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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
H2O
2O
X
OO
O O
X H
O
R
R
OHR
Vitamin E
Lipid
Ascorbate Chemistry Oxygen Society Education Program Buettner GR. (1993) Arch Biochem Biophys.
300:535-543.
C and E as Co-Antioxidants
C and E as Co-Antioxidants
Buettner GR. (1993) Arch Biochem Biophys.
300:535-543.
H2O
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
OR
AscH-
Vitamin E
Ascorbate Chemistry Oxygen Society Education Program
O
R
R
OHR
Asc F A-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
• 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 1 Intron
Normal cells:
Exon 2
Exon 1 Exon 2
Intron
Injected cells:
Exon 1 Exon 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 E4 E5Intron 3-4
In injected embryos:
GPx4b Morpholino
• Expected PCR product from normal embryos: 179 bp
• Expected PCR product from MO injected embryos: 2808 bp
E3 E4
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
Primer a a a a a b b b b b
8 h a 8 h a/b 8 h con 8 h non 24 h non 8 h b 8 h a/b 8 h con 8 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