Module Director: Jim Hurley
206 543-2871
jbhhh@u.washington.edu
A. Description
Overview:
G/P is a new module that provides NEI-funded investigators at the University of Washington with services that facilitate analyses of vision in normal and mutant animals. The G/P module consolidates routine genotyping and phenotyping analyses in order to free up investigators’ time to focus more on the aims of their specific research projects.
Location:
The equipment and personnel for this module are located in two rooms. Room RR802 is a ~200 sq. ft. wet lab which houses the PCR machines, incubator, freezer, electrophoresis equipment and other tools needed for genotype analyses. Room RR847 is a ~150 sq. ft. dark room that houses the ERG apparatus and water maze and is the site for phenotype analyses.
Services:
The following services are provided directly by staff from the genotype/phenotype module:
1. Genotyping of mice and zebrafish.
Tissue isolation (mouse tails or zebrafish fins), genomic DNA preparation, PCR, guidance and advice for protocol design, interpretation of results.
2. Phenotype analysis of mutant mice and zebrafish.
a. Electroretinography. Analyses of a-waves (photoreceptor function) and b-waves (synaptic transmission) and overall visual sensitivity. ERGs are used as functional indicators of retinal degeneration and delayed dark-adaptation.
b. Behavioral analysis. Visual sensitivity, light- and dark-adaptation, spatial and temporal resolution are measured by evaluating vision-dependent behavior in a water maze.
B. History of the G/P Module
The Genotyping/Phenotyping (G/P) Module replaces the Molecular Techniques Module. MT was productive but it was used by a limited number of investigators. A re-evaluation of the goals of the CORE grant led to the idea of replacing MT with a module that would be more widely used by vision researchers at the University of Washington. Many vision research labs at the UW now use mutant animals, in particular, mice and zebrafish. Therefore, a particularly useful module would be one that provides genotyping and phenotyping services to expedite routine analyses required to identify and characterize mutant animals. In the current proposal the G/P module replaces the MT module.
C. Publications that Used the G/P Module:
Jang G-F, McBee JK, Alekseev AM, Haeseleer F, Palczewski K: Stereoisomeric specificity of the retinoid cycle in the vertebrate retina. J Biol Chem 2000; 275:28128-38.
McBee JK, Kuksa V, Alvarez R, de Lera AR, Prezhdo O, Haeseleer F, Sokal I, Palczewski K: Isomerization of all-trans-retinol to cis-retinols in bovine retinal pigment epithelial cells: dependence on the specificity of retinoid-binding proteins. Biochemistry 2000; 39:11370-80.
Jang G-F, Van Hooser JP, Kuksa V, McBee JK, He Y-G, Janssen JJM, Driessen CAGG, Palczewski K: Characterization of a dehydrogenase activity responsible for oxidation of 11‑cis-retinol in the retinal pigment epithelium of mice with a disrupted RDH5 gene: a model for the human hereditary disease fundus albipunctatus. J Biol Chem 2001; 276:32456-65.
Sokal I, Li N, Klug CS, Filipek S, Hubbell WL, Baehr W, Palczewski K: Calcium-sensitive regions of GCAP1 as observed by chemical modifications, fluorescence, and EPR spectroscopies. J Biol Chem 2001; 276:43361-73.
Haeseleer F, Imanishi Y, Saperstein DA, Palczewski K: Gene transfer mediated by recombinant baculoviruses into mouse eye. Invest Ophthalmol Vis Sci 2001; 42:3294-3300.
McBee JK, Van Hooser JP, Jang G-F, Palczewski K: Isomerization of 11-cis-retinoids to all-trans-retinoids in vitro and in vivo. J Biol Chem 2001; 276:48483-93.
Sokal I, Hu G, Liang Y, Mao M, Wensel TG, Palczewski K: Identification of protein kinase C isozymes responsible for the phosphorylation of photoreceptor-specific RGS9-1 at SER475. J Biol Chem 2003; 278:8316-25.
Noorwez SM, Kuksa V, Imanishi Y, Zhu L, Filipek S, Palczewski K, Kaushal S: Pharmacological chaperone-mediated in vivo folding and stabilization of the P23H-opsin mutant associated with autosomal dominant retinitis pigmentosa. J Biol Chem 2003; 278:14442-50.
Maeda T, Van Hooser JP, Driessen CAGG, Filipek S, Manssen JJM, Palczewski K: Evaluation of the role of the retinal G-protein-coupled receptor (RGR) in the vertebrate retina in vivo. J Neurochem 2003; 85:944-56.
Imanishi Y, Batten M, Piston DW, Baehr W, Palczewski K: Noninvasive two-photon imaging reveals retinyl ester storage structures in the eye. J Cell Biol 2004; 164:373-382. Commentary: Alan W. Dove: New structures found in plain sight. J Cell Biol 2004; 164:332.
Batten ML, Imanishi Y, Maeda T, Tu D, Moise AR, Bronson D, Possin D, Van Gelder RN, Baehr W, Palczewski K: Lecithin: retinol acyltransferase (LRAT) is essential for accumulation of all-trans-retinyl esters in the eye and in the liver. J Biol Chem 2004, 279; 10422-10432.
