The gene targeting projects that GEF members have conducted in this year include the following scientific areas 1) Human disease modeling: 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. Mutations of the human Cep290 gene are associated with the most severe retinal dystrophy condition, Leber congenital amaurosis (LCA). The Cep290 gene has been deleted in the mouse ES cell genome creating a null mutation. Germline transmission of the Cep290 knockout allele was achieved, and homozygosity of the KO allele has confirmed to cause early onset of retinal degeneration, providing for the first time a good model system for Cep290 mutation-mediated LCA conditions. Other disease models which have been pursued in the core include RPGR point mutation knockin for Retinitis Pigmentosa, Zfp703 knockout for coloboma, cc2d2a for retinal degeration, and Rtbdn knockout for cataract. 2) Reverse genetic confirmation of disease candidate genes: The core supports the disease gene discovery programs by making knockouts and knockins 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, and KLHL7 for Retinitis Pigmentosa, which has been assigned as a disease candidate gene by linkage analyses by NEI researchers. 3) Functional genomic studies of genes with interesting expression patterns and predicted to be functionally important in physiology and pathology: Most of the current gene targeting projects are aimed at simply understanding the functions of various genes relevant to NEI, as well as other participating IC research programs. Examples of such include: growth/cytokine genes, which may be critically involved in lens morphogenesis such as KLPH knockout which has displayed phenotypes of cataract development, retinal vascular biology such as Cry-A3/A1, PDGF-C and PDGF-D conditional knockouts, which continue to generate interesting phenotypes in neural protection, ocular angiogenesis, and CNV development. Functions of miRNA have also been explored by making loss of function mutations in mice. There are currently 3 miRNA knockout projects at different stages of development in the core, all of which have achieved germline transmission and in phenotypic evaluations. The transgenic mouse projects in which the GEF has been involved this year can be divided into several major categories. Expression of normal or mutant proteins ectopically to help determine their roles in ocular physiology and structure. In this category, 5 constructs were microinjected to investigate retinal degenerations and diseases. Tissue-specific expression of cre recombinase for use in generating conditional gene knockout animal models. This category represented 2 constructs this year;one astrocyte-specific and one retiana-specific. Complex transgenes which include multiple functionalities. This category represented 3 constructs this year During the past year, we have: * worked on 17 different gene targeting projects at various stages * made 47 different constructs for gene targeting in ES cells or for over-expression gene in cells * made 75 endotoxin-free, large scale DNA preparations of the targeting constructs, and subsequently conducted electroporation experiments with each of the DNA preparations * picked, expanded and crypyopreserved 4400 ES colonies/clones * expanded 181 positive ES clones, 127 of which were thawed and grown in culture for karyotyping and microinjection * assisted in PCR screening of targeted clones by performing long range PCR (4957 reactions)) * assisted researchers in Southern blot confirmation of homologous recombination * injected 31 ES cell lines into mouse embryos to generate 221 chimeric mice * injected 8 DNA constructs into fertilized mouse oocytes to generate 28 transgenic mice * isolated DNA from 12,120 mouse tail biopsy samples * performed 13,946 PCR reactions to genotype mice in the facility * Set up 2,769 Matings to propagate mouse lines * Completed or oversaw weaning, tagging, and tail biopsy of 13,078 mice born in the facility * rederived 7 mouse lines * worked on cryopreservation of 96 mouse lines and 18 rat lines, freezing 1481 rat embryos and 19,382 mouse embryos at the two cell stage, and 1482 straws of sperm. * reconstituted for researchers 3 mouse lines from frozen germplasm stock * performed assisted reproduction to save 2 mouse lines from extinction. These services and collaborative services were performed for 19 PIs from 6 NEI labs (LI, LMDB, LRCMB, N-NRL, OGVFB, OSD), plus 4 PIs from two other institutes at NIH (NIDCD and NINDS). Additional publications to which the NEI Genetic Engineering Core Facility contributed: Zhengrong Wu, PhD;Soojin Lee, Ph.D.;Bryon Mahler, BS;Jodie Toward, BS;Blake Jones;Keith Wyatt, Ph.D.;Lijin Dong, Ph.D.;Graeme Wistow, Ph.D., 2009 A single destabilizing mutation (F9S) promotes concerted unfolding of an entire globular domain in S-crystallin to JMB Anil Kumar, Xu Hou, Chunsik Lee, Yang Li, Arvydas Maminishkis, Zhongshu Tang, Fan Zhang, Harald Franz Langer, Pachiappan Arjunan, Lijin Dong, Zhijian Wu, Linda Yijia Zhu, Lianchun Wang, Wang Min, Peter Colosi, Triantafyllos Chavakis, and Xuri Li, PDGF-DD targeting arrests pathological angiogenesis by modulating glycogen synthase kinase 3 beta (GSK3) phosphorylation J. Biol. Chem. jbc.M110.113787First Published on March 15, 2010, doi:10.1074/jbc.M110.113787 Zhongshu Tang, Pachiappan Arjunan, Chunsik Lee, Yang Li, Anil Kumar, Xu Hou1, Bin Wang, Piotr Wardega, Fan Zhang, Lijin Dong, Yongqing Zhang, Shi-Zhuang Zhang, Hao Ding, Kevin . Becker, Johan Lennartsson, Nobuo Nagai, Yihai Cao, Xuri Li1* Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3 phosphorylation. JEM 2010 in press Wang FE, Zhang C, Maminishkis A, Dong L, Zhi C, Li R, Zhao J, Majerciak V, Gaur AB, Chen S, Miller SS. MicroRNA-204/211 alters epithelial physiology. FASEB J. 2010 Jan 7.

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Fan, Jianguo; Lerner, Joshua; Wyatt, M Keith et al. (2018) The klotho-related protein KLPH (lctl) has preferred expression in lens and is essential for expression of clic5 and normal lens suture formation. Exp Eye Res 169:111-121
Lui, Julian C; Barnes, Kevin M; Dong, Lijin et al. (2018) Ezh2 Mutations Found in the Weaver Overgrowth Syndrome Cause a Partial Loss of H3K27 Histone Methyltransferase Activity. J Clin Endocrinol Metab 103:1470-1478
Wang, Herui; Shepard, Matthew J; Zhang, Chao et al. (2018) Deletion of the von Hippel-Lindau Gene in Hemangioblasts Causes Hemangioblastoma-like Lesions in Murine Retina. Cancer Res 78:1266-1274
Huang, Yanhan; Hill, Jennifer; Yatteau, Andrew et al. (2018) Reciprocal Negative Regulation Between Lmx1a and Lmo4 Is Required for Inner Ear Formation. J Neurosci 38:5429-5440
Imtiaz, Ayesha; Belyantseva, Inna A; Beirl, Alisha J et al. (2018) CDC14A phosphatase is essential for hearing and male fertility in mouse and human. Hum Mol Genet 27:780-798
Bhaskaran, Natarajan; Liu, Zhihui; Saravanamuthu, Senthil S et al. (2018) Identification of Casz1 as a Regulatory Protein Controlling T Helper Cell Differentiation, Inflammation, and Immunity. Front Immunol 9:184
Li, Jun; Han, Wenyan; Pelkey, Kenneth A et al. (2017) Molecular Dissection of Neuroligin 2 and Slitrk3 Reveals an Essential Framework for GABAergic Synapse Development. Neuron 96:808-826.e8
Veleri, Shobi; Nellissery, Jacob; Mishra, Bibhudatta et al. (2017) REEP6 mediates trafficking of a subset of Clathrin-coated vesicles and is critical for rod photoreceptor function and survival. Hum Mol Genet 26:2218-2230
Jiang, Jingjing; Wu, Xiaofei; Shen, Di et al. (2017) Analysis of RP2 and RPGR Mutations in Five X-Linked Chinese Families with Retinitis Pigmentosa. Sci Rep 7:44465
Fan, Jianguo; Jia, Li; Li, Yan et al. (2017) Maturation arrest in early postnatal sensory receptors by deletion of the miR-183/96/182 cluster in mouse. Proc Natl Acad Sci U S A 114:E4271-E4280

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