Transgenic expression of a GFP-rhodopsin COOH-terminal fusion protein in zebrafish rod photoreceptors.
To facilitate the identification and characterization of mutations affecting the retina and photoreceptors in the zebrafish, a transgene expressing green fluorescent protein (GFP) fused to the C-terminal 44 amino acids of Xenopus rhodopsin (Tam et al., 2000) under the control of the 1.3-kb proximal Xenopus opsin promoter was inserted into the zebrafish genome. GFP expression was easily observed in a ventral patch of retinal cells at 4 days postfertilization (dpf). Between 45-50% of the progeny from the F1, F2, and F3 generations expressed the transgene, consistent with a single integration event following microinjection. Immunohistochemical analysis demonstrated that GFP is expressed exclusively in rod photoreceptors and not in the UV, blue, or red/green double cones. Furthermore, GFP is localized to the rod outer segments with little to no fluorescence in the rod inner segments, rod cell bodies, or rod synapse regions, indicating proper targeting and transport of the GFP fusion protein. Application of exogenous retinoic acid (RA) increased the number of GFP-expressing cells throughout the retina, and possibly the level of expressed rhodopsin. When bred to a zebrafish rod degeneration mutant, fewer GFP-expressing rods were seen in living mutants as compared to wild-type siblings. This transgenic line will facilitate the search for recessive and dominant mutations affecting rod photoreceptor development and survival as well as proper rhodopsin expression, targeting, and transport.
Perkins
Brian D
BD
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA. bperkins@fas.harvard.edu
Kainz
Pamela M
PM
O'Malley
Donald M
DM
Dowling
John E
JE
D000818:Animals; D030801:Animals, Genetically Modified / Q000235:genetics; D001692:Biological Transport; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000235:genetics* / Q000378:metabolism; D018613:Microscopy, Confocal; D009154:Mutation / Q000502:physiology; D020419:Photoreceptor Cells, Vertebrate / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D017948:Retinal Rod Photoreceptor Cells / Q000502:physiology*; D012243:Rhodopsin / Q000737:chemistry / Q000235:genetics*; D012374:Rod Cell Outer Segment / Q000378:metabolism; D019076:Transgenes / Q000235:genetics*; D014212:Tretinoin / Q000494:pharmacology; D015027:Zebrafish
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D014212:Tretinoin; D012243:Rhodopsin