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

Embryonic expression of both GPx4a and GPx4b starts at 10 hpf

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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

Acknowledgements

• The Howard Hughes Medical Institute

• Maret Traber

• Galen Miller, Ed Labut & Carrie Barton

• Kevin Ahern