NDLI: A Cre-Lox P recombination approach for the detection of cell fusion in vivo.

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A Cre-Lox P recombination approach for the detection of cell fusion in vivo.

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A Cre-Lox P recombination approach for the detection of cell fusion in vivo.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Sprangers, Anthony J. Freeman, Brian T. Kouris, Nicholas A. Ogle, Brenda M.
Description	Country affiliation: United States Author Affiliation: Sprangers AJ ( Department of Biomedical Engineering, University of Wisconsin-Madison, USA.)
Abstract	The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including skeletal muscle, bone and placenta. Contemporary studies demonstrate fusion of cells of the same type confers enhanced function. For example, when the trophoblast cells of the placenta fuse to form the syncytiotrophoblast, the syncytiotrophoblast is better able to transport nutrients and hormones across the maternal-fetal barrier than unfused trophoblasts(1-4). More recent studies demonstrate fusion of cells of different types can direct cell fate. The 'reversion' or modification of cell fate by fusion was once thought to be limited to cell culture systems. But the advent of stem cell transplantation led to the discovery by us and others that stem cells can fuse with somatic cells in vivo and that fusion facilitates stem cell differentiation(5-7). Thus, cell fusion is a regulated process capable of promoting cell survival and differentiation and thus could be of central importance for development, repair of tissues and even the pathogenesis of disease. Limiting the study of cell fusion, is lack of appropriate technology to 1) accurately identify fusion products and to 2) track fusion products over time. Here we present a novel approach to address both limitations via induction of bioluminescence upon fusion (Figure 1); bioluminescence can be detected with high sensitivity in vivo(8-15). We utilize a construct encoding the firefly luciferase (Photinus pyralis) gene placed adjacent to a stop codon flanked by LoxP sequences. When cells expressing this gene fuse with cells expressing the Cre recombinase protein, the LoxP sites are cleaved and the stop signal is excised allowing transcription of luciferase. Because the signal is inducible, the incidence of false-positive signals is very low. Unlike existing methods which utilize the Cre/LoxP system(16, 17), we have incorporated a 'living' detection signal and thereby afford for the first time the opportunity to track the kinetics of cell fusion in vivo. To demonstrate the approach, mice ubiquitously expressing Cre recombinase served as recipients of stem cells transfected with a construct to express luciferase downstream of a floxed stop codon. Stem cells were transplanted via intramyocardial injection and after transplantation intravital image analysis was conducted to track the presence of fusion products in the heart and surrounding tissues over time. This approach could be adapted to analyze cell fusion in any tissue type at any stage of development, disease or adult tissue repair.
File Format	HTM / HTML
e-ISSN	1940087X
DOI	10.3791/3581
Journal	Journal of Visualized Experiments
Issue Number	59
Language	English
Publisher	MyJove Corp.
Publisher Date	2012-01-04
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Physical Sciences Discipline Life Sciences Discipline Medicine Cell Fusion Integrases Genetics Recombination, Genetic Animals Biosynthesis Luciferases, Firefly Luminescent Measurements Mesenchymal Stromal Cells Physiology Mice Mice, Inbred C57bl Transfection Research Support, N.i.h., Extramural Research Support, U.s. Gov't, Non-p.h.s. Video-audio Media
Content Type	Text
Resource Type	Article

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