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Hannah Breit, Jerome E. Roger, Debbie Cheng, Lijin Dong, Anand Swaroop; Function Of Pias3 In Developing Retina Determined By The Analysis Of Conditional Knockout Mice. Invest. Ophthalmol. Vis. Sci. 2012;53(14):424.
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Protein Inhibitor of Activated STAT3 (PIAS3) is a transcriptional modulator that directly binds to multiple factors to regulate activity. PIAS3 also functions as a SUMO (Small Ubiquitin-like Modifier)-E3 ligase, and covalent linkage of SUMO proteins can modify the function of target proteins. Recently, Blackshaw and our laboratory have demonstrated the importance of SUMOylation in modulating activity of Nrl and Nr2e3, essential regulators of photoreceptor differentiation. The importance of PIAS3 in rod and cone photoreceptor differentiation has been shown by in vivo electroporation in newborn retina. In order to analyze the consequence of the lack of PIAS3 expression early in retinal development and identify new PIAS3 targets, we have generated and analyzed conditional Pias3 knockout mice (CKO).
Pias3 floxed mice were produced by introducing LoxP sites flanking exon 2 to 5 of the Pias3 gene through homologous recombination. Long range PCR and Southern blots were used for screening the correct recombinants. Pias3-specific deletion in the forebrain and retina was obtained by crossing floxed mice with Rx-Cre mice. Immunohistochemistry (IHC) was performed on retinal sections and flat mount retina.
A specific deletion of Pias3 in the retina in Pias3f/f; Rx-Cre mice was confirmed by RT-PCR and PCR on retinal cDNA and genomic DNA respectively, with tail DNA used as control. Immunoblot analysis confirmed the complete absence of Pias3 protein in retinas of Pias3f/f; Rx-Cre mice. Preliminary analyses of different retinal cell types in 1-month old animals indicated decreased Muller cells (Sox9-positive cells) with displaced cell bodies in the inner nuclear layer compared to control. Absence of Pias3 expression in early retinal development did not affect M-cone differentiation. IHC with anti-S-opsin antibody and PNA on flat mount retina revealed an increased number of S-cones along the ventral-dorsal axis. No obvious change was observed in other retinal cell types based on IHC. In 6-month old Pias3 CKO, no signs of retinal degeneration were observed.
Initial analysis of Pias3 CKO showed an increase of S-cone photoreceptors. Quantification of each cell type is being performed to fully evaluate the effects of the lack of Pias3 expression during retinal development. Visual function will be analyzed by ERG.
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