Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.

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 undersco...

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Autores principales:	Manuel A F V Gonçalves, Josephine M Janssen, Maarten Holkers, Antoine A F de Vries
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/f3a2012abc124092af3a017d3399c3c0
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spelling	oai:doaj.org-article:f3a2012abc124092af3a017d3399c3c02021-12-02T20:21:12ZRapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.1932-620310.1371/journal.pone.0010954https://doaj.org/article/f3a2012abc124092af3a017d3399c3c02010-06-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20532169/pdf/?tool=EBIhttps://doaj.org/toc/1932-6203Cell-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.Manuel A F V GonçalvesJosephine M JanssenMaarten HolkersAntoine A F de VriesPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 6, p e10954 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Manuel A F V Gonçalves Josephine M Janssen Maarten Holkers Antoine A F de Vries Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.
description	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.
format	article
author	Manuel A F V Gonçalves Josephine M Janssen Maarten Holkers Antoine A F de Vries
author_facet	Manuel A F V Gonçalves Josephine M Janssen Maarten Holkers Antoine A F de Vries
author_sort	Manuel A F V Gonçalves
title	Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.
title_short	Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.
title_full	Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.
title_fullStr	Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.
title_full_unstemmed	Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.
title_sort	rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/f3a2012abc124092af3a017d3399c3c0
work_keys_str_mv	AT manuelafvgoncalves rapidandsensitivelentivirusvectorbasedconditionalgeneexpressionassaytomonitorandquantifycellfusionactivity AT josephinemjanssen rapidandsensitivelentivirusvectorbasedconditionalgeneexpressionassaytomonitorandquantifycellfusionactivity AT maartenholkers rapidandsensitivelentivirusvectorbasedconditionalgeneexpressionassaytomonitorandquantifycellfusionactivity AT antoineafdevries rapidandsensitivelentivirusvectorbasedconditionalgeneexpressionassaytomonitorandquantifycellfusionactivity
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Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.

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