WA Zebrafish Facility

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1 WA Zebrafish Facility ARC LIEF grant Applicants University of Western Australia David Hunt Shaun Collin Wayne Davies Ryan Lister Nathan Pavlos Edith Cowan University Ralph Martins Simon Laws Giuseppe Verdile

2 The zebrafish is an ideal model for the study of development and disease Adults are relatively small, so housing is relatively cheap Eggs and early embryos are transparent, thereby enabling the visualisation of early development Development is rapid - organs are made in 1-7 days Egg clutch size is large ENU mutagenesis screens have generated thousands of useful mutants, including an increasing number that accurately model human genetic diseases, and this is ongoing High-resolution imaging of RNA and protein expression in whole embryos is easy Drugs and chemicals can be easily tested for activities in zebrafish by adding them to the water Zebrafish are vertebrates and thus have a gene complement very similar to humans The use of zebrafish in research is estimated to be growing at three times the rate of the mouse

3 The origin of the zebrafish as a model organism The zebrafish, Danio rerio, a member of the family Cyprinidae in the Order Cypriniformes, is a robust tropical fish that is a popular addition to many home aquaria. Studies that demonstrated the utility of the zebrafish as a model organism were initiated in the 1970s by George Streisinger at the University of Oregon. Attributes considered important included small size, ease and low cost of maintenance, a large egg clutch size and transparency during embryonic development. The first zebrafish mutations came out of two chemical mutagenesis programmes implemented in the 1990 s, one led by Nobel prize winner Christiane Nüsslein-Volhard in Tübingen, Germany, and the other jointly by Wolfgang Driever and Mark Fishman in Boston, USA.

4 Life Cycle Fertilization to adult in 90 days

5 Zebrafish Technology Sequencing of the zebrafish genome Initiated in 2001 at The Wellcome Trust Sanger Institute in Cambridge, UK. This is essentially complete. Zebrafish have around 70% of genes in common with human and around 84% of disease genes. Molecular markers to follow cells and tissues during development Many transgenic lines are now available that mark cell lineages with fluorescent protein expression. As an extension to this, a zebrafish line has been genetically modified to produce a visible glow during periods of intense brain activity, thereby opening the potential of following cognitive processes in specific regions of the brain.

6 Zebrafish Technology Zebrafish reverse genetics The Zebrafish Mutation Project is now underway at the Wellcome Sanger Centre. This project aims to create a null allele in every protein coding gene in the zebrafish genome. The approach now used is based on the Targeted Induced Local Lesions in Genomes (TILLING) method using ENU-mutagenesis, followed by whole exome enrichment and Illumina next generation sequencing to identify mutations. All mutations are made freely available. To date (6 th November 2014), genes (45% of total) have been mutated, generating 24,088 mutant alleles.

7 Finding mutations

8 Morpholino oligonucleotides Widely used to knock-down the expression of specific genes. The oligonucleotide stably base pairs with the target RNA, thereby interfering with protein synthesis or RNA splicing. The use of Morpholinos has been extended to the study of micrornas (mirnas) by interfering with mirna maturation or the target 3 untranslated regions of interacting transcripts. Morpholinos can be caged such that they require photoactivation, thereby providing a method of light-regulation of gene activity. Site-specific gene targeting methods TALEN method TALE (Transcription Activator-Like Effector) Nucleases from Xanthomous, a plant pathogen CRISPR method Clustered regularly interspaced short palindromic repeats These latter two methods enable the knock-out of any zebrafish gene in your own laboratory.

9 Zebrafish use in biomedical research

10 Location of WA Zebrafish Facility at Biomedical Research Facility, Shenton Park

11 Layout of WA Zebrafish Facility Feeding robot Tank racks Tank racks Main room will house 16 racks, with each rack accommodating 50 x 3.5 litre tanks, to give a total capacity of 900 tanks One row will be serviced by a feeding robot Ancillary rooms Room 5 - Plant room with sentinel rack Room 1 - Quarantine room Room 2 - Food room Room 3 - Microscope/micromanipulator/injection room

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13 Time course 1. Order for Tecniplast now placed 2. Alterations to rooms at BRF to be completed by end of January Delivery of Tecniplast equipment to BRF in January Installation and commissioning in February and March 2015 Costings All routine husbandry of the fish will be provided. As far as possible, fish will be fed using the robot. This means that multiple feeds can be arranged with either wet or dry food. The robot will however only service one row of 5 racks = 250 tanks, which should be sufficient for the immediate future. Estimate of costings is based on the experience at UQ. On a cost recovery basis, UQ charges $3.95 per tank per week. Our costs should be a little lower, particularly our staff costs, but it might be prudent to use $4.00 per tank per week in grant applications. So NOW is the time to submit Zebrafish grants

14 Acknowledgements Melissa Lindemann Deidre Bourke Animal Welfare and Veterinary Advisors Simone Chapple Facilities Manager, Animal Care Services Marilyn Davies Diagnostic & Quality Control Manager, Animal Care Services