R01EY09769 Zack, Donald J
PROJECT NUMBER 5 R01 EY09769-04
This application proposes to study the molecular basis of retinal development using the regulation of rhodopsin gene expression as a model system. Preliminary transgenic mouse studies with fusion construct sconsisting of sequences upstream of the bovine rhodopsin gene ligated to the reporter gene lacZ (beta-galactosidase) have identified cis-acting DNA regulatory sequences that are capable of directing photoreceptor cell-specific gene expression. The finding of a discontinuous transgene expression gradient in 3 out of 3 transgenic lines with one of the constructs suggests the existence of a "topographical" regulatory element which defines spatial expression across the
retina. The finding of apparent integration site-independent expression with this same constructis suggestive of a rhodopsin locus control region (LCR), analogous to that seen with beta-globin. In order to pursue these results, additional transgenic lines will be generated to define more fully the regulatory elements involved and to characterize the properties of the putative topographical and LCR elements. DNA sequence comparisons of rhodopsin upstream regions from different species and in vitro DNA-protein binding studies (work for which funding is not being requested in this application) will be used to aid in the decision of which additional constructs to make.
To analyze the mechanisms responsible for the spatial gradient of transgene expression, photoreceptors that do and do not express lacZ will be isolated using the fluorescence activated cell sorter (FACS) and a fluorescent beta-galactosidase substrate. The two cell populations will then be compared with regards to a number of parameters including site-specific differences in DNase I hypersensitivity and methylation status. In vivo footprinting of the rhodopsin gene and the transgene will also be performed. A non-radioactive in situ hybridization assay will be used to look at the spatial and developmental pattern of expression of rhodopsin and other retinal genes in retinal whole mounts.
Due to the time and expense involved in transgenic studies, efforts will also be made to complement the transgenic studies with simpler methods for the in vivo study of retinal cis-acting elements. Approaches utilizing transfection of reporter constructs into primary retinal cultures and retinal cell lines and direct injection of DNA into the subretinal space of mice are proposed. (Direct injection of DNA into mouse skeletal and cardiac muscle has been shown to result in muscle expression.) Studies on the regulation of expression of retina-specific genes, in addition to their importance at the basic level, will also have implications for the understanding and treatment of human disease. The definition of retina-specific promoters will make possible targeted expression of heterologous and altered genes to the retina. This will facilitate the generation of animal models of human disease and may have implications for future attempts towards gene therapy, particularly in light of the finding that mutations in the rhodopsin gene are responsible for
some forms of autosomal dominant retinitis pigmentosa.