The authors would like to thank Dr. Peiman Hemmati (Department of Medicine, University of Wisconsin-Madison) for generously providing the H1 MSCs, and Dr. Tim Hacker, Dr. Gouqing Song and Ms. Jill Koch of the University of Wisconsin Cardiovascular Physiology Core Facility for performing mouse surgeries. This work was supported by the National Science Foundation through a Graduate Research Fellowship to Brian Freeman and NIH R21 HL089679.

1. Donor Cell Transfection
<ol>
	<li>Harvest mesenchymal stem cells (MSCs, derived from H1 embryonic stem cells kindly donated by Dr. Peiman Hematti; alternatively, any cell type of any species hypothesized to fuse in vivo could be employed) when 70 - 80% confluent with 1X trypsin (Mediatech, Manassas VA) for 5 min. Inactivate trypsin with &alpha;-MEM complete medium (antibiotic free, Invitrogen, Carlsbad CA)18. Centrifuge at 300 x g for 5 min.</li>
	<li>Carefully aspirate supernata...

<ol>
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The method described here allows, for the first time, discrete identification and temporal analysis of cell fusion in organisms, including small animals. The approach combines Cre-LoxP recombination with subsequent biophotonic image analysis. The approach is amenable to tracking not only cell-cell fusion, but also virus-cell fusion and so could prove useful for tracking viral infections. Image analysis is rapid and it is possible to image multiple small animals simultaneously. Detection of fusion is limited b...

<table border="1">
<tbody>
 <tr>
 <td>Name of reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>Neon Transfection System</td>
 <td>Invitrogen, Carlsbad, CA</td>
 <td>MPK5000</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Neon 100 &mu;L Kit</td>
 <td>Invitrogen, Carlsbad, CA</td>
 <td>MPK10025</td>
 <td>Contains R and E Buffer</td>
 </tr>
 <tr>
 <td>a-MEM powder</td>
 <td>Invitrogen, Carlsbad, CA</td>
 <td>12000-022</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (FBS)</td>
 <td>Hyclone, Logan UT</td>
 <td>SH30070.03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>B6.C-Tg(CMV-cre)1Cgn/J</td>
 <td>Jackson Laboratory, Bar Harbor, ME</td>
 <td>006054</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Trypsin 10X</td>
 <td>Fisher Scientific, Forest Lawn, NJ</td>
 <td>MT-25-054-Cl</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glutamine</td>
 <td>Fisher Scientific, Forest Lawn, NJ</td>
 <td>25030-081</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-Luciferin</td>
 <td>Caliper Life Sciences, Hopkinton, MA</td>
 <td>122796</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Xenogen Biophotonic Imaging System</td>
 <td>Caliper Life Sciences, Hopkinton, MA</td>
 <td>IVIS Spectrum </td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium Biocarbonate</td>
 <td>Sigma Aldrich, St. Louis, MO</td>
 <td>S6014-500G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Non-essential Amino Acids</td>
 <td>Invitrogen, Carlsbad, CA</td>
 <td>11140-050</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

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 trophoblasts1-4. 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 differentiation5-7. 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 vivo8-15. 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 system16, 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.

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

A method to track cell fusion in living organisms over time is described. The approach utilizes Cre-LoxP recombination to induce luciferase expression upon cell fusion. The luminescent signal generated can be detected in living organisms using biophotonic imaging systems with a sensitivity of detection of &tilde;1,000 cells in peripheral tissues.

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

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 ...

a-cre-lox-p-recombination-approach-for-detection-cell-fusion

Research

JoVE Journal

Bioengineering

18.7K 次观看.  University of Wisconsin-Madison. 一个在生活随着时间的推移的生物体细胞融合的方法来跟踪描述。该方法利用CRE - LoxP位重组荧光素酶表达诱导细胞融合后。产生荧光信号可以检测到使用生物光子成像系统的检测〜1000细胞周围组织的敏感性，在生活的有机体。