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George Inana, Christopher Murat, Weijun An, Margaret McLaren; Construction Of Conditional Over-Expression Transgenic Model of Casein Kinase 1 Epsilon, A Potential Therapeutic Target For Dry AMD. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4632.
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© ARVO (1962-2015); The Authors (2016-present)
Casein 1 Kinase Epsilon (Ck1e), a critical regulator of circadian oscillations, was identified as a potential therapeutic target for dry age-related macular degeneration (AMD) in our laboratory by a multi-pronged gene expression profiling approach combining gene expression in AMD, retinal pigment epithelium (RPE) phagocytosis, and cigarette smoking. The aim of the present work was to construct a Tet-On conditional over-expression transgenic mouse model of Ck1e, to screen and select the line with the best transgene expression, and to begin to analyze its phenotype.
The mouse Ck1e cDNA was cloned from mouse retinal mRNA by RT-PCR, verified by sequencing, subcloned into the pTRE-TIGHT-BI vector, and used for construction of transgenic mice. The resulting transgenics were bred with the pTet-Rosa26 transgenic mice to produce the dual transgenic mice of which 30 lines were obtained. They were screened for optimal expression by doxycycline induction and qRT-PCR. The best line was subjected to initial phenotypic analysis by doxycycline induction and OCT, immunostaining, and histopathological examination of the retina.
Variable degrees of inducible transgene expression were observed in the transgenic lines. Interestingly, many of the lines suffered spontaneous loss of the transgenes. Increased level of Ck1e expression was demonstrated in a stable transgenic line by immunostaining and qRT-PCR. Histopathological analysis of the transgenic retina induced for over-expression of Ck1e for different duration by doxycycline demonstrated dramatic abnormalities consisting of structural aberration of the photoreceptor layer consistent with retinal dystrophy.
A Tet-On conditional over-expression mouse transgenic model of Ck1e was obtained in which Ck1e can be over-expressed at will in the retina to study its effects. Preliminary results demonstrated a dramatic structural abnormality of the retina in the transgenic model induced to over-express Ck1e, consistent with the possible pathological role that elevated Ck1e plays in dry AMD that we have previously shown. This model should be very useful in studying the mechanism by which Ck1e is related to dry AMD, such as through outer segment phagocytosis by the RPE.
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