The gene targeting projects that GEC members have worked on 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 in the VHL locus can lead to Von Hippel Lindau disease which is often manifested in the eye as a condition called Angiomatosis. As a result, loss of vision is very common. Two frequently encountered disease-generating mutations, both of which are missense mutations, have been engineered into the equivalent locus in mouse to generate potential models for the disease. One of the two point mutation knockin lines has achieved germline transmission successfully and the other is still in the germline breeding process. Another disease model that was successfully accomplished this year is the engineering of the Zfp703 knockout 1st allele for coloboma conditions. 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 during the past year include continuation of the work on Fbn2 knockin for AMD, and Kcnj13 knockout 1st model for the human condition known as Snow Flake Vitroretinal Degeneration. 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. Work in this area currently include the completion of the miR183 cluster knockout, which is involved in sensory neuron function and deletion of which has displayed phenotypes of retinal dysfunctions;completion of the construction of the conditional knockout of the PNPLA2 gene, a putative receptor of PEDF in retinal pigment epithelium;and generating conditional alleles for the ganglion cell-specific genes Brn3a and Brn3b. During the past year, we have: * worked on 28 different gene targeting projects at various stages * made 33 different constructs for gene targeting in ES cells and mouse zygotes, or for over-expression gene in cells * made 70 endotoxin-free, large scale DNA preparations of DNA constructs, and conducted 10 electroporation experiments plus 7 transfection assays for making stable cell lines * picked, expanded and cryopreserved 1300 individual ES clones * Expanded 150 positive ES clones * Assisted in PCR screening of targeted clones by performing long range PCR (1100 reactions) *Derived 30 iPS cell clones using Yamanaka factors carried in a tet-inducible Lentiviral vector from 4 different lines of mice which carry fluorescence reporters under the control of specific loci for in vitro differentiation and small molecule screening purposes *Isolated 4 clones of MEF cells from 3 different lines of genetically engineered mice for phenotype analyses and direct reprogramming purposes *Established 3 lines of immortalized MEF cells using SV40 large-T antigen from two different lines of knockout mice for phenotypic analyses * injected 13 ES cell lines into mouse embryos to generate 65 chimeric mice * injected 4 DNA constructs into fertilized mouse oocytes to generate 8 transgenic mice * injected CRISPR/Cas9 constructs into fertilized mouse oocytes for 20 injection sessions * isolated DNA from 11,092 mouse tail biopsy samples * performed 5,842 PCR reactions to genotype mice in the facility * set up 3,117 matings to propagate mouse lines * completed or oversaw weaning, tagging, and tail biopsy of 11,874 mice born in the facility * rederived 25 mouse lines * worked on cryopreservation of 108 mouse lines freezing 8,027 mouse embryos at the two cell stage, and 1,320 straws of sperm. * cryopreserved 2 lines of zebrafish as frozen sperm and validated both lines. * performed assisted reproduction to save 24 mouse lines from extinction and/or reconstitute mouse lines from frozen stock. * worked on several special projects which do not fall neatly into the above categories. These services and collaborative services were performed for 17 PIs from 5 NEI labs (LI, LRCMB, N-NRL, OGVFB, OSD), plus 4 PIs from 3 other institutes at NIH (NIDCD, NHLBI, NINDS)

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National Eye Institute (NEI)
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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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