of Core projects active during FY2015: FY 2015 has been a busy and productive year at the Zebrafish Core. Below, we provide a brief description of the services offered by the zebrafish core: 1. Microinjections: We perform microinjections of plasmid DNA, mRNA, and morpholinos into 1-8 cell stage embryos to develop transient and stable models for loss or gain of function of candidate genes and microinjections of bacteria and dyes in 48-72 hpf embryos for evaluation of immune and kidney functions. 2. Generation of knockout mutants using targeted mutagenesis: We have developed efficient high-throughput protocols to apply targeted mutagenesis using genome-editing tools (ZFNs, TALENs and CRISPR/Cas9) to generate knockout mutant fish. The ease and flexibility of design for CRISPR/Cas9 nucleases combined with their low cost has increased our throughput for knocking out genes from <10 to >100 annually. We design mutagenesis experiments with the researchers and perform mRNA synthesis, microinjections, somatic analysis, founder screening and genotyping of carrier fish, leading to the generation of at least two mutant alleles for phenotype characterization. 3. Generation of knock-in mutants using targeted mutagenesis: We are testing several of the published strategies with different length homology arms to develop an efficient protocol for targeted knock-in of transgenes, and introduction of specific missense variants at the endogenous loci. We will know their efficiency in the near future when we outcross the injected fish. 4. Generation of transgenic lines: We maintain stocks of several plasmids for transgenesis along with the appropriate Materials Transfer Agreements. We help in the design and cloning of the transgenesis vectors, perform all steps of zebrafish transgenesis, and provide the researcher with two or more lines for further characterization. 5. Whole mount in situ hybridization (WISH): WISH is performed to determine the temporal and spatial expression of genes and comparison of gene expression in wildtype versus the mutant/morphant embryos. We perform the WISH protocol for projects requiring less than five probes and provide training and reagents for other projects. We also maintain a stock of plasmids for commonly used WISH probes and provide training to generate gene-specific probes. 6. Cryopreservation, in vitro fertilization and disaster plan: We perform cryopreservation of all mutant and transgenic lines generated in the Core by freezing testes of several healthy males. The samples are stored in duplicates at two locations: liquid nitrogen freezer in building 49 and at an off-site facility (Kamtek) in Frederick, MD. This allows us to store important lines for future use, ensures their safety in the case of a catastrophic event, and eliminates the need to maintain live fish for lines not actively studied. We perform in vitro fertilization to recover the lines as needed. 7. Training and educational tours: We provide training to all new users from various laboratories in zebrafish handling, breeding, microinjections, embryo care, fin clips, genotyping, anesthesia, euthanasia, phenotyping by WISH, imaging, histology, survival analysis, project-specific procedures and crossing with appropriate transgenic lines to monitor the desired tissues during development. This is one of our most time-consuming services. We also provide tours to students and teachers from schools and colleges throughout the country through the NHGRI Intramural Training Office, NHGRI office of education, NIH visitor center and NIH office of the Director. Below we provide a brief description of the projects we worked on during FY2015: Developed a new method, termed CRISPR-STAT, to screen sgRNAs for target-specific activity, reducing the husbandry costs on raising fish injected with inactive sgRNAs. Optimized protocols for CRISPR/Cas9 mediated knockout mutant generation in collaboration with Burgess lab Generated knockout mutants for 88 genes using CRISPR/Cas9 nucleases (Myung, Liu and Gahl labs) with 40 genes in progress (Chandrasekharappa and Burgess labs). Added 15 new users to the animal protocol and trained majority of them in microinjections, and all aspects of zebrafish handling, WISH, imaging and specific procedures to perform phenotype analysis of the mutant fish (Gahl, Liu, Myung, Burgess labs). Performed characterization of the kiaa0753 knockout fish to understand its function during embryogenesis (Gahl lab) Assisted in generation of adult diabetes model by STZ injections (Gahl lab). Generated Heat shock diploid fish by in vitro fertilization with UV inactivated sperm to develop homozygous fish. Performed cryopreservation of >100 mutant zebrafish lines (Core, Burgess, Liu, Gahl, Wilson, Ostrander labs). Optimization of protocols for targeted knock-in of transgenes and missense variants is under progress (Liu lab).
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