NHGRI/DIR Zebrafish Core

National Human Genome Research Institute

Investigators

Abstract

In FY2008, all 5 NHGRI wet laboratory branches and the office of clinical director used[unreadable] the services provided by the Core as described below.[unreadable] 1. Resequencing/TILLING:[unreadable] a) ENU mutagenesis and F1 collection: To reach our goal of archiving DNA[unreadable] and sperm from 5000 F1 males to ensure detection of functional mutations in[unreadable] any gene screened, we have performed another round of ENU mutagenesis.[unreadable] Currently, we are breeding 24 ENU-treated males to collect their progeny.[unreadable] b) Sequencing and recovery of mutations: The Core is currently screening 22[unreadable] genes for investigators from GMBB, GTB, GDRB and MGB. PCR and[unreadable] sequencing are performed at BGI. The remaining steps that include sequence[unreadable] data analysis, validation of potential mutations, in vitro fertilization (IVF)[unreadable] from the corresponding frozen sperm, and genotyping to identify mutation[unreadable] carriers are performed by the Core. A summary of on-going resequencing[unreadable] projects is provided in Table 1. Overall, we have received data for >60,000[unreadable] reactions and 20,000 reactions are in sequencing queue at BGI. To highlight[unreadable] a few of the outcomes, Dr. Liu has submitted a manuscript describing the[unreadable] three stages of hematopoiesis in zebrafish as revealed for the first time by the[unreadable] characterization of novel runx1 and gata1 mutants identified by the Core in[unreadable] previous years. Drs. Sood and Myung are analyzing a truncating mutation in[unreadable] shprh, a gene involved in DNA repair (presented at 2008 Zebrafish meeting in[unreadable] Madison). Drs. Liu and Hickstein are analyzing a truncation mutation in[unreadable] brca2 to further understand the role of BRCA2 in breast and ovarian cancers.[unreadable] 2. Microinjections: Despite personnel changes and training of new personnel in[unreadable] microinjections (a highly demanding technique), the Core has performed several[unreadable] microinjections during FY2008 (Table 2). Each project requires several rounds of[unreadable] microinjections and coordination with the researchers schedule, thus leading to a long[unreadable] period to complete.[unreadable] a) Morpholinos: The Core has performed several morpholino knockdown[unreadable] experiments both for previously on-going and new projects requested[unreadable] during FY2008. For each gene, two morpholinos are designed to block[unreadable] either translation or splicing. For each morpholino experiment, multiple[unreadable] rounds of microinjections are performed, first to determine the effective[unreadable] dose of the morpholino, followed by injections of the determined dose[unreadable] for phenotypic analysis. To prove specificity of the phenotype[unreadable] observed, morpholinos are then co-injected with mRNA for rescue.[unreadable] Thus each morpholino project is a long-term project and injections are[unreadable] scheduled depending on the availability of the requesting researcher to[unreadable] perform phenotypic analysis. At present, two projects are near[unreadable] completion and may lead to publications (cblC gene for Dr. Venditti[unreadable] and AK2 gene for Dr. Candotti).[unreadable] b) mRNA for Complementation studies: Dr. Yang has started a project[unreadable] to evaluate Lpp1 mutant mRNAs by their ability to rescue Van Gogh[unreadable] phenotype. This will help them determine important residues and[unreadable] 3 phosphorylation sites in Lpp1. For this project, we obtained embryos for[unreadable] the mutant line from ZIRC, genotyped adult carriers and optimized the[unreadable] microinjections using control Lpp1 mRNA. We will now be injecting[unreadable] mutant mRNAs as provided by the Yang lab.[unreadable] c) Plasmid DNA for generation of transgenics and evaluation of[unreadable] regulatory sequences: The Core has adopted the recently published[unreadable] method of generating transgenic zebrafish and generated a gata1-GFP[unreadable] transgenic line for proof of principle experiments. We are currently[unreadable] generating two transgenic lines for Dr. Liu to study cbfb-myh11 fusion[unreadable] protein involved in leukemia.[unreadable] d) Xenotransplantation: We started optimization of the transplantation[unreadable] technique in which 48 hour old embryos are injected with human cells[unreadable] to test their potential for causing melanoma for Dr. Samuels.[unreadable] 3. Whole mount in situ hybridizations: During FY2008, we determined the normal[unreadable] expression pattern of AK2 and also evaluated the morphants at different stages of[unreadable] development for hematopoietic markers by in situ hybridizations for Dr. Candotti.[unreadable] 4. Cryopreservation: In addition to the sperm from >3200 F1 males for resequencing[unreadable] project, the Core has archived several mutant lines for Drs. Liu and Feldman for backup[unreadable] and to eliminate the need to maintain live fish when they are not actively studied.[unreadable] 5. Educational tours: The Core has hosted several tour groups aimed at community[unreadable] outreach and promoting science among youth and minority groups.