of Core activities during FY2010 ENU mutagenesis and F1 collection: We have performed testes cryopreservation and DNA extraction from 300 males generated by ENU mutagenesis to bring our collection to a total of 3500 males. TILLING projects: Listed below are the genes sequenced and analyzed for identification of mutations in our TILLING collection: runx3, lmo2, pu.1, sox17, eomesa, mxtx, mxtx2, irx3a, irx3b, hnf4a, hnf4b, atoh1a, atoh1b, ak2, elg1, mmachc. We have identified a truncation mutation in elg1 and several missense mutations in other genes. Most of these mutations have been validated and recovered by IVF and phenotypic evaluations are currently underway. Zinc-finger mediated mutagenesis: ENU mutagenesis approach used in TILLING is random, knockout mutations are rare, and the chances of finding such mutations are further limited by the size of the coding exons and the number of F1 males screened. Complimentary approaches are required to identify knockout mutations in all genes of interest. Recently, the use of zinc-finger nucleases (ZFNs) has been demonstrated to specifically target a gene to generate null alleles. Zinc-finger nucleases provide sequence specificity, thus allowing targeting to a desired gene and introduce double strand breaks in DNA that result in in/dels when repaired by the non-homologous end joining repair pathway. As a pilot project to develop the downstream part of the technology in the Core, we used commercial source to order ZFNs for cbfb and mmachc, 2 genes that have been screened by TILLING, but no functional mutations were identified. We have injected mRNA encoding cbfb ZFNs and identified 5 founders with germline transmission of mutations. We have selected two additional genes to order ZFNs to continue the development of this technology. We have also acquired the ZFP pools from Massachusetts General Hospital to develop the ZFN generation in the Core as allowed by the personnel time and resources. Education and outreach: Zebrafish embryos 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 Intramural Training Office and office of education. We also host summer interns and give impromptu tours to several GMBB visitors, thus participating in the education and community outreach programs. Microinjection Projects: In FY2010, the Core worked on the following microinjection projects. 1. Dissecting the role of trilobite/Lpp1 in planar cell polarity: Purpose: Lpp1 acts in the planar cell polarity pathway to regulate convergent extension movement during gastrulation. The purpose of this project is to understand the structure function relationship of mouse Lpp1using the Van Gogh mutant fish (vangl2). Status: We have injected wildtype Lpp1 mRNA in vangl2 mutant embryos and demonstrated that it can rescue the mutant phenotype. Next we injected the mutant Lpp1 mRNAs with one or more of the phosphorylation sites mutated and evaluated their ability to rescue thus indicating the critical role of the phosphorylation sites. 2. To understand the function of elg1: Purpose: ELG1 is involved in DNA repair and mutated in tumor samples (unpublished data from Dr. Daphne Bell). The knockout mouse is embryonic lethal, therefore, a zebrafish model is being developed using both morpholinos and TILLING. Status: Translation and splice-blocking morpholinos were designed and injected into wildtype, p53-/- and Tg(HuC:eGFP) embryos. Results showed defective early embryonic development and apoptosis rescued by p53. 3. To develop zebrafish model of cobalamine C deficiency: Purpose: Cobalamin C deficiency (cblC) is caused by mutations in the MMACHC gene whose cellular function is unknown. The patients display a wide spectrum of phenotypic manifestations. The goal oof this project is to develop an animal model to understand the pathophysiology of the disease. Status: Two morpholinos have been used to generate the disease model and metabolic studies have been performed. 4. To understand the function of igf2bp2a and igf2bp2b: Purpose: SNPs in IGF2BP2 locus confer increased susceptibility to type 2 diabetes. The function of IGF2BP2 is not known and no animal model has been generated. Status: Identification of the zebrafish orthologs: igf2bp2a and igf2bp2b by bioinformatic approaches, whole-mount in situ hybridization showing that igf2bp2a is expressed ubiquitously at first and becomes restricted to the head and brain region by 24hpf and to the developing pancreas and neuromasts by 5dpf, obtained two transgenic lines with GFP expression driven by pancreasspecific promoters, insulin and elastase, performed knock-down of igf2bp2a using splice blocking morpholino and observed a phenotype in a dose-dependent manner, confirmed by RT-PCR 5. Planar cell polarity in left-right asymmetry: Purpose: Using two mutants of planar cell polarity and left-right asymmetry: vangl2 and seahorse, Dr. Yangs goal is to understand their relationship to each other and the other pathways involved. Status: seahorse mutant embryos obtained from ZIRC, grown to adulthood and genotyped to identify carriers. Heterozygous vangl2 fish were crossed to hererozygous seahorse fish to generate compound heterozygotes. 6. Functional studies of RBM10: Purpose: Resequencing of exons from X chromosome led to the identification of mutations in RBM10 in two families with a very rare genetic disorder. The goal of the project is to validate the role of RBM10 in diseases phenotype by knock-down in zebrafish. Status: Zbrafish homolog of RBM10 is not annotated and bioinformatics search did not identify the 5end. Therefore two splice-blocking morpholinos were designed and tested. One morpholino gave a specific phenotype while second morpholino did not. A third morpholino was designed to validate the phenotype. However, no phenotype was observed while RT-PCR data confirmed knock down of the gene in both cases. From these data we concluded that compensatory mechanisms exist for rbm10 knock-down in zebrafish. 7. Transgenic enhancer screen: Purpose: To test computationally identified cis-regulatory elements (enhancers) using an in vivo system. 10 cis-regulatory elements will be cloned into a zebrafish enhancer detection vector which has RFP as internal control and GFP expression driven by the cloned fragment (Bessa et al., 2009). The trangenesis is mediated by tol2. Plasmid DNA mixed with tol2 mRNA will be injected into 300 embryos each (due to high toxicity associated with this vector) followed by evaluation of GFP and RFP expression at 24 and 48hpf in the surviving embryos. Second round of injections may require sub-sections of the potential elements to be tested to further define the sequence conferring the regulatory function. Status: Established the technique using a positive construct and tol2 transposase mRNA, 8 constucts have been injected multiple times. Two of them gave very specific pattern of GFP expression indicating their potential regulatory function in those tissues. 8. Understanding the function of RMRP, a RNA gene causing CHH: Purpose: Mutations in the enzymatic site of RMRP or in the promoter leading to the decreased level of expression are found in Cartilage Hair Hypoplasia (CHH). There is no animal model of CHH and, therefore, knock-down by morpholinos targeting the highly conserved enzymatic site of rmrp was thought to be an ideal model. Status: Two morpholinos were designed to target the two highly conserved regions in the enzymatic site. Microinjections of both morpholinos at several doses have been performed. Injected embryos showed cranial abnormalities and alcian blue staining showed dysmorphology of the pharyngeal arches.
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