Zebrafish (Daniorerio)

Transcription

1 Zebrafish (Daniorerio) Connecting genotype to phenotype Photo credit: Robert Tanguay

2 Somefactsaboutzebrafish Originate from North India(Ganges), slow streams Freshwaterfish, tolerate12-39 C (optimal25 31 C) Adultsizeca. 4 cm Sexes easily distinguishable Eggs/pair: ca. 200 eggs/week Tetraploid ManyWtstrains

3 Whyis zebrafish usedas a modelorganism? Powerfulmodeltostudyvertebrate, development, evolution, genetics, and diseases Small size Highnumbers (50 fishper 10 litretank) Relatively cheap to grow Generationtime2-4 month Regenerate fins, organs, fast wound healing Manyhuman drugsworkin zebrafish

4 Whyis zebrafish usedas a modelorganism? Easytobread manyeggs External fertilisation Fast development

5 Zebrafish embryos are see-through!

6 Zebrafish Genome Sequenced genome protein-coding genes 70% of human geneshavezf orthologues 47% human geneshaveonezf orthologue Somehuman geneswhichhaveseveralzf orthologues(2.28 average) No ZF orthologues: LIF, OSM or IL6, BRCA1 (but BARD1) repeat content 52.2%, type II DNA transposable elements(39% of genome) Proteome. Howe et al. 2013, Nature, 496,

7 Geneticwork LargescaleMutagenesis: for examplewithethylnitrosourea(enu), Nüsslein-Volhard, Tübingen 1996 Morpholinos(2000) 25nt synthetic RNA block translation or splicing(5 day development) Mutations in the gene of interest: ZFN (2008), TALEN (2011), CRISPR (2013) Knockout mutation Specific mutations in active site etc. CRISPR Hwang et al. 2013, Nature Biotechnology

8 TheZebrafish core-facilityin Iceland At theuniversityof Reykjavik Karl Ægir Karlsson zebrafish as a model system for studying sleep

9 Zebrafish Core facility What do we provide? Wild type Zebrafish eggs Assistance with microinjections, genotyping and crossing fish Assisting to take videos, perform behavioural analysis Protocols for molecular and biochemical methods Daily maintenance of the fish (feeding and cleaning) Keeping the fish lines Cost/tank/month: 1.200kr

10 vhm1 Microinjections Pulling and loading needle

11 Slide 10 vhm1 Valerie;

12 Zebrafish eggs Daily Several hundred - thousand eggs Timing of egg production To hold eggs in place:

13 Microinjections Injection: Into the cell CRISPR Into the yolk -MO

14 Growing up First 2-3 weeks: 28 C Incubator Then flowing system

15 Zebrafish husbandry-food Aim to grow zebrafish with: a. Short generation time b. Maximal egg production Larvae Live: paramecium ( µm), rotifers ( µm) Dry: algae powder smaller than 100 µm Adults Live: Artemia(brine shrimp) (800µM +) Dry: Algae, yeast etc.

16 Time. Day 1-3: Microinjection Day 2-5: Genotyping Day ca. 90: Crossing with WT Day : F1 Growing up Day 170: Genotyping F1 Day 180: Crossing F1 Day : F2 Growing up Experiments: heterozygous and homozygous 6 month after injection, onwards Yu et al. 2014, PlosONE

17 DNA, PCR, restriction digest Genotyping Surveyor or T7 endonuclease assay High resolution melt analysis (HRMA) Sequencing Talbot and Amacher, 2014, Zebrafish Fluorescent PCR for CRISPR-mediated mutagenesis in zebrafish: CRISP-STAT (somatic tissue activity test)

18 Carrington et al., 2015, Nucleic Acid Research CRISP-STAT evaluating sgrna Uninjected Injected + F0 50% Somatic mosaic mutation 30% 0% High somatic score, more chance of germline mutation! Screen fish before raising the animals, saves time!

19 The Charr group -Askja Some on-going projects Arctic charr morphs in Lake Thingvallavatn What is the molecular background for the observed differences in craniofacial morphology in charr?

20 - Genes in extracellular matrix organisation - Glucocorticoid signalling network genes - AhR2 pathway Possible roles of -mir206 -mir199a

21 Charr Functional analysis in Zebrafish mrna AhR2 hu 3335 mutant zebrafish Send from Oregon (R.L.Tanguay) via Berlin (P.Alves) -Stain for cartilage and bone - Gene expression of candidates found in charr (qpcr) MicroRNA use mirna inhibitors and mimics to down-and up-regulate mirna candidates (mir-199a and mir-206) in zebrafish - Check target gene expression - Phenotype (bone and cartilage formation) control 5 days dexamethasone

22 CRISP-CAS9: STE-20 serine/threonine kinase family member Magnús Karl Magnússon/ DeCode Ser/thr kinase Icelandic Mutation (IM) in protein (1 base: 1aa) in active site of kinase Associated with with a specific type of cancer In progress: generate zebrafish with IM or kinase dead (CRISPR) so far 28% success for KD (one homozugous mutant, 2.7%) Plan to: Screen the fish Are they different in developing cancer? Effects on apoptosis, mtor signalling etc

23 The zebrafish model organism database - ZFIN Zebrafish International Resource Center(ZIRC) Oregon, USA European Zebrafish Resource Center(EZRC) Karlsruhe, Germany

24 Publications using zebrafish -Pubmed 3000 Each year Year

25 Come to visit! Plan experiment with zebrafish!