of Core projects active during FY2017: During FY2017, we completed several on-going projects and started new projects. We also participated in presenting the work at scientific conferences and writing of resulting manuscripts. A brief description of the services offered by the zebrafish core: 1. Microinjections: We perform microinjections of DNA, RNA and/or morpholinos into yolk or nucleus of 1-cell stage embryos for functional genomics and transgenesis projects. We also perform microinjections of fluorescent dyes, bacteria and cells in blood, brain or yolk of older embryos for evaluation of immune and kidney functions and cell migration assays. 2. Generation of gene knockout mutants using genome-editing nucleases: Currently, we are using CRISPR-Cas9 for the generation of genetic knockout mutants. This is a multi-step process requiring 8 months for completion/gene. Each project starts with a consultation meeting to design the optimum target site for their gene of interest. The Core staff performs guide RNA design and optimization, mRNA synthesis, microinjections, primer design, somatic analysis by fluorescent PCR, founder screening, identification of heterozygous fish and sequencing to determine the exact nature of selected indel mutations. 3. Characterization of mutant fish: The mutant fish generated from the above pipeline are transferred to the investigator for phenotype analysis. We help them perform preliminary characterization by microscopic observations during embryonic development and survival to adulthood. For extensive phenotype characterization, we provide guidance, training and help with imaging, fin clips and genotyping. 4. Generation of transgenic lines: The Core provides guidance in the design and cloning of desired genomic fragments into transgenesis vectors, and performs microinjections and founder screening to generate stable transgenic lines. 5. Whole mount in situ hybridization (WISH): The Core maintains several plasmids for WISH probes. The Core staff performs and trains researchers for the WISH protocol (multi-step one week protocol requiring extensive pipetting) and imaging. 6. Cryopreservation and in vitro fertilization (IVF): We perform cryopreservation of all in-house generated mutant and transgenic lines by dissecting testes from 3-5 healthy males per line. The samples are stored in duplicates at an on-campus and off-campus locations as per NHGRI disaster plan. Cryopreservation also eliminates the need to maintain live fish when a project is completed and saves husbandry costs. We perform IVF by obtaining eggs from healthy females to recover the lines as needed. 7. Training and Educational tours: We provide training to new users in zebrafish handling, breeding, embryo care, fin clips, genotyping, anesthesia, euthanasia, WISH, imaging, project-specific procedures. The Core is a favorite spot for tours organized by NHGRI Education and Community Involvement Branch, Intramural Training Office, NIH visitor center, NIH office of the Director etc. Tour groups consist of students and teachers from a variety of settings starting from the middle school level to university level and NIH staff and trainees. A brief description of the projects that the Core staff worked on during FY2017: Performed 300 microinjections to help NHGRI investigators in generation of mutant and transgenic lines Completed generation of knockout mutants for 16 genes and started the process for 17 genes Performed CRISPR sgRNA design and evaluation for genes for the Undiagnosed Diseases Program group Provided hands-on training to 10 users in microinjections, zebrafish handling, breeding, euthanasia, anesthesia, fin clips, genotyping, sequence analysis, sgRNA and primer design, CRISPR-STAT, WISH, imaging, cryopreservation and IVF Performed microinjections and imaging to evaluate the role of mfsd12a in pigmentation Participated in characterization of the phenotypes of knockout fish for 27 genes involved in a variety of diseases and biological processes (split hand and foot deformity, Fanconi anemia, inflammatory diseases, hematopoiesis, and RNA splicing) by monitoring embryo development, survival to adulthood and larval phenotypes using WISH, imaging, histology and genotyping Provided help in fin clips and genotyping of 3000 adult fish Provided help in screening of 300 founders by setting-up crosses and embryo care Analyzed Heat shock diploid fish for polymorphisms using sequencing at four loci to identify the homozygous fish for PacBio sequencing. Extracted high molecular weight DNA from heat shock diploid fish and goldfish Performed cryopreservation and IVF of 110 zebrafish lines Facilitated importing and exporting fish lines for collaborations Developed guidelines for characterization of mutants by basic embryonic phenotype and adult survival analysis to narrow down the mutants for deep phenotyping. New developments: This year we successfully adopted two new technologies that will be extremely useful in success of the zebrafish model for human genetic diseases. First, <10% of zebrafish null mutants display clear embryonic phenotypes. For the remaining mutants, the question always remains whether the mutation causes a true null. The zebrafish field lacks good antibodies to use for confirmation of the truncated proteins. Therefore, we adopted a recently published method to confirm whether the selected indel mutations cause loss of function of the protein. In this method, mutant and WT RT-PCR products are cloned into a reporter plasmid with GFP. Co-injections of the reporter plasmid with a control plasmid expressing RFP are followed by scoring of the injected embryos for fluorescent reporters. Robust RFP expression but no GFP expression is the desired outcome, indicating the mutant protein is non-functional. We performed the assay on dhx15 alleles and confirmed that both alleles are non-functional. Second, exact missense mutations seen in the human patients can be modeled by targeted knock-in via homology directed repair. However, the efficiency of homologous recombination in zebrafish is extremely low. After testing several published methods for donor DNA design, we obtained successful germline transmission of the desired knock-in event in one gene as a proof-of principle. Screening for two additional genes is underway and if successful, we will be able to offer knock-in as a service in the near future.
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