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Published on: July 14, 2020
Off-Target Retinal Pigment Epithelium (RPE) Expression of Cre in the Rhodopsin-iCre75 Mouse Line
David G Ball1,2, Eleanor Ostafin1,3, William J Spencer1,4
1Ophthalmology and Visual Sciences, SUNY Upstate Medical University, Syracuse, New York, United States.
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.
Insights
The rhodopsin-iCre75 mouse line, widely used for targeting rod photoreceptors, shows unintended Cre expression in retinal pigment epithelium (RPE) cells. This off-target recombination impacts the interpretation of studies using this Cre mouse line.
Area of Science:
- Genetics and Genomics
- Developmental Biology
- Ophthalmology
Background:
- Transgenic Cre mouse lines are crucial for cell-specific gene manipulation.
- Off-target Cre expression can compromise study validity.
- The rhodopsin-iCre75 line has been a standard for rod photoreceptor research.
Purpose of the Study:
- To re-evaluate the cell-type specificity of the rhodopsin-iCre75 mouse line.
- To determine if Cre expression is restricted to rod photoreceptors.
- To assess potential off-target recombination in other ocular cell types.
Main Methods:
- Crossed rhodopsin-iCre75 mice with the Ai14 Cre reporter strain (tdTomato expression).
- Analyzed Cre recombination in ocular tissues using confocal microscopy.
- Validated Cre protein presence via western blotting.
Main Results:
- Cre expression was not specific to rod photoreceptors in the rhodopsin-iCre75 line.
- Significant Cre recombination observed in retinal pigment epithelium (RPE) cells.
- Approximately 6% of RPE cells showed Cre recombination by postnatal day 4, with higher central concentrations.
Conclusions:
- The rhodopsin-iCre75 mouse line exhibits unintended Cre recombination in RPE cells.
- These findings necessitate re-interpretation of previous studies using this line.
- Future research must account for this RPE-specific off-target activity.
