of Core projects active during FY2013: 1. Characterization of gene function using WISH and morpholinos: This year, we analyzed the function of the following candidate genes identified by genomic approaches using WISH and morpholinos: cecr1b, gne, itga11, sccpdha, vps45 and zak. In all cases, gene-specific morpholinos were designed to target translation start site and/or splicing junctions. Microinjections were performed to determine the optimal dose of each morpholino, followed by microinjections for phenotype analysis using imaging, WISH and transgenic lines. In some cases, activity of the wildtype and mutant human mRNAs was assesses using complementation of the morpholino phenotype. 2. Generation of knockout mutants using ZFNs and TALENs: In collaboration with the NHGRI Genomics Core, we developed high throughput method of fluorescent PCR for founder screening and genotyping to complete these projects in a cost-effective and timely fashion. We have generated multiple mutant alleles for ten genes and additional six genes are in progress. A brief summary of the various projects using targeted mutagenesis is given below: 2.1 Role of AK2 in reticular dysgenesis: To model reticular dysgenesis in zebrafish and investigate the mechanisms underlying its pathophysiology, we generated multiple ak2 mutants: a missense mutation (L124P) by TILLING and null mutants by ZFNs. Dr. Rissone has performed a comprehensive study of the effects of ak2 deficiency on hematopoiesis using morpholinos and both mutants, demonstrating a critical role of ak2 in erythroid development during primitive hematopoiesis and hematopoietic stem cell (HSC) development during definitive hematopoiesis. 2.2 Role of c-Met signaling in the posterior lateral line development: To investigate the role of met in the migrating lateral line primordium, we generated loss-of-function alleles using ZFNs that are being characterized by the Burgess lab. 2.3 Genes involved in regulation of hematopoiesis: To assist Liu lab in their investigation of the regulatory network involved in hematopoiesis, we have generated loss-of-function alleles using ZFNs and TALENs for sox17, runx1 and cbfb. Liu lab is currently analyzing expression of specific hematopoietic markers by WISH and transgenic lines in these mutants. 2.4 Modeling metabolic diseases in zebrafish: To model disorders of vitamin B12 metabolism, Cobalamin C (cblC) disease and methylmalonic academia, for insights into their underlying pathology and assessment of the efficacy of potential therapeutics, we generated loss-of-function alleles in mmachc and mut using ZFNs. Their characterization is currently underway by Venditti lab. 2.5 Candidate genes identified by genomic approaches: We have generated knockout mutants for the following genes identified as putative candidate genes in various studies undergoing at NHGRI: kctd7 (progressive myoclonic epilepsy), hint3 (distal myopathy with weakness and atrophy of distal muscles and high arched palate), ubqln4 (neurological phenotype), aifm2 (new immune disorder), pus3 (Kabuki-like syndrome), and bmp3 (craniofacial anomalies). Mutant generation for additional candidate genes is in progress. 3. Generation of stable transgenic lines: We have generated stable transgenic lines using tol2 mediated transgenesis to analyze effects of over-expression and specific mutations in hint3 and bmp3 genes. In addition, we are generating transgenic lines for use in cell tracking experiments by marking nuclei and membranes by photo-convertible fluorophores. 4. Evaluation of conserved non-coding sequences for enhancer activity using tol2 mediated transgenesis and ZED vector: Evaluation of highly conserved 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). We are currently evaluating wildtype and variant forms of two conserved elements associated with idiopathic scoliosis and sagittal craniosynostosis. Transient expression is variable due to mosaicism, therefore, we generated stable lines for the conserved element localized in the IRX cluster region and identified founders with germline transmission. 5. Chemical screening: The Core is facilitating characterization of the role of elg1 and msh6 in DNA repair by Myung laboratory using previously generated knockout fish lines and testing their sensitivity to various DNA damaging agents. In addition, we provide aliquots of the spectrum collection from Microsource Discovery Systems for chemical screening projects. 6. 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. We have held many tours organized by the ITO, office of education, and NIH visitor center.
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