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CHI SUN, Shiming Chen; Generation of a transgenic mouse for inducible ectopic CRX expression. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2374.
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The Cone-rod homeobox (CRX) protein is a key transcription factor essential for photoreceptor function and survival. Mutations in human CRX gene cause distinct photoreceptor diseases that are not incurable. This research aimed to develop a therapeutic strategy to rescue sick photoreceptors by an exogenously controlled gene regulation system that depends on the administration of a drug to turn transgene expression on. The results will guide future translational research.
Tet-On system was used to induce wildtype CRX expression in the presence of doxycycline (dox). A transgene (TRE-hCRX) construct is designed to carry tetracycline-responsive element (TRE)-driven FLAG-tagged human CRX (hCRX) cDNA. Inducibility of the transgene and activity of the induced hCRX protein were validated in transfected HEK293T cells by Western blots and Rhodopsin promoter-driven luciferase reporter assays. For generating the transgenic mouse, the transgene was targeted to the mouse H11 locus, a commonly used insert site. hCRX induction in the TRE-hCRX transgenic mouse was examined by immunohistochemistry on cultured retinal explants supplied with dox.
In TRE-hCRX transgenic system, binding of dox triggers a conformational change in rtTA, which allows the resulted complex bind to TRE to drive hCRX expression. We detected induced hCRX protein by Western blots of an anti-CRX antibody in HEK293T cells transfected with TRE-hCRX, pCAG-Nrl-Cre, pCAG-LSL-rtTA plasmids. Dual luciferase assays with the Rho promoter-driven luciferase reporter showed a linear dox-response curve of hCRX regulatory activity on the target promoter, confirming the success in the transgene design. For further testing this Tet-On system in retina, postnatal (P) day 0 retinae of TRE-hCRX transgenic mice were co-electroporated with pCAG-LSL-rtTA and pCAG-Cre plasmids. We supplied dox in the culture medium at P7 and harvested retinal explants for retinal sections at P8. Induced hCRX protein was detected by an anti-FLAG antibody in tissue sections, which demonstrated the in vivo application of this Tet-ON system.
We have generated a new transgenic mouse for expressing inducible ectopic CRX expression in the retina. Future research will employ this system to develop a new treatment strategy for CRX-linked diseases. The results also help to establish a work model for treating diseases associated with mutations in a regulatory gene.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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