Abstract
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.
Highlights
Diverse biological phenomena involve fusion between either the same or different cell types
Another example is the occurrence of low-level heterotypic cell fusion between stem or progenitor cells from one tissue and parenchymal cells from another tissue especially under conditions disrupting the homeostasis of the recipient organ
By deploying the bipartite cell fusion monitoring system together with short-hairpin RNAs and the specific p38a/b inhibitor SB 203580 we showed the involvement of the p38 mitogen-activated protein kinase (MAPK) pathway in human myotube formation
Summary
Diverse biological phenomena involve fusion between either the same or different cell types (i.e. homotypic and heterotypic cell fusion, respectively). We started by testing whether lentivirus vector particles encoding a FLP-dependent gene switch with the structural organization depicted in Fig. 1B could be generated and, following indicator cell transduction, express the reporter gene in a recombinase-dependent manner.
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