1 / 22

Anna Troedsson-Wargelius Research group : Reproduction and growth

Knockdown of salmon dead-end affect gonad morphology in 10 months old Atlantic salmon ( Salmo Salar L.). Anna Troedsson-Wargelius Research group : Reproduction and growth Institute of Marine Research, Bergen, Norway

fynn
Download Presentation

Anna Troedsson-Wargelius Research group : Reproduction and growth

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Knockdown of salmon dead-end affect gonad morphology in 10 months old Atlantic salmon (SalmoSalar L.) Anna Troedsson-Wargelius Research group: Reproduction and growth Institute of Marine Research, Bergen, Norway 1st International Conference on Integrative Salmonid Biology, 18th of June, 2012

  2. Why alternative sterility methods? • Escaped aquacultured salmon is considered a major threat against wild salmon populations • 2. Early sexual maturation is a major economic, welfare and sustainability problem in salmon farming, due to negative impacts of maturation on growth, flesh quality, osmoregulation and immune function. • 3. Triploid are currently used but are more prone to develop • skeletal deformities, cataract and perform bad during sub- • optimal condition (low oxygen, temp etc.), and also in the end fish mature. • 4. “New” and “old” biotechnology becomes much more applicable with the existence of a salmon genome

  3. Primordialgermcells (PGC)- precursors of adult germcells Dome stage (top view) One somite Twelve somites Eighteen somites 24 hpf Ablation of the primordial germ cells = sterile fish χ Modified from Prabhat et al 2003

  4. CodPGCs Control MO Nanos MO

  5. Some of the most studied ”PGC” survival genes in vertebrates

  6. Gene expression in salmon Embryo (100 day˚) vertebrae Muscle Testes Ovary Heart Liver dead-end Eye Fins Skin Gill vasa nanos3a nanos3b β-actin

  7. Spatial expression in oocytes

  8. dead end • Essential for survival and migration of PGC’s inzebrafish , medaka, pond loach and mouse • RNA binding protein

  9. Sex determination Model 1 PGC necessary for sex determination χ No PGC’s -all sterile males Model 2 PGC’s Do not affect sex determination No PGC’s -sterile males and females

  10. How do we specifically target sterility genes • Destroy mRNA Morpholino knock downtargets mRNA • Targeted genome editing specifically mutates gene of interestGMO-fish

  11. Morpholinos-Destroythetranscript

  12. Workflow for functionalanalysis Fluroscein-labeled morpholinos against dead-end (dnd), and control (C) 100 day º 10 months embryos 15 min-1hour old PCR to identify Splice effect on mRNA Histology of gonads on dnd, and control

  13. dead end in salmon dndMo1 dndMo2 dnd Mo1 Control Mo dnd Mo1 Control Mo Control Mo dndMo1 dndMo2

  14. Effect of dead-end morfolino in salmon

  15. Phenotypehemisterile male and female

  16. Genome editing -Zinc finger nucleases (ZFN,2008) -TALENs (transcription activator-like effectors, 2010)

  17. ZFN binding regions in salmon dead end

  18. Zinc finger experiments in salmon FO Zinc fingers targeting dead-end 100 day º 10 months male 15 -60 min after fertilization PCR and sequencing Identifes mutation in eggs Screen sperm for mutations

  19. To make homozygoussalmon F0 F1 F2 * ZFN Inj. homozygote fish mosaicfish heterozygote fish Fertilisation Sperm from Parr wt Fertilisation Double haploid * Temperature and photoperiodinduced males (Fjelldal et al Aquaculture 2011)

  20. Conclusions • It is possible to knock down genes essential for primordial germ cells survival in Atlantic salmon • Weak dead end morpholino phenotype in salmon increase MO • Genome editing using ZFN nucleases is currently being investigated

  21. Acknowledgements Institute of Marine Research Øyvind Drivenes Rolf Edvardsen Geir Lasse Taranger Ole Tørrisen Eva Andersson Trine Haugen Per Gunnar Fjelldal Ketil Malde Anne Torsvik Stig Mæhle Lise Dyrhovden Marine Harvest Hans Øyvind Svensvik Roy Hjelmeland University of Utrecht Rüdiger Schulz Jan Bogerd

  22. - . Thanks for your Attention!

More Related