Identification of a zebrafish cone photoreceptor-specific promoter and genetic rescue of achromatopsia in the nof mutant.
OBJECTIVE
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 (TalphaC) 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 TalphaC 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 TalphaC promoter fragment replicates the temporal and spatial pattern of endogenous TalphaC 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.
Kennedy
Breandán N
BN
UCD Conway Institute and UCD School of Biomolecular and Biomedical Sciences, University College Dublin, Dublin, Ireland. brendan.kennedy@ucd.ie
Alvarez
Yolanda
Y
Brockerhoff
Susan E
SE
Stearns
George W
GW
Sapetto-Rebow
Beata
B
Taylor
Michael R
MR
Hurley
James B
JB
D000818:Animals; D030801:Animals, Genetically Modified; D018389:Codon, Nonsense; D003117:Color Vision Defects / Q000235:genetics / Q000628:therapy*; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005434:Flow Cytometry; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D015316:Genetic Therapy; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D017120:Nystagmus, Optokinetic / Q000502:physiology; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017949:Retinal Cone Photoreceptor Cells / Q000378:metabolism*; D015293:Transducin / Q000235:genetics*; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics*
D018389:Codon, Nonsense; D029961:Zebrafish Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D015293:Transducin