Document Detail


Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.
MedLine Citation:
PMID:  20532169     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
Cell-to-cell fusion is involved in multiple fundamental biological processes. Prominent examples include osteoclast and giant cell formation, fertilization and skeletal myogenesis which involve macrophage, sperm-egg and myoblast fusion, respectively. Indeed, the importance of cell fusion is underscored by the wide range of homeostatic as well as pathologic processes in which it plays a key role. Therefore, rapid and sensitive systems to trace and measure cell fusion events in various experimental systems are in demand. Here, we introduce a bipartite cell fusion monitoring system based on a genetic switch responsive to the site-specific recombinase FLP. To allow flexible deployment in both dividing as well as non-dividing cell populations, inducer and reporter modules were incorporated in lentivirus vector particles. Moreover, the recombinase-inducible transcription units were designed in such a way as to minimize basal activity and chromosomal position effects in the "off" and "on" states, respectively. The lentivirus vector-based conditional gene expression assay was validated in primary human mesenchymal stem cells and in a differentiation model based on muscle progenitor cells from a Duchenne muscular dystrophy patient using reporter genes compatible with live- and single-cell imaging and with whole population measurements. Using the skeletal muscle cell differentiation model, we showed that the new assay displays low background activity, a 2-log dynamic range, high sensitivity and is amenable to the investigation of cell fusion kinetics. The utility of the bipartite cell fusion monitoring system was underscored by a study on the impact of drug- and RNAi-mediated p38 MAPK inhibition on human myocyte differentiation. Finally, building on the capacity of lentivirus vectors to readily generate transgenic animals the present FLP-inducible system should be adaptable, alone or together with Cre/loxP-based assays, to cell lineage tracing and conditional gene manipulation studies in vivo.
Authors:
Manuel A F V Gonçalves; Josephine M Janssen; Maarten Holkers; Antoine A F de Vries
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2010-06-03
Journal Detail:
Title:  PloS one     Volume:  5     ISSN:  1932-6203     ISO Abbreviation:  PLoS ONE     Publication Date:  2010  
Date Detail:
Created Date:  2010-06-10     Completed Date:  2010-09-01     Revised Date:  2010-09-28    
Medline Journal Info:
Nlm Unique ID:  101285081     Medline TA:  PLoS One     Country:  United States    
Other Details:
Languages:  eng     Pagination:  e10954     Citation Subset:  IM    
Affiliation:
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, The Netherlands. m.goncalves@lumc.nl
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MeSH Terms
Descriptor/Qualifier:
Cell Fusion*
Cells, Cultured
Gene Expression*
Genetic Vectors*
Humans
Lentivirus / genetics*
Mesenchymal Stem Cells / metabolism
Comments/Corrections

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