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J. Greenwood, M. Fruttiger, S. E. Moss; Inducible Genetic Targeting of Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2527.
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The retinal pigment epithelium (RPE) has many functions, such as formation of the posterior blood retina barrier, metabolic support of photoreceptors and phagocytosis of photoreceptor outer segments. Failure of these functions causes visual loss and is at the centre of many blinding diseases. In order to study the molecular mechanisms that underlie RPE function we have developed transgenic mice that allow inducible genetic targeting of RPE cells in vivo.
We have recombined a sequence coding for a tamoxifen-inducible form of Cre recombinase (CreER) into the open reading frame of the MCT3 gene (also known as Slc16a8) in a bacterial artificial chromosome (BAC) and used the resulting BAC to generate transgenic mice (MCT3-CreER mice) by pronuclear injection.
Tests of MCT3-CreER mice in a ROSA-lacZ reporter background demonstrated recombination activity specifically in RPE and choroid plexus epithelium upon systemic tamoxifen administration. Whole mount analysis showed that recombination efficiency was variable across the retina and higher in neonatal than adult mice. In the absence of tamoxifen no recombination activity could be detected. We also crossed MCT3-CreER mice with a strain that conditionally expresses diphtheria toxin A-chain (ROSA-DTA mice), and found that in this background Cre recombination activity induces cell death and leads to RPE cell depletion after tamoxifen administration. Surviving, non-affected RPE cells upregulated alpha smooth muscle actin. In areas of the retina with low RPE depletion, remaining RPE cells increased their size and maintained expression of tight junction molecules at cell boundaries. However, in areas more strongly affected, RPE cells detached from each other and tight junction components were no longer detectable.
We show in our transgenic model that RPE cells can maintain a continuous monolayer by cell-spreading when individual RPE cells are genetically depleted. Furthermore, MCT3-CreER mice will be useful for functional studies of individual genes in RPE cells because our system allows for normal development and is only activate by tamoxifen. Thus RPE functions in adult animals can be addressed.
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