Establishment of conditional reporter mouse lines at ROSA26 locus for live cell imaging

Abstract

A series of conditional reporter mouse lines were established in which specific organelles were labeled with fluorescent proteins. Subcellular localization and intensity of 28 fluorescent fusion-protein constructs were surveyed in cell lines, and 16 constructs then were selected to generate mouse lines. The fusion cDNAs were inserted into the ROSA26 genomic locus next to the stop sequences flanked with loxP so that fluorescent proteins were expressed under the ubiquitous ROSA26 transcriptional machinery when the loxP sequences were recombined with Cre. The subcellular localization and intensity of the fusion product in each reporter mouse line were examined by ubiquitously expressing them in E7.5 embryos. Twelve reporter lines, that mark nucleus, mitochondria, Golgi apparatus, plasma membrane, microtubule, actin filament, and focal adhesion, were found suitable for live imaging. Distinct double staining was demonstrated for nucleus and plasma membrane or Golgi apparatus; clear time-lapse live images were obtained for nucleus and plasma membranes; conditional expression was confirmed on Lyn-Venus and H2B-mCherry lines in notochord with Not-Cre.