Genomic manipulation projects at GEC this year are from 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, one such model was developed at GEC in collaboration with Dr. Michael Redmond's laboratory at NEI. Mutations in the RPE65 locus can lead to Leber's congenital amaurosis (LCA) in human, which is often manifested as vision loss. One frequently encountered disease-generating point mutation in human was engineered into the RPE65 locus in mouse to generate potential models for the disease. This mutant line was created through the CRISPR/Cas9 technology, with which, the desired mutation was directly introduced into the mouse genome in oocytes with no ES cell manipulation necessary. In collaboration with Dr. J. Fielding Hejtmancik's laboratory at NEI, we participated in characterization of the conditional and knockout-first alleles of the Kcnj13 gene, a candidate for dominant and recessive forms of snowflake vitreoretinal degeneration in human. 2) Reverse genetic confirmation of disease candidate genes: The core supports the disease gene discovery programs by making knockout and knockin models of the candidate genes to establish direct linkage between the genes identified in forward genetic screens and the phenotypic manifestations against which the screens were conducted. During the past year, one project was conducted in this area, the generation of knockout mutation in Asph1 (Aspartate beta-hydroxylase domain-containing protein 1) locus for a distinct form of syndromic ectopia lentis in human. 3) Functional genomic studies of genes predicted to be 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 includes the characterization 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 knockouts of the miR204 and miR211 genes, which have shown, in preliminary studies, to be involved in maintenance of RPE normal functions; and generating inducible Cre lines under the control of Nrl or Crx locus. Additional targets in this area include Pcp4lI, Cdr2, Il12a, Frmpd1, Crh, B3glct, and Admts9. During the past year, we have worked on 41 different gene targeting projects of total 15 distinct loci at various stages. In order to achieve mutagenesis goals of all the projects above, we have conducted the following experimental procedures: * Designed and constructed 76 recombinant DNA clones for gene targeting in ES cells, CRISPR/Cas9-mediated gene targeting in mouse zygotes, and lentiviral construction. * Performed 53 in vitro transcription assays for guide RNA production in CRISPR-mediated genome editing. * Made 10 endotoxin-free, large scale DNA preparations of DNA constructs. * Conducted over 141 transfection assays for making mutant ES cell lines, picked 4228 ES clones, expanded and cryopreserved over 4228 individual ES clones, expanded 32 positive ES clones and in addition, conducted at least 4,779 genomic PCR reactions for screening of targeted mutations. * injected 3 ES cell lines into mouse embryos to generate 5 chimeric mice * Injected 2 DNA constructs into fertilized mouse oocytes to generate 14 transgenic mice * injected 32 CRISPR/Cas9 constructs into fertilized mouse oocytes for 65 injection sessions to produce 53 mutant mice For the purposes of colony maintenance, we conducted following procedures: * isolated DNA from 12,973 mouse tail biopsy samples * performed 8,738 PCR reactions to genotype mice in the facility * set up 3,003 matings to propagate mouse lines * completed or oversaw weaning, tagging, and tail biopsy of 11,766 mice born in the facility * made 843 mouse deliveries to researchers' labs * provided 181 consultations to researchers on breeding strategies * rederived 10 mouse lines * worked on cryopreservation of 77 mouse lines freezing 6346 mouse embryos at the two cell stage, and 1280 straws of sperm * cryopreserved 7 lines of zebrafish as frozen sperm and validated 8 lines. Cryopreserved testes from 3 lines of zebrafish * performed assisted reproduction to save 16 mouse lines from extinction and/or reconstitute mouse lines from frozen stock 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 (NHLBI, NINDS, NICHD)
Showing the most recent 10 out of 50 publications
