Gt(ROSA)26Sor^{tm1(CAG-ECFP/EYFP*)Phep}

The inserted sequence encodes a cre recombinase-inducible, sensitive, non-invasive fluorescent reporter of intracellular chloride ion concentration [Cl-] (Kapp ~30 mM) and pH ((pKalpha 7.1-8.0 pH units at different [Cl-]) under physiologic conditions. This reporter molecule, which the authors call Cl-Sensor, takes advantage of the quenching of EYFP by Cl- and permits fluorescence resonance energy transfer- (FRET-) based ratiometric measurement of [Cl-] in living cells. The chimeric reporter protein comprises enhanced cyan fluorescent protein (ECFP) joined by a 20-amino acid linker to enhanced yellow fluorescent protein (EYFP) containing three amino acid substitutions (His148Gln, Ile152Leu and Val163Ser) that increase its [Cl-] sensitivity five-fold. The targeting construct contained the CAG promoter (consisting of the cytomegalovirus immediate early (CMV-IE) enhancer and the chicken beta-actin promoter); the adenovirus splice donor and splice acceptor sites; a neomycin resistance (neor) gene flanked 5' by an inverted loxP site and a Kozak consensus sequence and 3' by two copies of the polyadenylation site from the bovine growth hormone gene and a mutant loxP2272 site; the Cl-Sensor cDNA followed by the SV40 polyadentylation site, in reverse transcriptional orientation relative to neor; a loxP site and an inverted mutant loxP2272 site. This construct was inserted into the original Gt(ROSA)26Sor targeting vector at the unique XbaI site approximately 300 bp 5' of the original gene trap integration site. (J:209636, J:211025)