Genetics Survey of Common Eye Diseases in Laboratory Mouse Strains Bo Chang, Ron Hurd, Jieping Wang, and Patsy Nishina The Jackson Laboratory, Bar Harbor, Maine Correspondence: Bo Chang, The PURPOSE. As in human populations, in which founder mutations have been identified in groups Jackson Laboratory, 600 Main Street , of families, a number of founder mutations have been observed across strains in mice. In this Bar Harbor, ME 04609; report, we provide a phenotype and genotype survey of three common eye diseases in the [email protected]. collection of JAX mice strains at The Jackson Laboratory (JAX). These eye diseases are retinal Submitted: April 24, 2013 degeneration 1 (Pde6brd1), retinal degeneration 8 (Crb1rd8), and cone photoreceptor Accepted: June 20, 2013 function loss 3 (Gnat2cpfl3). Citation: Chang B, Hurd R, Wang J, METHODS. Ocular lesions for rd1 and rd8 were evaluated by fundus examination and fundus Nishina P. Survey of common eye photography, and the abnormal retinal function observed in mice homozygous for cpfl3 was diseases in laboratory mouse strains. assessed by ERG. Genotyping protocols for rd1, rd8, and cpfl3 mutations were performed by Invest Ophthalmol Vis Sci. 2013;54:4974–4981. DOI:10.1167/ PCR with appropriate primers. iovs.13-12289 RESULTS. We have actively screened retired breeders for surface dysmorphologies, and for intraocular defects by indirect ophthalmoscopy, slit-lamp biomicroscopy, and ERG to discover new spontaneous mutations in strains from the Genetic Resource Science (GRS) production colony. Through this process, we have found that of the strains screened, 99 strains carried the rd1 mutation, 85 strains carried the rd8 mutation, and 20 strains carried the cpfl3 mutation. CONCLUSIONS. Of the 1000 of strains screened during this study, 204 carried one of three founder mutations in Pde6b, Crb1,orGnat2. Since these three retinal mutations occur commonly in various mouse strains, genotyping for these mutations, and/or avoiding mouse strains or stocks carrying these mutant alleles when studying new retinal disorders is recommended. The robust PCR genotyping protocols to test for these common alleles are described herein. Keywords: retinal degeneration, fundus photography, spontaneous mutation, animal model, eye diseases iscovery of human retinal degenerations generally is our understanding of the function of specific genes and how Didentified by self-reports of visual problems with subse- they lead to disease when disrupted. Studies of mouse models quent eye examinations. However, determining the etiology of human retinal degeneration have contributed to our and the disease progression, elucidating the mechanisms understanding of the pathophysiology, as well as the etiology, underlying the disease, and exploring methods for delaying or of these diseases, and allowed for development of gene-based ameliorating retinal degenerations can be impeded by ethical therapy to delay the progression of disease and increase visual considerations of exploratory experimentation on humans. function.3,4 Also, human eye tissue (including biopsies) seldom is available Historically, the first retinal degeneration model was for study because it is difficult to obtain tissue from the eye described by Clyde E. Keeler more than 80 years ago. It without risk of further damage to the patient’s vision, and if subsequently was identified as a disruption in phosphodiester- samples are available, they usually are at the end stages of the ase 6 beta (Pde6brd1, formerly rd1, rd, identical to Keeler disease process. rodless retina, r).5–7 The second retinal degeneration model Animal models have advanced our understanding of eye was discovered 50 years later, and named retinal degeneration- diseases significantly as they allow for sampling of tissue slow (Rds or Rd2).8 The third retinal degeneration was throughout development and disease progression, for invasive discovered in 1993 and named retinal degeneration 3, rd3.9 studies, and for rapid genetic analysis. Use of mouse models, in Subsequently, during the last 20 years, many additional mouse particular, has increased through the past decades due to their models of retinal degeneration have been discovered as the ability to be modified genetically. In addition to manipulation by tools used commonly in human clinical eye examinations have genetic engineering, a large number of spontaneous and been adapted for use with the smaller mouse eyes.10,11 chemically-induced mutations have been identified by screen- Interestingly, through screening many retired breeders of ing mice using indirect ophthalmoscopy and ERG at The different mouse strains at JAX, mutations in Pde6b, Crb1, and Jackson Laboratory (JAX; Bar Harbor, ME), where many mouse Gnat2 have been found to recur in high frequency. Because the models of retinal degeneration and diseases have been strains examined all bore the same respective mutations within discovered.1,2 These mouse models have increased significantly these particular genes, this suggested that they were founder Copyright 2013 The Association for Research in Vision and Ophthalmology, Inc. www.iovs.org j ISSN: 1552-5783 4974 Downloaded from iovs.arvojournals.org on 09/28/2021 Common Eye Diseases in Mice IOVS j July 2013 j Vol. 54 j No. 7 j 4975 FIGURE 1. Fundus photographs of normal pigmented (A) and albino (C) mice as well as fundus photographs of mice bearing the Pdeb6rd1 mutation in pigmented (B) and albino (D) background. mutations that had been propagated across multiple strains in accordance with the ARVO Statement for the Use of Animals through the years of model development and inbreeding.12–14 in Ophthalmic and Vision Research. As significant retinal pathology occurs in the presence of rd1 rd8 Pde6b and Crb1 , and functional deficits occurs in the Clinical Retinal Evaluation presence of Gnat2cpfl3, interpretation of disease phenotypes resulting from different gene/mutations and experimental Mouse eyes, dilated with 1% atropine (Alcon Laboratories, Inc., studies where behavioral performance is an end point, Fort Worth, TX), were evaluated by indirect ophthalmoscopy respectively, may be confounded. Knowledge of the genotype with a 90 diopter lens. If a mouse strain showed signs of early for the common disease alleles in these experimental models and rapid retinal degeneration, such as attenuation of retinal will aid in the proper selection of mouse strains for use in vessels or areas of depigmentation (Fig. 1B), the mouse strain research studies and interpretation of data generated from was genotyped for the rd1 mutation. If a mouse strain in the them. C57BL/6 genetic background or related background showed a retinal spotting phenotype (Fig. 2A), the mouse strain was typed for the rd8 mutation. Fundus photographs were taken MATERIALS AND METHODS with the Micron III retinal imaging microscope from Phoenix Research Laboratories (Phoenix, AZ). Mice The mice screened in this study were bred and maintained in Simplified Screening for Functional ERG standardized conditions at the Production and Research Animal Alterations Facilities at JAX. They were maintained on a NIH31 6% fat chow diet and provided acidified water, in a pathogen-free Attenuation of retinal vessels and disturbances in the retinal vivarium environment with a 14-hr light/10-hr dark cycle. One pigment epithelium are detected easily by indirect ophthal- pair of retired breeders from the JAX Mouse Repository was moscopy. However, even if the retina has a normal screened for each strain. All experiments were approved by the appearance, retinal function still may be impaired. Therefore, Institutional Animal Care and Use Committee, and conducted all strains with a normal fundus appearance by indirect Downloaded from iovs.arvojournals.org on 09/28/2021 Common Eye Diseases in Mice IOVS j July 2013 j Vol. 54 j No. 7 j 4976 FIGURE 2. Fundus photographs of mice homozygous for the Crb1rd8 mutations with the typical pattern of white retinal spots (A), with a few white retinal spots (B), and with no retinal white spots (C). ophthalmoscopy were screened for functional deficits by base of the tail for grounding, a gold loop electrode was placed ERG. Because the standard ERG test is time consuming, between the gum and cheek, and the active gold loop requiring approximately 30 to 60 minutes per mouse (not electrode was placed gently on the cornea slightly below the including the time necessary for dark adaption), we have middle of the eye. A drop of 2.5% Gonioscopic Prism Solution developed a simple screening ERG test that takes less than 10 (Wilson Ophthalmic, Mustang, OK) was placed on the cornea minutes per mouse to detect for functional deficits, such as and electrode to assure a good contact and to prevent drying of 14,15 retinal cone photoreceptor function loss (achromatopsia) the eye surface. In the simplified test, 10 flashes of the highest 16,17 andnob-wave(nob)mutations. light intensity (light intensity ¼ 1.9 log cd/m2) were averaged. Briefly, one eye per mouse was dilated with 1% cyclopen- If a mouse had a photopic b-wave amplitude at or above 100 tolate hydrochloride ophthalmic solution (Bausch & Lomb, lV and implicit times at approximately 50 ms, the mouse was Inc., Tampa, FL). A group of 5 to 10 mice could be dilated at considered normal (Fig. 3A), whereas a mouse with a b-wave the same time. Mice were weighed to determine the proper dosage of anesthesia (0.1 mL/20 g body weight of a mixture of amplitude at or below 50 lV and/or implicit times longer than 0.8 mL ketamine [Ketamine KCL injection USP, 100 mg/mL;