Morpholino Knockdown of Glutathione Peroxidase 4 in embryonic zebrafish Megan Liu Mentor: Maret Traber Text

Vitamin E Vitamin E in humans functions primarily as an antioxidant Protects fats in low density lipoproteins (LDLs) from oxidation Intercepts free radicals and prevents the lipid peroxidation chain reaction

Lipid peroxidation can cause cell damage in a variety of ways 1) Impair membrane fluidity and function 2) Lipid hydroperoxides can undergo lipid peroxidation, generating reactive aldehydes Lipid Peroxidation

The accumulation of oxidative damage is thought to a cause of many diseases Believed to be especially pivotal in aging. Lipid Peroxidation

H 2 O 2 O X OO O O XH O R R OH R Vitamin E Lipid Ascorbate Chemistry Oxygen Society Education Program Buettner GR. (1993) Arch Biochem Biophys. 300: C and E as Co-Antioxidants

Buettner GR. (1993) Arch Biochem Biophys. 300: H 2 O OOH Lipid Vitamin E removes the peroxyl radical; ascorbate can recycle E; enzymes then remove the damaged fatty acid and insert a new one, repairing the lipid. O R R O R AscH - Vitamin E Ascorbate Chemistry Oxygen Society Education Program O R R OH R Asc FA-CoA PLA2 GPx GPx4

Glutathione Peroxidase 4 (GPx4) is a selenoprotein that reduces lipid hydroperoxides, using glutathione (GSH) as the reducing agent GPx4 also senses and translates oxidative stress into apoptotic pathways GPx4

GPx4 is the only glutathione peroxidase that reduces phospholipid hydroperoxides in membranes (Roveri et al., 1994; Thomas et al., 1990), and Considered the primary enzymatic defense system against oxidative damage to cell membranes (Brigelius-Flohe et al., 1999) Reduces hydroperoxides in lipoproteins and complex lipids (Ran et al., 2007) GPx4

GPx4 Expression Embryonic expression of both GPx4a and GPx4b starts at 10 hpf Text

GPx4 Expression GPx4a is expressed primarily in the yolk sac GPx4b is expressed in the retina, brain, somites, and pronephric duct Shown on right by RNA in situ hybridization Both are expressed in the liver Image: Thisse et. al., 2003 GPx4a GPx4b

Hypothesis Both GPx4a and GPx4b are essential for normal development in zebrafish. We can knock down GPx4a and GPx4b expression with morpholino injections Morpholino knockdown of GPx4a and GPx4b in embryonic zebrafish will cause abnormal development

Morpholinos Morpholinos are molecules used to modify gene expression Morpholinos bind by base pairing to mRNA, and, in our case, prevent mRNA from being properly spliced Exon 1Intron Normal cells: Exon 2 Exon 1Exon 2 Intron Injected cells: Exon 1Exon 2Intron Morpholino

Experimental Design We injected antisense morpholinos at varying concentrations to knock down GPx4a and GPx4b in embryonic zebrafish at the mRNA level Checked for morpholino incorporation at 8 and 24 hpf with a fluorescein tag and for morpholino efficacy with a change of product size of mRNA Injection Process 6 hpf

Experimental Design Some embryos were injected with a morpholino for a human gene to control for the injection process Non injected controls were also collected to control for spawn quality Embryo morphology was assessed by stereo microscopy at 24, 48, and 120 hpf Samples for RNA extraction were collected at 8, 24 and 48 hpf

GPx4a Morpholino Expected PCR product from normal embryos: 207 bp Expected PCR product from MO injected embryos: 703 bp MO E3 E4E5 Intron 3-4 In injected embryos:

GPx4b Morpholino Expected PCR product from normal embryos: 179 bp Expected PCR product from MO injected embryos: 2808 bp E3E4 MO Intron 3-4 In injected embryos:

Splice Blocking Confirmation: Expected Splice blocking can be confirmed with RT- PCR Amplified DNA with primers flanking intron 3-4 in GPx4a and GPx4b Image: cDNA and gDNA amplified with GPx4a and GPx4b primers

Splice Blocking Partial splice blocking was seen in 2.5 mM gpx4a morpholino injected embryos at 8 hpf Injections at lower concentrations (1mM) did not affect splice blocking or embryo morphology cDNA Primeraaaaabbbbb 8 h a 8 h a/b 8 h con8 h non 24 h non 8 h b8 h a/b8 h con8 h non 24 h non 500 bp

Conclusions/What’s Next GPx4a Morpholino was only partially effective at knocking down GPx4a expression GPx4b morpholino was not effective Next: try a different morpholino Observe embryo morphology for 2.5 mM injections

Acknowledgements The Howard Hughes Medical Institute Maret Traber Galen Miller, Ed Labut & Carrie Barton Kevin Ahern