Spector David L, Goldman Robert D
Cold Spring Harb Protoc. 2010 Nov 1;2010(11):pdb.prot5517. doi: 10.1101/pdb.prot5517.
Fluorescent protein fusions (FPFs) have been used to address a wide range of questions in individual cells as well as in specific tissues of particular organisms. However, investigators must take extreme care when using FPFs to ensure that the resultant fusion protein is expressed at or close to the endogenous level of the parent protein, and also that it is full length, localizes correctly, and behaves normally once incorporated in the cell. Although transient transfection methods can be used to introduce DNA coding for FPFs, in many cases it is beneficial and/or essential to develop stable cell lines expressing the fusion protein of interest. In addition to providing more native levels of expression, the individual clones can be generated from single cells, the integration site of the plasmid can be mapped, and the copy number can be determined. Moreover, because every cell in the population is expressing the fusion protein, cell cycle analyses and biochemical fractionation are significantly easier to accomplish. This article presents a protocol for generating, selecting, and screening stable cell lines expressing FPFs.
荧光蛋白融合体（FPFs）已被用于解决单个细胞以及特定生物体特定组织中的一系列问题。然而，研究人员在使用FPFs时必须格外小心，以确保产生的融合蛋白在亲本蛋白的内源性水平或接近该水平表达，并且它是全长的，定位正确，一旦整合到细胞中行为正常。虽然可以使用瞬时转染方法来引入编码FPFs的DNA，但在许多情况下，开发表达感兴趣融合蛋白的稳定细胞系是有益的和/或必不可少的。除了提供更接近天然水平的表达外，单个克隆可以从单细胞产生，可以绘制质粒的整合位点，并可以确定拷贝数。此外，由于群体中的每个细胞都在表达融合蛋白，因此细胞周期分析和生化分级分离更容易完成。本文介绍了一种生成、选择和筛选表达FPFs的稳定细胞系的方案。
