NEI Genetic Engineering Core

National Eye Institute

Investigators

Linked publications & trials

Abstract

The gene targeting activities that GEF members have participated in this year include the following: [unreadable] [unreadable] 1) Human disease modeling:[unreadable] It is often desirable to have mutations of human genetic conditions replicated in mouse so that the diseases can be modeled. In the last year, several such models have been made or are being developed in the NEI Genetic Engineering Core, in collaboration with NEI researchers. Point mutations of the human RPE65 gene are associated with the most severe retinal dystrophy condition, Leber congenital amaurosis (LCA). Three point mutations, which are frequently found in LCA patients, have been separately introduced into the mouse ES cell genome, and one of them has been microinjected, resulting in germline transmission from chimeric mice. These engineered mutant mice will serve as a model system for studying the disease, and possibly participate in preclinical therapy trails. Other disease models which have been pursued in the core, and are at different stages of development include: Cep290 knockout for retinal degeneration, RPGR point mutation knockin for Retinitis Pigmentosa, and Zfp503 knockout for coloboma.[unreadable] [unreadable] 2) Reverse genetic confirmation of disease candidate genes:[unreadable] The core supports the disease gene discovery programs by making knockouts of the candidate genes to establish direct linkage between the genes identified in forward genetic screens and the genetic conditions against which the screens were conducted. Projects in this area include: Cyp4v3 knockout for Bietti crystalline corneoretinal dystrophy, which has been linked to the human Cyp4v2 locus (mouse Cyp4v3) by NEI researchers, and, in collaboration with NIDCD researchers, transmembrane channel-like (TMC) genes 3, 6 and 8, mutations of which have been linked to hearing loss. [unreadable] [unreadable] 3) Functional genomic studies of genes with ocular specific expression or functions:[unreadable] Most of the current gene targeting projects are aimed at simply understanding of the functions of various genes relevant to institute research programs. Examples of such include: growth/cytokine genes, which may be critically involved in lens morphogenesis (KLPK knockout), retinal vascular biology (Cry-A3/A1, PDGF-C and PDGF-D conditional knockouts), and ocular micro RNA functional analyses (there are currently 6 miRNA knockout projects at different stages of development).[unreadable] [unreadable] The transgenic mouse projects in which the GEF has been involved this year can be divided into four major categories. [unreadable] [unreadable] 1) Expression of normal proteins ectopically to help determine their roles in ocular physiology and structure. In this category, 9 constructs were microinjected to investigate glaucoma, factors possibly mediating autoimmune uveitis, and several types of retinal degenerations and diseases.[unreadable] [unreadable] 2) Expression of mutant proteins known to be involved in human diseases. The 9 constructs in this category included mutant proteins involved in: age related macular degeneration (AMD), human cataract, retinal diseases, protein turnover, and neuronal and vascular patterning in retina and CNS.[unreadable] [unreadable] 3) Tissue-specific expression of cre recombinase for use in generating conditional gene knockout animal models. This category represented only 2 constructs this year; one in which cre is targeted to the trabecular meshwork (to be used in making glaucoma models), and one in which cre expression is directed to photoreceptor cells (for modeling retinal diseases and degenerations).[unreadable] [unreadable] 4) In depth study of genetic promoter regions, often referred to as "promoter bashing", to gain a detailed understanding of how gene expression is regulated, and to determine optimal promoter sequences to use in transgene constructs when designing new transgenic animal models. This category included 13 constructs for studying corneal expression and 7 constructs for studying lens expression.[unreadable] [unreadable] [unreadable] During the past year, we have:[unreadable] * worked on 32 different gene targeting projects at various stages[unreadable] * made 17 different constructs for gene targeting in ES cells [unreadable] * made 23 endotoxin-free, large scale DNA preparations of the targeting constructs, and subsequently conducted electroporation experiments with each of the DNA preparations[unreadable] * picked, expanded and crypyopreserved 6,284 ES colonies/clones [unreadable] * expanded 144 positive ES clones, 34 of which were thawed and grown in culture for karyotyping and microinjection[unreadable] * assisted in PCR screening of targeted clones by performing long range PCR (3,012 reactions) [unreadable] * assisted researchers in Southern blot confirmation of homologous recombination[unreadable] * injected 29 ES cell lines into mouse embryos to generate chimeric mice[unreadable] * received 26 new DNA constructs for transgenic mouse production[unreadable] * injected 40 DNA constructs into fertilized mouse oocytes to generate transgenic mice[unreadable] * isolated DNA from 10,492 mouse tail biopsy samples[unreadable] * performed 14,913 PCR reactions to genotype mice in the facility[unreadable] * set up 779 matings to propagate mouse lines[unreadable] * overseen weaning, tagging and tail biopsy of 9,079 mice born in the facility[unreadable] * rederived 67 mouse lines[unreadable] * worked on cryopreservation of 12 mouse lines and 14 rat lines, freezing 2 cell stage embryos and sperm[unreadable] * cryopreserved one naturally-occurring mouse line, freezing morula stage embryos[unreadable] * reconstituted for researchers two mouse lines from frozen germplasm stock[unreadable] * performed assisted reproduction to save 35 mouse lines from extinction.[unreadable] [unreadable] These services and collaborative services were performed for 16 PIs from 6 NEI labs (LI, LMDB, LRCMB, N-NRL, OGVFB, OSD), plus 17 PIs from 5 other institutes at NIH (NIDCD, NHLBI, NICHD, NIDCR, NINDS) plus one PI from another DHHS agency (FDA). [unreadable] [unreadable] [unreadable] Additional publications to which the NEI Genetic Engineering Core Facility contributed:[unreadable] 4. Tuo J, Bojanowski CM, Zhou M, et al. (2007) Murine ccl2/cx3cr1 deficiency results in retinal lesions mimicking human age-related macular degeneration. Invest Ophthalmol Vis Sci 48(8):3827-36.[unreadable] 5. Swamynathan SK, Davis J, Piatigorsky J (2008) Identification of Candidate Klf4 Target Genes Reveals the Molecular Basis of the Diverse Regulatory Roles of Klf4 in the Mouse Cornea. Invest Ophthalmol Vis Sci 49:3360-3370.[unreadable] 6. Fujimoto C, Shi G, Gery I (2008) Microbial products trigger autoimmune ocular inflammation. Ophthalmic Res 40:193-199.[unreadable] 7. Ross RJ, Zhou M, Shen D, et al. (2008) Immunological protein expression profile in Ccl2/Cx3cr1 deficient mice with lesions similar to age-related macular degeneration. Exp Eye Res 86(4):675-83.[unreadable] 8. Chan CC, Ross RJ, Shen D, et al. (2008) Ccl2/Cx3cr1-deficient mice: an animal model for age-related macular. Ophthalmic Res 40(3-4):124-8.[unreadable] 9. Zhou Y, Grinchuk O, Tomarev SI (2008) Transgenic Mice Expressing the Tyr437His Mutant of[unreadable] Human Myocilin Protein Develop Glaucoma. Invest Ophthalmol Vis Sci 49:1932-1939.