NHGRI/DIR Zebrafish Core

National Human Genome Research Institute

Investigators

Linked publications & trials

Abstract

In FY2022, the Core was used by 12 investigators from nine NHGRI branches for a variety of projects as described below. 1. Microinjections of mRNA and protein for various isoforms of UBA1 followed by microscopic observations, imaging and genotyping of 1,000 embryos to establish a rescue assay with the goal of understanding the contribution of different UBA1 isoforms to pathophysiology of VEXAS syndrome. 2. Microinjections of fanca and fanco mRNAs alone and in combinations with knock-in reagents followed by CRISPR-STAT to evaluate their roles in homologous recombination. 3. Microinjections of wildtype and mutant versions of runx1 mRNAs to establish a variant evaluation assay by phenotype rescue of runx1 mutant embryos. 4. Treatment of wildtype and uba1 mutant embryos with a drug of interest followed by microscopic observations, imaging and genotyping of 1,000 embryos to evaluate a possible treatment option for patients with VEXAS syndrome. 5. Generation of 8 knockout mutant zebrafish lines for aldh6a1, mcee and lmbrd. 6. Phenotypic characterization of knockout fish for zrsr2 and gba1 and knock-in fish for gba1 and gata2b by microscopic observations, imaging, WISH, and histology. 7. Design, microinjections and CRISPR-STAT for multiple sgRNAs for knock-in of epitope tags in seven genes, design of donor oligos based on the most active sgRNA for 4 genes. Microinjections with donor oligos and CRISPR-STAT analysis is currently in progress. 8. Evaluation of 15 sgRNAs to tyr gene with different modifications to evaluate their effect on targeting efficiency. Each sgRNA is injected into 100 embryos followed by microscopic observations for pigmentation phenotype and CRISPR-STAT analysis to test its activity. 9. Processing of 37,000 samples for genotyping and 10,000 samples for sequencing 10. Cryopreservation of 19 new mutant lines. 11. In vitro fertilization to recover 3 lines. 12. Fin clips, genotyping and breeding for colony management of 6 mutant lines. 13. Training of 3 new users in microinjections, imaging, bone and cartilage staining and genotyping. 14. Maintenance of >25 lines (WT, mutant and transgenics) by breeding and genotyping. 15. Technology development for high-resolution analysis of gene and protein expression using RNAScope and immunohistochemistry