The ROSAnT-nG allele consists of a CMV enhancer/chicken beta-actin core promoter, a loxP-flanked nT cassette, and a nG cassette, all inserted into the Gt(ROSA)26Sor locus.
Prior to exposure to Cre recombinase, strong red fluorescence is observed in the nuclei of cells/tissues in a widespread fashion. No EGFP expression is reported prior to cre-mediated recombination. Superficially, ROSAnT-nG mice may be distinguished from wildtype by tail or whole body epifluorescence (tdTomato fluorescence). The red fluorescence occurs in bands around the tail (reflecting the distribution of the nuclei).
When bred to Cre recombinase expressing mice, offspring will have the floxed nT cassette deleted in the cre expressing cells/tissues; this allows expression of the downstream nG cassette and results in strong green fluorescence in the nuclei of cre expressing cells and future cell lineages derived from these cells.
This double-fluorescent system allows direct live visualization of both Cre recombined and non-recombined cells at single cell resolution, offering an internal control for phenotypic analysis of Cre-induced mosaic mutants and providing a second marker for cell lineage tracing applications. In addition, localization of fluorescent proteins to nuclei is well-suited for examination of proliferating differentiated cell types (e.g., hepatocytes), tissues refractory to whole-cell flow cytometry analyses (e.g., liver, muscle, brain, kidney) and tissues with ploidy transitions (e.g., cancers, trophectoderm derivatives), as well as immuno-co-localization studies for cell type-specific transcription factors.
The C-terminal SRm160 domain effectively targets the fluorescent protein to the nucleus. While slow diffusion out of isolated nuclei is observed at room temperature in less than one hour, the use of cold temperatures, viscous buffers or mild fixation allows longer/permanent nuclear retention. Mice homozygous for the ROSAnT-nG allele are viable and fertile.
