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Wataru Otsu, Jen-Zen Chuang, Ching-Hwa Sung; Establishment of a novel cone transfection technique for cone-rod dystrophy mouse models. Invest. Ophthalmol. Vis. Sci. 2018;59(9):984.
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Our current understanding of the cell biology of cones significantly lags behind our knowledge about rods. The etiology of several cone-rod dystrophies and macular degenerative diseases that predominantly affect cones remains elusive. We aimed to modify the current retinal transfection protocol, which mainly targets rods, to improve the efficiency and versatility in manipulating cone-expressed proteins in mouse in vivo.
We performed plasmid transfection in embryonic mouse retinas in utero. We performed transfection using a single plasmid which encodes tetracycline operator (tetO)-miniCMV promoter-directed “transgene” (or short hairpin RNA) and rtTA3. We characterized the types of the transfected retinal cells, and the efficiency of gene induction and suppression using doxycycline, either via diet or i.p. injection.
Many of our transfected retinal cells harvested at 3-weeks of age had morphology characteristic for cones and expressed cone opsin. Transfected bovine peripherin2/rds was predominantly expressed in the cone outer segment, indicating that the transfected proteins follow a similar transport pathway to that of their endogenous counterparts. The exogenously expressed fluorescent protein allows the identification of cell-autonomous phenotype(s) of transfected cones. Our TetOn induction system is tight, synchronized, and highly efficient. Furthermore, the transgene expression level is tunable according to the dose of doxycycline.
We greatly improved the cone targeting efficiency. We pioneered the TetOn inducible system in transfected cones to perform conditional gene expression and gene suppression. These technical advancements are powerful in uncovering the function of the cone-expressed proteins that involved the etiology of various cone-rod dystrophy.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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