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The Knockdown of 12-lipoxygenase in Embryonic Zebrafish Causes Abnormal Development

The Knockdown of 12-lipoxygenase in Embryonic Zebrafish Causes Abnormal Development

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The Knockdown of 12-lipoxygenase in Embryonic Zebrafish Causes Abnormal Development

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  1. The Knockdown of 12-lipoxygenase in Embryonic Zebrafish Causes Abnormal Development Amber Bannon Mentor: MaretTraber, PhD

  2. Relevance • 12-lipoxygenase may have a role in angiogenesis • Lipoxygenase plays important roles in inflammatory diseases • Atherosclerosis • Cancer • Osteoporosis • Diabetes Kuhn and O’Donnell. Progress in lipid research 2006 • Role in embryonic development has not yet been studied

  3. Hypothesis • We believe 12-lipoxygenase is necessary for normal embryonic development in zebrafish. Therefore, when it is knocked down, there will be an abnormal phenotype and altered gene expression.

  4. Zebrafish model • Able to separate embryo from mother • Genes are generally homologous to those in humans • Rapid embryonic development 48 hpf 12 hpf *hpf = hours post fertilization

  5. Study design • Treat embryos at the single cell stage • Inject with LOX morpholino • Inject with control morpholino (random oligomer) • Non-injected control • Observe individual embryos over a 5 day period • Collect embryo samples for RNA extraction at 24 hpf and 48 hpf to analyze gene expression

  6. Morpholino injections • Short anti-sense oligomers with high mRNA binding affinity • Labeled with fluorescein tag to verify incorporation • Knock down protein expression by blocking exon/intron splice site

  7. In normal cells… EXON 1 INTRON EXON 2 EXON 1 EXON 2 PROTEIN

  8. Morpholino injected… EXON 1 INTRON EXON 2 MORPHOLINO EXON 1 INTRON EXON 2 MORPHOLINO Control Injected 250bp ABNORMAL PROTEIN Confirmation of block by RT-PCR/gel electrophoresis 171bp

  9. LOX knockdown malformations LOX morpholino injected Noninjected control Control morpholino injected

  10. LOX morpholino injection increases the number of embryonic malformations

  11. LOX morpholino injection increases the number of embryonic malformations

  12. LOX morpholino injection increases the number of embryonic malformations

  13. Quantitative Reverse Transcriptase- Polymerase Chain Reaction (qRT-PCR) • Utilizes reverse transcriptase to synthesis cDNA from mRNA • Amplifies and quantifies cDNA in real time • The number of copies of cDNA measure the relative gene expression • Using control and morpholino injected samples we are able to determine changes in gene expression

  14. Proposed pathway for lipoxygenase activity PL-Arachidonic LOX PL-HPETE GPx4 PLA2 PL-HETE HPETE GPx4 PLA2 HETE

  15. 12-lipoxygenase (12-LOX) • Catalyzes the addition of oxygen to the 12th carbon of arachidonic acid • Produces hydroperoxides (HpETE) in phospholipids • Important role in normal biological functions • Byproducts cause the lipid-oxidation chain reaction OOH 12-HpETE Arachidonic acid

  16. 12-lipoxygenase

  17. Proposed pathway for lipoxygenase activity PL-Arachidonic LOX PL-HPETE GPx4 PLA2 PL-HETE HPETE GPx4 PLA2 HETE

  18. Gluatathioneperoxidase 4 (GPx4) • Catalyzes the reduction of the hydroperoxides(HpETE) created by lipoxygenase • Produces hydroxy products (HETE) • Important signaling molecule for normal biological function • Phospholipid antioxidant that utilizes glutathione and selenium OOH OH 12-HpETE 12-HETE

  19. Glutathione Peroxidase 4

  20. Phospholipase A2 (PLA2) • Cleaves molecules from the phospholipid at the sn-2 position • Produces HpETE and HETE from PL-HpETE and PL-HETE

  21. Proposed pathway for lipoxygenase activity PL-Arachidonic LOX PL-HPETE GPx4 PLA2 PL-HETE HPETE GPx4 PLA2 HETE

  22. Phospholipase A2

  23. Conclusion • Knock down of 12-lipoxygenase expression causes an abnormal phenotype in zebrafish embryos • There is a change in mRNA expression observed at 24 hpf • This change appears to be linked to the abnormal morphology seen in lipoxygenase knock down embryos • Lipoxygenase is necessary for normal embryogenesis in zebrafish

  24. What’s next? • This project provides an essential stepping stone for future research in the Traber lab on the molecular function of vitamin E in development • Knock down the other genes in the proposed pathway • Obtain vitamin E deficient fish and observe their development • Knock down LOX in vitamin E deficient fish

  25. Acknowledgements • MaretTraber, Ph.D. • Traber lab • Galen Miller • Ed Labut • Robert Tanguay, Ph.D. • Tanguay Lab • Kevin Ahern, Ph.D. • Howard Hughes Medical Institute • Cripps Scholarship Fund, College of Science