A Cre-based double fluorescence indicator system for monitoring cell fusion events and selection of fused cells.
We have established an in vitro Cre/loxP-based assay for monitoring cell fusion events that specifically traces the transport of cytoplasm from one cell to its fusion partner. Cells with a double fluorescence vector indicate fusion with cells expressing Cre recombinase by switching expression from red to green fluorescent protein through a Cre-mediated recombination event that simultaneously activates puromycin-acetyltransferase expression. This strategy allows for both the observation and puromycin selection of indicator cells that have undergone fusion with a Cre recombinase-expressing partner. A fusion protein of Cre with estrogen receptor (ER) can be used to control Cre recombinase activity through the tamoxifen-induced translocation of the Cre-ER fusion protein to the nucleus. Here we have established a new methodology that not only allows the monitoring of the transport of cellular contents, but also enables the purification of fused cells using puromycin.
Pfannkuche
Kurt
K
Institute for Neurophysiology, University of Cologne, Cologne, Germany. akp59@uni-koeln.de
Spitkovsky
Dimitry
D
Wunderlich
Frank Thomas
FT
Abd El Aziz
Osama Mohamed
OM
Saric
Tomo
T
Hescheler
Jürgen
J
Sachinidis
Agapios
A
D000818:Animals; D002459:Cell Fusion / Q000379:methods*; D002460:Cell Line; D005822:Genetic Vectors; D006801:Humans; D007150:Immunohistochemistry; D019426:Integrases / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008856:Microscopy, Fluorescence / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*; D011691:Puromycin; D011960:Receptors, Estrogen; D015203:Reproducibility of Results; D000097573:Red Fluorescent Protein
D019426:Integrases; D008164:Luminescent Proteins; D011691:Puromycin; D011960:Receptors, Estrogen; D000097573:Red Fluorescent Protein; C045073:Cre recombinase