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 genes in ciliogenesis are associated with the most severe retinal dystrophy conditions such as Leber congenital amaurosis (LCA) and Retinitis Pigmentosa (RP). This year, two new engineered alleles within this category have had successful germline transmissions which are Cp110 conditional knockout and RPGR C-terminus deletion mutation respectively, bringing the total number of engineered alleles for studying conditions in cilioopathy to 4. The previously built two models in this category include the Cep290 knockout and the Cc2d2a knockout, both have given interesting phenotypes in ciliogenesis of photoreceptor cells. Another disease model which was successfully accomplished this year is the work on Zfp503 knockout for coloboma conditions. Zfp703 knockout first allele, another project related to conditions in coloboma, is still under development. 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 VHL knockin of point mutations for Von Hippel-Lindau, both are disease candidate mutations. 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 Reep6 knockout, which is involved in intracellular protein trafficking and deletion of the gene has displayed phenotypes of delayed retinal degeneration;continuation in the projects of structural and functional analysis of the RPE65 gene by generating point mutations of the gene in mouse;and the conditional knockout of the PNPLA2 gene, a putative receptor of PEDF in retinal pigment epithelium;and generating conditional alleles for ganglion cell-specific genes Brn3a and Brn3b. The 6 transgenic mouse projects in which the GEC has been involved this year can be divided into several categories. Expression of normal or mutant proteins ectopically to help determine their roles in ocular physiology and structure. In this category, 1 construct was microinjected. Tissue-specific expression of cre or dre recombinase for use in generating conditional gene knockout animal models. This category represented 3 constructs this year. Generation of models of specific human gene defects to study the genetic disease in mouse. This category represented 2 constructs this year. During the past year, we have: * worked on 22 different gene targeting projects at various stages * made 13 different constructs for gene targeting in ES cells or for over-expression gene in cells * made 250 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 2000 ES colonies/clones * expanded 160 positive ES clones * assisted in PCR screening of targeted clones by performing long range PCR (1250 reactions)) * injected 14 ES cell lines into mouse embryos to generate 105 chimeric mice * injected 6 DNA constructs into fertilized mouse oocytes to generate 57 transgenic mice * isolated DNA from 12,216 mouse tail biopsy samples * performed 6,197 PCR reactions to genotype mice in the facility * Set up 3,455 Matings to propagate mouse lines * Completed or oversaw weaning, tagging, and tail biopsy of 13,075 mice born in the facility * rederived 51 mouse lines * worked on cryopreservation of 375 mouse lines and 2 rat lines, freezing 7 rat embryos and 8,084 mouse embryos at the two cell stage, and 2,250 straws of sperm. * performed assisted reproduction to save 35 mouse lines from extinction and/or reconstitute mouse lines from frozen stock. * worked on 4 special projects which do not fall neatly into these categories. These services and collaborative services were performed for 18 PIs from 6 NEI labs (LI, LMDB, LRCMB, N-NRL, OGVFB, OSD), plus 6 PIs from 3 other institutes at NIH (NIDCR, NIDDK and NINDS).
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