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...
| Main Authors: | , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2010-06-01
|
| Series: | PLoS ONE |
| Online Access: | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20532169/pdf/?tool=EBI |
| _version_ | 1818422779859435520 |
|---|---|
| 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 |
| collection | DOAJ |
| 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. |
| first_indexed | 2024-12-14T13:31:41Z |
| format | Article |
| id | doaj.art-f3a2012abc124092af3a017d3399c3c0 |
| institution | Directory Open Access Journal |
| issn | 1932-6203 |
| language | English |
| last_indexed | 2024-12-14T13:31:41Z |
| publishDate | 2010-06-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj.art-f3a2012abc124092af3a017d3399c3c02022-12-21T22:59:42ZengPublic Library of Science (PLoS)PLoS ONE1932-62032010-06-0156e1095410.1371/journal.pone.0010954Rapid and sensitive lentivirus vector-based conditional gene expression assay to monitor and quantify cell fusion activity.Manuel A F V GonçalvesJosephine M JanssenMaarten HolkersAntoine A F de VriesCell-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.https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20532169/pdf/?tool=EBI |
| spellingShingle | 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. PLoS ONE |
| title | 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_short | 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 |
| url | https://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20532169/pdf/?tool=EBI |
| work_keys_str_mv | AT manuelafvgoncalves rapidandsensitivelentivirusvectorbasedconditionalgeneexpressionassaytomonitorandquantifycellfusionactivity AT josephinemjanssen rapidandsensitivelentivirusvectorbasedconditionalgeneexpressionassaytomonitorandquantifycellfusionactivity AT maartenholkers rapidandsensitivelentivirusvectorbasedconditionalgeneexpressionassaytomonitorandquantifycellfusionactivity AT antoineafdevries rapidandsensitivelentivirusvectorbasedconditionalgeneexpressionassaytomonitorandquantifycellfusionactivity |