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S. Seo, Y. Le, W. Hauswirth, A. Lewin; Early AMD Mouse Model by Reduction of MnSOD2 Using the Cre-LoxP Recombination System. Invest. Ophthalmol. Vis. Sci. 2009;50(13):781.
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Oxidative stress in the retinal pigment epithelium (RPE) complex is thought to be a leading cause in the development of age-related macular degeneration (AMD). Our hypothesis is that reduction of RPE specific manganese superoxide dismutase (MnSOD) will cause increased levels of reactive oxygen species in the retina/RPE/choroid complex leading to pathogenesis similar to the early signs of AMD.
To reduce MnSOD2 in the RPE, we employed two different strategies for implementing the Cre-loxP system. First is the introduction of Cre recombinase through a subretinal injection of AAV1 virus expressing VMD2-CRE into mice bearing a floxed-SOD2 allele to initiate RPE-specific recombination. Alternatively, we bred mice to introduce a Tet-On RPE65 Cre allele into SOD2f/f mice and then triggered Cre expression, and thereby recombination, by including doxycycline in mice’s drinking water (5 % sucrose + 5 mg/ml doxycycline). To verify that the AAV1-VMD2-CRE virus was capable of inducing recombination, the virus was subretinally injected into a reporter mouse strain which contains a floxed PGK-neo cassette and a yellow fluorescent protein gene inserted into the ROSA26 locus. The presence of EYFP was measured by immunohistochemical staining. In both models, the expression of Cre was evaluated with RT-PCR and the expression of MnSOD2 was measured by immunohistochemical staining for MnSOD2 in isolated RPE flat mounts. The progression of the disease was measured by staining for the presence of oxidative stress in the RPE using dihydroethidium (DHE) staining and by electron microscopy (EM) and histological analysis.
In the reporter mouse line, YFP expression was detected only in the RPE layer after injection with AAV-VMD2-CRE. Reduced expression of MnSOD was confirmed by staining in the RPE-flat mount. However, the doxycycline-induced cre expression was not consistent in SOD2f/f-RPE65-Cre+/- mouse. As expected, the reduced expression of SOD2 led to the increased oxidative stress in the RPE as shown by DHE staining. By 15 months, retinas exhibited deposits in RPE layer, shortened outer and inner segments of photoreceptors, and the thinning of outer nuclear layer indicating loss of photoreceptor cells.
RPE-specific down-regulation of SOD2 by using Cre-mediated recombination leads to increased oxidative stress in the RPE and histological changes similar to those observed in AMD.
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