Document Detail


Transfection of mammalian cells with fluorescent protein fusions.
MedLine Citation:
PMID:  21041390     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
INTRODUCTION 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.
Authors:
David L Spector; Robert D Goldman
Publication Detail:
Type:  Journal Article     Date:  2010-11-01
Journal Detail:
Title:  Cold Spring Harbor protocols     Volume:  2010     ISSN:  1559-6095     ISO Abbreviation:  Cold Spring Harb Protoc     Publication Date:  2010 Nov 
Date Detail:
Created Date:  2010-11-02     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  101524530     Medline TA:  Cold Spring Harb Protoc     Country:  United States    
Other Details:
Languages:  eng     Pagination:  pdb.prot5517     Citation Subset:  IM    
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