cell of colossal size (>1 mm diameter) that is highly amenable for microinjection and a stalwart model for heterologous expression. Oocytes are easily obtainable, robust in vitro, and faithfully express injected constructs. Their large size translational capacity provides a huge canvas for observing and recording integrated cellular responses-from studies of single molecules within single cells to medium-throughput drug-screening applications. Most eukaryotic promoters suffice for expression, and the oocyte can functionally express proteins from many diverse organisms. This protocol provides a basic introduction for scientists keen to perform nuclear microinjections of cDNA constructs. These are easy methods to master, do not require elaborate equipment, and make accessible a wonderful model cell system for studying signaling, transport, cell architecture, and protein function.
卵母细胞是一种特殊的单细胞,体积巨大(直径>1毫米),非常适合显微注射,是异源表达的可靠模型。卵母细胞很容易获得,在体外健壮,能忠实地表达注射的构建体。其较大的尺寸和翻译能力为观察和记录整合的细胞反应提供了广阔的空间——从单细胞内单分子的研究到中通量药物筛选应用。大多数真核启动子足以实现表达,卵母细胞能够功能性地表达来自许多不同生物体的蛋白质。本方案为热衷于对cDNA构建体进行核显微注射的科学家提供了基本介绍。这些方法易于掌握,不需要精密的设备,并且为研究信号传导、转运、细胞结构和蛋白质功能提供了一个出色的模型细胞系统。