NHGRI/DIR Zebrafish Core

National Human Genome Research Institute

Investigators

Linked publications, trials & patents

Abstract

In FY2023, the Core staff worked on 11 new or previously on-going projects for NHGRI investigators. In addition, we performed colony management, IVF, processing of samples for genotyping and sequencing, and training of new users. A brief description of these projects is given below. Projects completed: 1.Evaluation of 15 sgRNAs to tyr gene with different modifications to evaluate their effect on targeting efficiency: Each sgRNA was injected into 100 embryos twice followed by microscopic observations for pigmentation phenotype and CRISPR-STAT analysis for activity analysis. Three guide RNAs were determined to meet the desired criteria for targeting efficiency. 2.Management of 6 mutant lines in genes involved in melanoma metastasis: We performed fin clips, genotyping and breeding of these mutant lines for colony management, cryopreservation and shipping to collaborators. New projects started: 1.Generation of a knock-in zebrafish line with patient-specific point mutation in h3f3a (K27M) for evaluation of its role in a mosaic skeletal disorder: We have completed the design, microinjections and somatic knock-in confirmation. Founder screening will be performed in FY2024. 2.Generation of knock-in zebrafish lines with patient-specific splice site mutations in runx1 in FPDMM patients: We have completed the design, microinjections and somatic knock-in confirmation. Founder screening will be performed in FY2024. 3.Generation of cylbl KO zebrafish lines to evaluate its role in an adult neurological phenotype: We have designed sgRNAs in the desired functional domain and are in the process of microinjections and CRISPR-STAT analysis. Once an active sgRNA is identified, injected embryos will be grown for founder screening to establish mutant lines. 4.Evaluation of DNA damage in whole kidney marrow (WKM) cells of runx1/fancd2 KO fish using immunohistochemistry with GammaH2AX antibody: We Used WKM cells of gata2a KO fish as a positive control and wildtype (WT) fish as a negative control to streamline the protocols for dissection and dissociation of kidneys into viable cells on slides and appropriate antibody dilutions for specific staining of nuclear foci formed by the DNA damage. We will perform these experiments using kidneys from runx1/fancd2 KO fish at different time points (4-12 months) in FY2024. Progress on other on-going projects: 1.Testing RUNX1 variants identified in FPDMM families for pathogenicity and mechanism of action: The goal of this project is to establish a rescue assay by injections of WT RUNX1 mRNA in runx1 KO embryos for evaluation of patient-specific mutations. runx1 KO embryos display lack of hematopoietic stem cells (HSCs) in their caudal hematopoietic tissue. Therefore, we are using WISH with c-myb probe for presence or absence of HSCs as a proxy for rescue. However, constitutive expression of WT RUNX1 mRNA led to generalized toxicity. Therefore, we are now evaluating the use of heat shock promoter to induce RUNX1 expression after 24 hpf. Injected embryos are exposed to higher temperature after 24 hpf, screened for GFP and incubated until 3.5dpf for WISH, imaging, and genotyping. Once we determine the exact timing and duration of induced expression required for a detectable rescue, we will start injecting the mutant versions of mRNA. 2.Generation of fish lines with epitope tags inserted at 3 ends of sox2, sox10, sox4b, six1a, six1b, six4b, sox21a and atoh1a for proteomics analysis using tag-specific antibodies: Previously, we identified active sgRNAs for 4 genes, designed donor DNA oligos and performed CRISPR-STAT analysis. This year, we performed microinjections and founder screening for these 4 genes. Next, we identified Cpf1 target sites in 2 genes, designed donor oligos and performed knock-in experiments. So far, we have successfully generated 2 fish lines with precise knock-in: sox2-HA and sox2-FLAG. Founder screening for sox10, sox4b and six1a is on-going. Although somatic knock-in was confirmed in these genes, efficiency of germline transmission is low, most likely due to the embryonic lethality of injected embryos. 3.Characterization of fanca knockout fish for hematopoietic defects: We performed fin clips, genotyping and out-crossing of fanca KO fish with cd41-GFP and gata1-DSRed transgenic lines. Embryos were screened for the presence of transgene before growing to adulthood. We will continue to help in this project as needed. 4.Phenotypic characterization of gba1 knock-in and knockout lines: We established a new genotyping technique for embryos, so that they can be kept alive for phenotype analysis after determining their genotype. This technique reduces the number of embryos required for the phenotype analysis. Other Core activities: 1.Maintenance of >30 WT, mutant and transgenic lines by breeding and genotyping for colony management, cryopreservation and submission to ZIRC. 2.Processing of 50,000 samples for genotyping and sequencing on our ABI sequencing machine. 3.In vitro fertilization to recover frozen mutant lines. 4. Training of new users. 5.Technology development for high-resolution analysis of gene and protein expression using RNAScope and immunohistochemistry: RNAScope did not work on whole mount embryos. 6.Technology development for knock-in when somatic knockout causes embryonic lethality: As a proof of principle, we are performing knock-in using heterozygous for a deleterious mutation to correct the mutation while making the desired point mutation in dhx15. 7.Generation of knockout and knock-in mutant lines in 5 genes for collaborators from NCI and NHLBI.