Transgenic GFP-Rhodopsin Zebrafish Model System for Study of Retinal Disease
Summary Background: Rhodopsin is a G-protein coupled receptor that initiates the light response upon absorption of a photon in the outer segment of the retina (ROS). Rhodopsin constitutes at least 90% of the total protein in the outer segment and as a consequence the efficient transport of this molecule is essential for the proper functioning of rod photoreceptors. More than 70 mutations in the rhodopsin gene have been identified as leading to inherited retinal disease retinitis pigmentosa and faulty trafficking of rhodopsin to the outer segment in animal models. While it is known that the C-terminal tail of rhodopsin contains the signals that are necessary and sufficient for proper sorting and transport of rhodopsin to the rod outer segment, very little is known, however, about the genes and their products required for proper rhodopsin transport to the outer segment. In recent years, the zebrafish has emerged as a powerful model organism with which transgenic methods can be utilized to create animals that express reporter genes such as GFP.Invention: A transgenic stable line of zebrafish that expresses a GFP marker in the rod photoreceptors of the eye. The GFP protein is fused to C-terminal 44 amino acids (CT44) of Xenopus rhodopsin, which contains the outer segment localization signal for proper targeting of the rhodopsin protein to the outer segments of the rod photoreceptors. The transgene expression occurs exclusively in rod photoreceptors--control verification studies demonstrated that the GFP protein in the zebrafish line localized to the ROSs and is not observed in the inner segments.
Applications Advantages: One advantage of the zebrafish is its transparent nature during embryonic and larval stages, which proves quite useful when studying transgenic zebrafish that express GFP reporter genes. By generating transgenic zebrafish that express a targeted GFP molecule in rods, the location of GFP within individual photoreceptor cells can be studied in living larval fish by confocal microscopy as well as in frozen sections without the need for specific antibody staining. Another advantage is that the transgene expression is stable through multiple generations in a predictable Mendelian fashion. This is in contrast to previous reports that found a low percentage of fish with an inherited transgene don’t express the transgene in subsequent generations. Most important of all, the transgene expression is similar to endogenous rhodopsin. For example, the transgene is expressed in a cell-specific manner and follows the same spatial expression pattern seen with the endogenous rhodopsin gene.
Applications: This model will allow researchers to study specific genetic mutations and/or chemicals or drugs that may alter the movement of the protein to the outer segments or as a model to study the targeting of certain proteins in the rod photoreceptors. For example, in the human disease retinitis pigmentosa, abnormal targeting of certain proteins within the rods leads to blindness. Using the lazy eyes mutation as an initial example, the researchers have shown that rod degeneration can be seen in living animals and this phenotype can be used to identify mutant larvae. It is also possible to analyze the progression of certain aspects of these mutations in living embryos using time-lapse imaging techniques. For example, one could perform time-lapse imaging of developmental processes and examine its perturbation in response to pharmacological manipulations. Mutations affecting RA signaling pathways may also be characterized in this manner, as RA was shown to increase both the number of photoreceptors and the level of transgene expression in the model system.
Publications:
Transgenic expression of a GFP-rhodopsin COOH-terminal fusion protein in zebrafish rod photoreceptors. Visual Neuroscience (2002), 19, 257–264 For Further Information Please Contact the Director of Business Development Laura Brass Email: [email protected] Telephone: (617) 495-3067
Inventor(s): Dowling, John E.
Type of Offer: Licensing
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