of Core projects active during FY2016: During FY2016, we completed several on-going projects and started new projects. In addition, we developed high throughput protocols to streamline the generation of mutant fish with minimal time and cost. We also participated in presenting the work at scientific conferences and writing of resulting manuscripts. Below, we provide a brief description of the services offered by the zebrafish core: 1. Microinjections: Genetic material in the form of DNA, RNA or morpholinos is injected into 1-cell stage embryos to develop transient and stable models for loss or gain of function of candidate genes. We also perform microinjections of fluorescent dyes, bacteria and tumor cells in older embryos (24-72hpf) for xenotransplantation of tumor cells and evaluation of kidney and immune functions. 2. Generation of knockout mutants using genome-editing nucleases: In collaboration with the Burgess lab, we have developed an efficient high-throughput pipeline to generate knockout mutant fish using genome-editing nucleases, i.e. CRISPR-Cas9, TALENs and ZFNs. Implementation of this pipeline along with the low cost of CRISPR-Cas9 has increased our throughput for knocking out genes from <5 to >50 annually. We consult with the researchers to design the optimum target site for their gene of interest. We perform all steps of the mutant generation pipeline, i.e. target design, mRNA synthesis, microinjections, somatic analysis, founder screening and identification of heterozygous fish from the F1 generation of injected founder fish. The researcher receives two different mutant alleles for phenotype characterization. 3. Generation of transgenic lines: We provide guidance in the design and cloning of the transgenesis vectors, perform all steps of zebrafish transgenesis (i.e. microinjections, scoring by fluorescence, founder screening and generation of stable lines), and provide the researcher with two or more lines for further characterization. 4. 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 embryos. We provide training for the generation of gene-specific probes. The Core staff performs the WISH protocol and provides researchers training on imaging and genotyping for analysis. 5. Cryopreservation, in vitro fertilization (IVF) 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 not actively studied mutants to save husbandry costs. We perform IVF to recover the lines. 6. Characterization of mutant fish and training: We provide training to all new users in zebrafish handling, breeding, 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 participate in characterization of the mutant fish by performing phenotype analysis during embryonic development, imaging, survival to adulthood, and project-specific procedures. 7. Educational tours: We host tour groups (Middle and high school students, School and community college teachers, NIH staff and summer interns) 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 FY2016: Performed screening of guide RNAs (gRNA) with known germline transmission efficiency to develop CRISPR-STAT for quick identification of efficient gRNAs (Carrington et al., Nucl Acids Res 2015). Using this method, we found 70% of new gRNAs to show desired target-specific activity, thus reducing the husbandry costs on raising and screening fish for remaining 30% of inactive gRNAs. Developed high throughput protocols for CRISPR-Cas9 mediated knockout mutant generation in collaboration with Burgess lab (Varshney and Carrington et al., Nature Protocols, accepted for publication). Generated knockout mutants for 30 genes and 7 genes are in progress Performed CRISPR gRNA and primer design for difficult to design genes for UDP Performed microinjections (200) to generate mutant and transgenic lines Completed identification of founders and shipping of embryos from 200 founders transmitting mutations in 80 DNA repair genes to Myung lab at the Institute for Basic Science in Korea. Dr. Myungs lab will perform phenotypic characterization of the recovered mutant lines, thus providing insights into the entire DNA repair pathway. Provided hands-on training to 12 users in microinjections, zebrafish handling, breedings, euthanasia, anesthesia, fin clips, genotyping, sequence analysis, CRISPR-STAT, WISH and imaging Participated in characterization of the phenotypes for aifm2, dhx15, gata2b, kiaa0753, mmachc, mut, pus3 and trnt1 knockout fish by monitoring embryo development, WISH, imaging, kidney clearance assay, histology Performed survival analysis by fin clips and genotyping of 2000 adult fish generated by crossing of heterozygous mutant fish in the Fanconi Anemia pathway Provided help in fin clips and genotyping of 1500 adult fish Generated Heat shock diploid fish by in vitro fertilization with UV inactivated sperm to develop homozygous fish line without polymorphisms for PacBio sequencing. Currently, ten heat shock diploid fish are growing after multiple rounds of IVF (extremely poor fertilization and survival). We have also tested several protocols to extract high molecular weight DNA. We will perform SNP analysis to identify the true homozygous fish and extract DNA in August. Performed cryopreservation and IVF of 20 zebrafish lines Facilitated importing and exporting fish lines for collaborations
Showing the most recent 10 out of 16 publications
