Gt(ROSA)26Sor^{tm1(CAG-mCherry/GFP)Ganl}

The targeting vector consists of (from 5' to 3') an FRT3 site, PGK neo polyA cassette, a CAG promoter, a Kozak sequence (GCCACC), the mCherry-GFP-mtFIS1<101-152> coding sequence (details below), a human growth hormone polyA sequence (hGHpA), an additional polyA sequence and a FRT site. Recombination mediated cassette exchange (RMCE) is used to insert the CAG promoter and the open reading frame for the mCherry-GFP-mtFIS1<101-152> fusion protein into the Gt(ROSA)26Sor locus. The mCherry-GFP-mtFIS1<101-152> fusion protein (mito-QC) is a cDNA sequence composed of (from 5' to 3') a monomeric red fluorescent protein (mCherry) fused in-frame to a green fluorescent protein (GFP), then an in-frame outer mitochondrial membrane targeting sequence (amino acids 101-152 from the human FIS1 locus [fission, mitochondrial 1]). Under steady-state conditions, the mitochondrial network exhibits both mCherry (red) fluorescence and GFP (green) fluorescence. Upon mitophagy, mitochondria are delivered to lysosomes where mCherry fluorescence remains stable, but GFP fluorescence becomes quenched by the acidic (low pH) microenvironment. (J:237975)