David G Ball, Eleanor Ostafin, William J Spencer
These findings indicate that the rhodopsin-iCre75 line has unintended Cre recombination in RPE cells providing important implications for the interpretation of prior and future studies using this line.
PURPOSE: Cre mouse lines are an important tool to manipulate gene expression in specific cell types and at distinct developmental timepoints. Overlooked and off-target expression of Cre is a common issue with transgenic Cre lines that has confounded the interpretation of many studies. The rhodopsin-iCre75 mouse line expresses Cre behind a rod opsin promoter and has been the most widely used line for targeting rod photoreceptor cells over the past two decades. Here, we re-evaluated the specificity of the rhodopsin-iCre75 mouse line for rod photoreceptors.
METHODS: We crossed the rhodopsin-iCre75 mouse line with the fluorescent Cre reporter strain, Ai14, which expresses tdTomato in Cre-lox recombined cells. We identified recombined cells in mouse eye tissues by confocal microscopy. Independent of this reporter strain and as validation, we detected the presence of Cre protein by western blotting.
RESULTS: We report that Cre expression in the rhodopsin-iCre75 mouse line is unexpectedly not rod photoreceptor specific. We show that Cre is expressed in approximately 6% ± 2% (mean ± SD) of retinal pigment epithelium (RPE) cells by postnatal day 4, a timepoint preceding rhodopsin expression in rods. Recombined RPE cells are found in patches and are concentrated centrally where approximately 17% ± 6% (mean ± SD) of RPE cells are Cre positive.
CONCLUSIONS: These findings indicate that the rhodopsin-iCre75 line has unintended Cre recombination in RPE cells providing important implications for the interpretation of prior and future studies using this line.