Document Detail


Quantitative assays for cell fusion.
MedLine Citation:
PMID:  18979254     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Cell fusion would seem to be obviously recognizable upon visual inspection, and many studies employ a simple microscopic fusion index to quantify the rate and extent of fusion in cell culture. However, when cells are not in monolayers or when there is a large background of multinucleation through failed cytokinesis, cell-cell fusion can only be proven by mixing of cell contents. Furthermore, determination of the microscopic fusion index must generally be carried out manually, creating opportunities for unintended observer bias and limiting the numbers of cells assayed and therefore the statistical power of the assay. Strategies for making assays dependent on fusion and independent of visual observation are critical to increasing the accuracy and throughput of screens for molecules that control cell fusion. A variety of in vitro biochemical and nonbiochemical techniques have been developed to assay and monitor fusion events in cultured cells. In this chapter, we briefly discuss several in vitro fusion assays, nearly all based on systems of two components that interact to create a novel assayable signal only after cells fuse. We provide details for the use of one example of such a system, intracistronic complementation of beta-galactosidase activity by mutants of Escherichia coli lacZ, which allows for either cell-by-cell microscopic assay of cell fusion or quantitative and kinetic detection of cell fusions in whole populations. In addition, we describe a combination of gene knock-down protocols with this assay to study factors required for myoblast fusion.
Authors:
Jessica H Shinn-Thomas; Victoria L Scranton; William A Mohler
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Publication Detail:
Type:  Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Methods in molecular biology (Clifton, N.J.)     Volume:  475     ISSN:  1064-3745     ISO Abbreviation:  Methods Mol. Biol.     Publication Date:  2008  
Date Detail:
Created Date:  2008-11-03     Completed Date:  2008-11-24     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9214969     Medline TA:  Methods Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  347-61     Citation Subset:  IM    
Affiliation:
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Biological Assay / methods*
Cell Fusion / methods*
Cells, Cultured
Chemiluminescent Measurements
Fluorescence
Fluorescence Resonance Energy Transfer
Hemagglutinins / metabolism
Integrases / metabolism
Mice
Peptides / metabolism
RNA, Small Interfering / metabolism
Temperature
beta-Galactosidase / metabolism
Grant Support
ID/Acronym/Agency:
HD43156/HD/NICHD NIH HHS
Chemical
Reg. No./Substance:
0/Hemagglutinins; 0/Peptides; 0/RNA, Small Interfering; EC 2.7.7.-/Cre recombinase; EC 2.7.7.-/Integrases; EC 3.2.1.23/beta-Galactosidase

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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