Transfection of mammalian cells with fluorescent protein fusions

Spector, D. L., Goldman, R. D. (2010) Transfection of mammalian cells with fluorescent protein fusions. Cold Spring Harbor Protocols, 2010 (11). ISSN 1559-6095

URL: http://www.ncbi.nlm.nih.gov/pubmed/21041390
DOI: 10.1101/pdb.prot5517

Abstract

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.

Item Type: Paper
Uncontrolled Keywords: Animals *Eukaryotic Cells Humans Luminescent Proteins/genetics/*metabolism Mammals Plasmids Recombinant Fusion Proteins/genetics/*metabolism Selection, Genetic Staining and Labeling/*methods Transfection/*methods
Subjects: organs, tissues, organelles, cell types and functions > cell types and functions > cell types
organs, tissues, organelles, cell types and functions > cell types and functions > cell types
organs, tissues, organelles, cell types and functions > cell types and functions > cell types

bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > green fluorescent protein
organism description > animal > mammal
organism description > virus > viral transfection
CSHL Authors:
Communities: CSHL labs > Spector lab
Depositing User: Matt Covey
Date Deposited: 26 Mar 2014 20:23
Last Modified: 26 Jan 2015 15:48
Related URLs:
URI: http://repository.cshl.edu/id/eprint/29712

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