Advances in high-throughput DNA sequencing has led to the discovery of many new human disease associated gene mutations.
Validating the function of these variants, however, particularly in rare diseases, is an ongoing challenge. The SickKids Zebrafish Genetics and Disease Models Core aims to bridge human disease modeling to drug discovery using zebrafish as a model system.
Zebrafish are ideal for this type of translational biology because of their large clutch size, rapid early development, and transparent embryos, making them perfect for both CRISPR-Cas genome editing and high-throughput drug screening.
But why zebrafish?
We’re passionate about genome engineering and our model organism, the zebrafish.
What makes the zebrafish our choice as a model system?
Zebrafish have key properties that make them an amazing model system to study human diseases:
- many human disease genes are conserved in the zebrafish genome;
- they produce a lot of transparent (beautiful!) embryos amenable to the latest imaging and genetic techniques; and
- they’re easy to work with!
Work on your human disease modeling project at the SickKids Zebrafish Core!
We are curious researchers driven by a desire to understand the molecular basis of human diseases and strategies to cure them – so we always look forward to potentially working with you on your disease modeling research projects!
We would be happy to discuss your next project – send us an email with a brief overview of your project, and we’ll get in touch as soon as we can.
Take advantage of our expertise in genome engineering and zebrafish biology to advance your team’s human disease modeling and candidate drug discovery.
You’ll have access to state-of-the-art CRISPR-Cas9 techniques to generate zebrafish knockouts and knockins, and tol2 transposon transgenesis to generate zebrafish models that can be used for high-throughput screens to identify small molecules, which may be future candidate therapeutics for devastating human diseases.
The SickKids Zebrafish Genetics and Disease Models Core offers several services and benefits to research teams.
- generation of germline zebrafish mutants either insertion-deletion (1 guide RNA) or big deletion (2 guide RNAs flanking the region to be deleted: we have already done respectively ~5kb and 600bp deletions and ~2kb deletion of transcriptional start sites (TSS) (per Goudarzi et al. 2019 eLife);
- validation of guide RNA (gRNA) function via high resolution melt (HRM) analysis and Sanger Sequencing (any model organism);
- human candidate disease gene functional validation in F0 zebrafish by injection of four single gRNAs targeting the same gene candidate (according to the method of Wu et al. 2018 Dev Cell 46(1):112-125);
- tissue-specific knockouts; and
- point mutation knockins using plasmid donors with 1 and 1.1 kb homology arms flanking the target (Hoshijima et al 2016).
We clone (via rapid and scar-less isothermal assembly cloning) and prepare tol2 plasmid constructs for injection, integration, and generation of stable transgenic zebrafish lines expressing any DNA of interest.
Upon request, we use molecular biology techniques:
- to clone (via rapid and scar-less isothermal assembly cloning) and propagate any plasmid DNA;
- to verify gene expression by end point reverse-transcription (RT) PCR and quantitative real-time RT-PCR (any template RNA/cDNA);
- to validate organ and tissue expression of genes in zebrafish embryos via colorometric in situ hybridization (ISH).
We inject morpholinos (and the corresponding rescue constructs) for clients interested in validating their human candidate disease genes by knocking down orthologous genes in zebrafish.
Upon request, we can chemically suppress observed phenotypes in the CRISPR-Cas9 mutants we have generated.
Under the Canadian Health of Animals Act, it’s illegal to import research zebrafish from sources outside of Canada without prior quarantine, inspection, and clearance for spring viremia virus by the Canada Food Inspection Agency (CFIA). The SickKids Zebrafish Quarantine Room is a CFIA-certified holding facility that allows us to import zebrafish (fee for service), from sources outside of Canada, on behalf of Toronto or other Canadian laboratories.
Depending on your project needs, our experimental set ups, and timelines, we offer customized projects not listed above on a case-to-case basis