The NICHD zebrafish core was established on May 1st 2012. During its first four months of operation (5/1/2012 through 8/31/12, the time of writing this report), the core director (B. Feldman) has engaged in active research projects with seven NICHD labs and one laboratory from the National Childrens Hospital. The core has provided consultation to an additional three laboratories (one NICHD and two from other NIH ICs). These research projects fall into the following broad categories. Category 1: Optimizing RNA staining protocols Clients: Harold Burgesss laboratory (NICHD). Goal: to optimize procedures for handling and performing whole-mount RNA stains on very small tissue samples specifically dissected zebrafish brains. Progress: We have established a protocol that is labor saving, highly sensitive and with an excellent signal-to-noise output. Category 2: Standardizing TALEN-based genome editing for zebrafish gene knockouts. Clients: Forbes Porter, Constantine Stratakis, Thomas Sargent, Brant Weinstein, Harold Burgess, Igor Dawid and Ajay Chitniss laboratories. Goal: to (A) implement and streamline protocols for designing and synthesizing DNA constructs to be used for generating targeted zebrafish mutants and (B) implement and streamline protocols for rapidly quantifying the degree of mutagenesis in individual animals. Progress: (A) From a list of eight genes to be targeted, constructs for two have been built and one set was used to establish mutagenized fish. Designs for the other six constructs have been completed and construction has commenced for three. (B) A robust mutant detection procedure has been established for a ninth gene for which constructs were commercially purchased and mutant detection protocols have been partially established for an additional two genes. Category 3: Pilot studies for high-throughput phenotyping Clients: Mendel Tuchman (National Childrens Hospital of Washington). Goals: To establish conditions of neurotoxicity in a format amenable to a screen aimed at identifying novel neuroprotectants in a small molecule screen. Progress. We have established a regimen of exposure to ammonia that reliably produces neurotoxicity. We have also established the incubation solutions and multi-sample chambers that are optimal for high-throughput phenotyping. Category 4: Determining overexpression phenotypes of zebrafish orthologs to human candidate-disease genes. Clients. Goal. To identify zebrafish orthologs to human genes whose misimpression leads to alterations in growth or signaling in the Sonic Hedgehog signaling pathway. Progress. We have obtaining the basic reagents and established the basic design for these experiments.
|Gore, Aniket V; Athans, Brett; Iben, James R et al. (2016) Epigenetic regulation of hematopoiesis by DNA methylation. Elife 5:e11813|
|Trivellin, Giampaolo; Bjelobaba, Ivana; Daly, Adrian F et al. (2016) Characterization of GPR101 transcript structure and expression patterns. J Mol Endocrinol 57:97-111|
|Feldman, B; Tuchman, M; Caldovic, L (2014) A zebrafish model of hyperammonemia. Mol Genet Metab 113:142-7|
|Roessler, Erich; Hu, Ping; Hong, Sung-Kook et al. (2012) Unique alterations of an ultraconserved non-coding element in the 3'UTR of ZIC2 in holoprosencephaly. PLoS One 7:e39026|