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(Investigative Ophthalmology and Visual Science. 2007;48:522-529.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-0975
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Identification of a Zebrafish Cone Photoreceptor–Specific Promoter and Genetic Rescue of Achromatopsia in the nof Mutant

Breandán N. Kennedy,1 Yolanda Alvarez,1 Susan E. Brockerhoff,2 George W. Stearns,2 Beata Sapetto-Rebow,1 Michael R. Taylor,2 and James B. Hurley2

1From the UCD Conway Institute and UCD School of Biomolecular and Biomedical Sciences, University College Dublin, Dublin, Ireland; and the 2Department of Biochemistry, University of Washington, Seattle, Washington.

PURPOSE. To identify in vivo a promoter fragment that specifically directs transgene expression in all zebrafish cone photoreceptors. This promoter subsequently enables GFP labeling of cones for facile morphologic analysis and purification and genetic rescue of achromatopsia.

METHODS. Promoter fragments of the zebrafish cone transducin {alpha} (T{alpha}C) gene were subcloned upstream of EGFP and microinjected into one- to two-cell–stage embryos. Promoter activity was assessed by fluorescence microscopy in wholemounts and retinal cryosections, and cone photoreceptors were purified by flow cytometry. Visual physiology was assessed by the optokinetic response (OKR) assay.

RESULTS. A 3.2-kb promoter fragment from zebrafish T{alpha}C specifically directed robust transgene expression in retinal cone photoreceptors and pineal photoreceptors. With this promoter, a stable transgenic line expressing EGFP in all zebrafish cone photoreceptors types was generated, and populations of cones were purified. Achromatopsia in the nof mutant was rescued using the identified promoter fragment to direct transgenic expression of wild-type cone transducin in mutant cones.

CONCLUSIONS. A 3.2-kb T{alpha}C promoter fragment replicates the temporal and spatial pattern of endogenous T{alpha}C expression. The integrity of cones can be readily assessed in an EGFP transgenic line generated with this promoter, enabling downstream genetic and chemical screens for cone determinants.




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 J. Biol. Chem.Home page
V. A. Smyth, D. Di Lorenzo, and B. N. Kennedy
A Novel, Evolutionarily Conserved Enhancer of Cone Photoreceptor-specific Expression
J. Biol. Chem., April 18, 2008; 283(16): 10881 - 10891.
[Abstract] [Full Text] [PDF]


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