An efficient inducible RPE-Selective cre transgenic mouse line

Area of Science:

• Genetics
• Ophthalmology
• Molecular Biology

Background:

• Current mouse models for retinal pigment epithelium (RPE)-specific gene manipulation exhibit limitations in recombination efficiency and expression patterns.
• Developing a reliable and temporally regulated Cre driver line is crucial for advancing research into RPE biology and disease.

Purpose of the Study:

• To generate a novel transgenic mouse line enabling efficient and temporally controlled Cre-mediated gene recombination specifically in the murine RPE.
• To validate the RPE-selectivity and efficiency of the new driver line using a reporter system and a gene ablation model.

Main Methods:

• Utilized ϕC31 integrase for targeted insertion of a BEST1 promoter-driven Cre recombinase estrogen receptor fusion (BEST1-CreERT2) construct into the Rosa26 locus.
• Generated Rosa26^{BEST1-CreERT2} mice and bred them with tdTomato reporter and Cre-conditional Tfam mutant lines.
• Administered 4-hydroxytamoxifen to induce recombination and assessed tdTomato expression and gene ablation outcomes.

Main Results:

• Robust and efficient tdTomato expression was observed in the RPE of Rosa26^{BEST1-CreERT2} mice post-tamoxifen induction, with high percentages in both sexes at different ages.
• Minimal off-target recombination was detected in Muller glia (<0.6%) and sparse foci in the testis, with no expression in other ocular or non-ocular tissues.
• RPE-specific ablation of Tfam using this model resulted in expected retinal thinning and diminished electroretinogram responses, confirming model functionality without observed retinal toxicity.

Conclusions:

• The generated Rosa26^{BEST1-CreERT2} mouse line provides a powerful tool for highly efficient, tamoxifen-inducible, and RPE-selective gene manipulation.
• This model overcomes limitations of previous RPE Cre lines, offering improved temporal control and reduced mosaicism for studying RPE function and related pathologies.