The NICHD zebrafish core was established on May 1st 2012. During its first complete reporting year of operation (10/1/2012 through 9/3/13, the time of writing this report), the core director (B. Feldman) has engaged in consultation and/or active research projects with eight NICHD labs, one group from NIEHS, one lab from NIAID and one lab from the National Childrens Hospital. These research projects fall into the following broad categories. Category 1: Standardizing TALEN-based genome editing for zebrafish gene knockouts. With consultation and support from the core, alleles have been successfully generated and detected in the Porter, Dawid, Weinstein, Burgess, Stratakis and Sargent labs. Additional TALEN-based mutation projects are underway for the Stratakis and Chitnis labs. Category 2: Standardizing CRISPR/Cas9-based genome editing for zebrafish gene knockouts. The core acquired reagents and performed feasibility experiments using CRISPR/Cas9 technology. It is currently our genome-editing method of choice. We are planning CRISPR/Cas9 disruptions for three genes with the Stratakis lab and we have assisted the Weinstein and Dawid lab with reagents for several other genes. Category 3: Pilot studies for high-throughput phenotyping. With the Tuchman lab from Childrens National Medical Center, we are working to identify neuroprotectants in a small molecule screen. Following on a regimen of exposure to ammonia that reliably produces neurotoxicity that we developed in the last reporting year, we are now optimizing conditions for doing this in 96-well format so as to screen a library of small molecules. The compound library has been acquired. The preliminary data helped with the successful K01 application by Drs. Tuchman and Caldovic. Together with a team lead by Dr. Weis of NIEHS, we have also determined phenotypes for zebrafish embryos exposed to various flame retardants, data that was presented at the 2013 Society for Toxicology meeting. Category 4: Determining overexpression phenotypes of zebrafish orthologs to human candidate-disease genes. Goal. To identify zebrafish orthologs to human genes whose misimpression leads to alterations in growth or kidney tumor formation. Progress. With the Stratakis lab, we have identified transient overexpression phenotypes for two genes associated with kidney cancer and one gene associated with acromegaly. Whole-mount in situ hybridization studies have been performed to explore the affected pathways. We are planning to build transgenic constructs for these three genes to make more stable overexpression lines. An analogous project has been planned with the Kaler lab and a mutant strain of fish for this project has been obtained. Category 5: Transient antisense. Stratakis, DeCherney and Lenardo (NIAID) labs. Antisense morpholino oligonucleotides targeting two genes implicated in juvenile kidney cancer were obtained and phenotypes determined with the Stratakis lab. For one of these, an RNA-seq project was performed, leading to a set of implicated pathways that are being investigated in follow-up studies. The Lenardo lab tested one morpholino for a gene of interest, no phenotype was observed. The DeCherney lab used PNAs to investigate the function of several small tRNA-like molecules they discovered. Phenotypes were obtained, but due to the novelty of this project, a number of ongoing controls are being performed.
|Feldman, B; Tuchman, M; Caldovic, L (2014) A zebrafish model of hyperammonemia. Mol Genet Metab 113:142-7|