A Cre-based double fluorescence indicator system for monitoring cell fusion events and selection of fused cells

Biotechniques. 2010 Feb;48(2):113-20. doi: 10.2144/000113352.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Fusion / methods*
  • Cell Line
  • Genetic Vectors
  • Humans
  • Immunohistochemistry
  • Integrases / genetics
  • Integrases / metabolism*
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Mice
  • Mice, Transgenic
  • Microscopy, Fluorescence / methods*
  • Molecular Biology / methods*
  • Puromycin
  • Receptors, Estrogen
  • Red Fluorescent Protein
  • Reproducibility of Results

Substances

  • Luminescent Proteins
  • Receptors, Estrogen
  • Puromycin
  • Cre recombinase
  • Integrases