| Quantitative assays for cell fusion. | |
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MedLine Citation:
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PMID: 18979254 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Jessica H Shinn-Thomas; Victoria L Scranton; William A Mohler |
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Publication Detail:
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Type: Journal Article; Research Support, N.I.H., Extramural; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Methods in molecular biology (Clifton, N.J.) Volume: 475 ISSN: 1064-3745 ISO Abbreviation: Methods Mol. Biol. Publication Date: 2008 |
Date Detail:
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Created Date: 2008-11-03 Completed Date: 2008-11-24 Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 9214969 Medline TA: Methods Mol Biol Country: United States |
Other Details:
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Languages: eng Pagination: 347-61 Citation Subset: IM |
Affiliation:
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Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT, USA. |
Export Citation:
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APA/MLA Format Download EndNote Download BibTex |
| MeSH Terms | |
Descriptor/Qualifier:
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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:
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HD43156/HD/NICHD NIH HHS |
| Chemical | |
Reg. No./Substance:
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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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