Detailed information on projects performed is the Core during FY2011 is provided below. 1. Characterization of gene function using WISH and morpholinos: The Core has completed on-going projects from FY2010 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. Cancer Genetics Branch (CGB) and Undiagnosed Disease Program (UDP) have been our major new users during this year. 2. Generation of genetic mutants using TILLING: New requests to identify missense and truncation mutations were received for 4 genes. Twelve new amplicons were designed and added to the on-going TILLING projects. BGI has performed 16,000 sequencing reactions this year. Analysis of these data led to the identification of a truncation mutation in socs3a. IVF was performed and mutation carriers were identified by fin clips and transferred to Dr. Burgess for phenotypic evaluation. Sequencing and data analysis for stat3, ak2 and gne are in progress. 3. Generation of knockout mutants using Zinc-finger nucleases: 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. Recently, the use of zinc-finger nucleases (ZFNs) has been demonstrated to specifically target a gene to generate null alleles. ZFNs 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. We developed optimized protocols for various steps involved in ZFN-mediated mutagenesis. A progress report on the status of mutant generation for these 4 genes follows: i) cbfb: Seven germ-line transmitting founders were identified by screening 50 ZFN-injected embryos grown to adulthood. Eight different in/del mutations causing frame shift with premature truncation of the protein have been identified in these founders. Heterozygous carriers for 2 independent mutations have been provided to Dr. Liu for phenotypic analysis. Testes from heterozygous males for other mutations have been cryopreserved.. ii) mmachc: Two sets of ZFNs were evaluated by injecting mRNAs at several doses, somatic analysis of the injected embryos and screening of the progeny from injected embryos grown to adulthood. These experiments were repeated in a different genetic strain to rule out the polymorphisms as the cause for lack of specificity. It is unclear at the moment why these ZFNs did not generate any mutations at the target locus. Their efficiency might be really low, below the detection limits of our screening protocol. Sigma is designing new ZFNs for mmachc and will provide us with 3 top candidates out of their in vitro screen for injections into zebrafish embryos. We will perform the injections and evaluation when these ZFNs become available. iii) igf2bp2a: Two sets of ZFNs were obtained from Sigma in November 2010. Injections of the first set at 3 doses were performed in December 2010. Somatic analysis of the injected embryos showed very high frequency of targeting. Founder screening identified multiple in/del mutations being transmitted by each fish screened. We are currently growing F1 generation of these founders. Heterozygous carriers of two deleterious mutations from 2 independent founders will be identified and evaluated for phenotype. iv) eomesb: Two sets of ZFNs were obtained from Sigma in November 2010. Simialr to igf2bp2a, the first set of ZFNs showed high frequency of targeting. Injections were repeated at lower doses to avoid off-target effects and founder screening is currently underway. 4. Generation of stable transgenic lines: The purpose of this project is to develop stable zebrafish lines expressing oncogenic fusion protein CBFB-MYH11 and an internal deletion form that removes an important domain to generate leukemia models for identification of novel therapeutic agents via chemical screening. We are using an inducible system with heat-shock promoter driving the oncogene. The vector also contains 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), followed by exposure to higher temperatue to induce the transgene (RFP as marker). 5. Evaluation of conserved non-coding sequences for enhancer activity using tol2 mediated transgenesis and zebrafish enhancer detector (ZED) vector: Several labs are interested in testing computationally identified cis-regulatory elements (enhancers) using an in vivo system. These elements are cloned into a zebrafish enhancer detection vector that has RFP as internal control and GFP expression driven by the cloned fragment (Bessa et al., Developmental Dynamics 238: 2409-2417, 2009). The trangenesis is mediated by tol2 transposon. Plasmid DNA mixed with tol2 mRNA is injected into one-cell stage embryos and evaluated by GFP and RFP expression at several stages of development. We signed MTA and obtained the ZED plasmid and a positive control fragment cloned into the ZED plasmid from Dr. Bessa (Spain). We have established the technique using the positive control construct and injected several of the experimental constructs in both orientations multiple times to obtain consistency between injections and statistical data on the number of embryos showing a particular GFP pattern. 6. Evaluation of new proposals for zinc-finger mediated mutagenesis: Currently, there are two sources of ZFNs: a commercial source costing $22,500/target gene and an academic resource, termed CoDA costing $1000/gene (Sander et al., Nature methods 8: 67-70, 2011). In the first approach, Sigma performs zinc-finger selection using proprietary algorithm and assembles candidate ZFN pairs using ZFP modules from the proprietary ZFN archive, clone them into the appropriate vectors, and performs validation by mismatch sensitive nuclease assay and provides us with the ZFN pair with the highest DNA-cleavage efficiency. In the second approach, zinc finger modules are identified by bioinformatic analysis of the target sequence, synthesized by any DNA synthesis company and cloned into the appropriate vectors. This year, we had funds for one ZFN from Sigma. We received 3 proposals from Faculty, got them evaluated by Sigma and chose ak2 (Candotti lab) for ZFN targeting using Sigma approach. We decided to explore CoDA and another new approach of gene targeting, termed TALEN (Miller et al., Nature Biotech 29: 143-148, 2011) for the other two targets. Table 2 in the paper shows the results of the bioinformatic analysis of the 3 genes for each approach. We are currently analyzing target regions for polymorphisms to ensure specificity of ZFNs or TALENs. 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 ITO 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.
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