of Core activities during FY2012 The Core has completed many on-going projects from FY2011 and initiated several new projects involving characterization of gene function using manipulations of gene expression and generation of genetic mutants. The purpose of these projects is to evaluate function of unannotated genes that are discovered using genomic technologies to harbor potential causal variants for disease susceptibility and to understand disease pathology. 1. Characterization of gene function using WISH and morpholinos: We are working on several projects involving characterization of candidate genes identified by exome sequencing at UDP, Gahl lab (MGB) and Ostrander lab (CGB) using WISH and morpholinos. In particular, Gahl lab is characterizing candidate genes for Hurmansky-Pudlak syndrome. We have established an assay using Tg(cd41:EGFP) fish and morpholinos against HPS1 gene to analyze the effect on thrombocytes (equivalent to platelets in mammals). We are using this assay to test candidate genes for HPS8 and HPS10. We are using Tg(lysC:GFP) fish to analyze effect on neutrophils in vps45 morphants. For UDP group, we are currently working on analysis of sccpdha, atp6vih, and ubqln4 genes using morpholinos. For Ostrander lab, we are helping in characterization of candidate genes identified by GWAS to be involved in canine skull shape variation. 2. Generation of genetic mutants using TILLING: Due to the successful implementation of ZFN-mediated mutagenesis, we have limited the use of TILLING to genes where missense mutations are desirable as the nonsense mutations are expected to be embryonic lethal. This year, 8,000 sequencing reactions were performed by BGI. Analysis of these and previous data led to the identification of three new mutations: a splice site and a missense mutation in gne (Dr. Huizing/MGB) and a missense mutation in ak2 (Dr. Candotti/GMBB). We have recovered the mutant fish by IVF and analysis of their phenotypes is in progress. 3. Generation of knockout mutants using ZFNs and TALENs: To date, we have performed targeted mutagenesis of 10 genes, 5 using Sigma-ZFNs, 4 using CoDA-ZFNs (new cheaper method for assembling ZFNs available through ZFN consortium) and 1 through TALENs. 4. Generation of stable transgenic lines: We are generating stable transgenic lines to analyze effects of over-expression and specific mutations in bmp3 and hint3 genes for Ostrander lab and UDP group, respectively. Wildtype and mutant forms of both human and zebrafish hint3 genes were cloned into Tol2 vector containing an internal transgenesis control. Injections of the plasmid DNA mixed with transposase RNA have been performed. We are currently screening founders by expression of the transgenesis marker (green heart), and have identified founders expressing human wildtype and mutant HINT3. We are using an inducible system with heat-shock promoter to drive bmp3 after gastrulation at specific time points during development. Injections have been performed and founder screening is underway. 5. Evaluation of conserved non-coding sequences for enhancer activity using tol2 mediated transgenesis and zebrafish enhancer detector (ZED) vector: Evaluation of non-coding sequence variants, often detected as putative mutations and associations in human genetic diseases is challenging. Furthermore, computationally identified conserved elements are presumed to function as enhancers. Zebrafish provides an ideal in vivo system to test their role as enhancers by tol2 transgenesis and a minimum promoter plus the fragment to be tested driving GFP. ZED vector with RFP expression in muscle at 48hpf as an internal control of transgenesis was developed recently (Bessa et al., Developmental Dynamics 238: 2409-2417, 2009). Plasmid DNA is mixed with tol2 mRNA, injected into one-cell stage embryos and evaluated by GFP and RFP expression at several stages of development. We have obtained the ZED plasmid and a positive control fragment cloned into the ZED plasmid from Dr. Bessa (Spain). We are currently evaluating wildtype and two variant forms of a 293 bp conserved element associated with idiopathic scoliosis for Dr. Wilson/IDRB. 6. Chemical screening: The Core is facilitating characterization of the role of elg1 in DNA repair by Myung laboratory. Previously, we had generated elg1 knockout fish using TILLING. These fish have no obvious embryonic phenotype but do not survive to adulthood. Myung lab is currently testing their sensitivity to various DNA damaging agents. They also have plans to test mutants for other DNA repair genes for sensitivity to DNA-damaging agents and we are in the process of obtaining these mutants from different sources. In addition, we have purchased the spectrum collection from Microsource Discovery Systems, which contains 2900 unique small molecules for chemical screening projects by Burgess, Liu, Myung labs and UDP group. This chemical library will be maintained in the Core and copies at appropriate dilutions provided to users for screening projects. 7. Evaluation of new proposals for generating genetic mutants: This year we received 4 proposals from Faculty for generation of knockout mutants. We have also re-evaluated bmp3 as TALENs have been unsuccessful so far. We have ordered Sigma-ZFNs for 3 genes and TALENs from Dr. Ekkers lab at Mayo Clinic for the other 2 genes (set-up as a collaboration by Dr. Burgess). Retroviral mutagenesis: Another approach for generating genetic mutants in zebrafish is retroviral integrations, and is currently undergoing in Burgess laboratory. They have generated mutations in >3,000 genes and plan to generate mutations in 10,000 genes in next 3-5 years. The Core screens their collection for mutations in genes requested for mutant generation. This resource will also help in providing a second, independent allele to complement the studies from TILLING and ZFN-targeted mutants. Education and outreach: Zebrafish embryos at different stages of development and transgenic lines with GFP showing circulating blood, blood vessels, or other specific organs make an attractive visual teaching tool. The Core is thus a popular stop for tours organized by the ITO, office of education, and NIH visitor center. We also host summer interns and give impromptu tours to several GMBB visitors, thus participating in the education and community outreach programs. This year, we helped students from Governors school at George Mason University in setting-up zebrafish experiments in their own lab through webinar and hands-on-demonstrations during their visit to our lab. We also participated in NHGRI Take Your Child to Work day by demonstrating the role of animals in research to 6 groups of students. We provided a tour to NHGRI summer interns through ITO.
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