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 condition, Leber congenital amaurosis (LCA). Last year, we successfully produced Cep290 knockout, and generated the first mouse model in this category. This year, addional two loci related to ciliopathy have been engineered in mouse to produce models of human LCA. They are the Cp110 conditional knockout, and the knockout of Cc2d2a. While Cp110 conditional knockout model still being verified for germline transmission of the targeted allele, the Cc2d2a knockout mouse line has given clear phenotypes of early retinal degeneration attributable to the failure of ciliogenesis and subsequent disruption in Shh signaling pathway. Other disease models which have been pursued in the core include RPGR C-terminus deletion mutation for Retinitis Pigmentosa, continuation of the work on Zfp703 knockout for coloboma 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 during the past year include continuation of the work on Cyp4v3 knockout for Bietti crystalline corneoretinal dystrophy and KLHL7 for Retinitis Pigmentosa, both of which were assigned as disease candidate genes 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 is aimed at simply understanding the functions of various genes relevant to NEI, as well as other participating IC research programs. Examples of such include knockout of Reep6 which is involved in intracellular protein trafficking and deletion of which has displayed phenotypes of delayed retinal degeneration;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. The transgenic mouse projects in which the GEC 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 32 different gene targeting projects at various stages * made 18 different constructs for gene targeting in ES cells or for over-expression gene in cells * made 152 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 1,246 ES colonies/clones * expanded 339 positive ES clones * assisted in PCR screening of targeted clones by performing long range PCR (756 reactions)) * injected 24 ES cell lines into mouse embryos to generate 160 chimeric mice * injected 4 DNA constructs into fertilized mouse oocytes to generate 5 transgenic mice * isolated DNA from 9,500 mouse tail biopsy samples * performed 9,761 PCR reactions to genotype mice in the facility * Set up 2,539 Matings to propagate mouse lines * Completed or oversaw weaning, tagging, and tail biopsy of 13,610 mice born in the facility * rederived 31 mouse lines * worked on cryopreservation of 110 mouse lines and 5 rat lines, freezing 154 rat embryos and 17,047 mouse embryos at the two cell stage, and 1,580 straws of sperm. * reconstituted for researchers 3 mouse lines from frozen germplasm stock * performed assisted reproduction to save 2 mouse lines from extinction * worked on 3 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 4 other institutes at NIH (NIAAA, NIDCD, NIDDK and NINDS).
Showing the most recent 10 out of 50 publications
