amber bannon mentor: maret traber, phd the knockdown of 12-lipoxygenase in embryonic zebrafish...
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Amber BannonMentor: Maret Traber, PhD
The Knockdown of 12-lipoxygenase in Embryonic Zebrafish Causes Abnormal
Development
Relevance
12-lipoxygenase may have a role in angiogenesis
Lipoxygenase plays important roles in inflammatory diseasesAtherosclerosisCancerOsteoporosis Diabetes
Kuhn and O’Donnell. Progress in lipid research 2006
Role in embryonic development has not yet been studied
HypothesisWe 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.
Zebrafish model Able to separate embryo from motherGenes are generally homologous to those
in humansRapid embryonic development
48 hpf12 hpf
*hpf = hours post fertilization
Study designTreat embryos at the single cell stage
Inject with LOX morpholinoInject 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
Morpholino injectionsShort 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
Morpholino injected…EXON 1 INTRON EXON 2
MORPHOLINO
EXON 2EXON 1 INTRONMORPHOLINO
ABNORMAL PROTEIN
Confirmation of block by RT-PCR/gel electrophoresis
171bp
250bp
Control
Injected
LOX morpholino injection increases the number of embryonic malformations
LOX
Co
ntr
ol
No
nin
j
LOX
Co
ntr
ol
No
nin
j
LOX
Co
ntr
ol
No
nin
j
48hpf 72hpf 120hpf
0
2
4
6
8
10
12
14
16
18
Trial 1
MalformedDead
Nu
mb
er
of
fish
LOX morpholino injection increases the number of embryonic malformations
LOX
Co
ntr
ol
No
nin
j
LOX
Co
ntr
ol
No
nin
j
LOX
Co
ntr
ol
No
nin
j
48hpf 96hpf 120hpf
0
5
10
15
20
25
30
35
40
Trial 2
MalformedDead
Nu
mb
er
of
fish
LOX morpholino injection increases the number of embryonic malformations
LOX
Co
ntr
ol
No
nin
j
LOX
Co
ntr
ol
No
nin
j
LOX
Co
ntr
ol
No
nin
j
LOX
Co
ntr
ol
No
nin
j
48hpf 72hpf 96hpf 120hpf
0
2
4
6
8
10
12
14
16
18
Trial 3
MalformedDead
Nu
mb
er
of
fish
Quantitative Reverse Transcriptase- Polymerase Chain Reaction (qRT-PCR)Utilizes reverse transcriptase to
synthesis cDNA from mRNAAmplifies and quantifies cDNA in real
timeThe number of copies of cDNA
measure the relative gene expressionUsing control and morpholino injected
samples we are able to determine changes in gene expression
HPETE
PL-Arachidonic
LOX
PL-HPETE
GPx4 PLA2
PLA2
PL-HETE
GPx4
HETE
Proposed pathway for lipoxygenase activity
12-lipoxygenase (12-LOX)Catalyzes the addition of oxygen to the 12th
carbon of arachidonic acid Produces hydroperoxides (HpETE) in
phospholipidsImportant role in normal biological functions
Byproducts cause the lipid-oxidation chain reaction
Arachidonic acid
OOH12-HpETE
HPETE
PL-Arachidonic
LOX
PL-HPETE
GPx4 PLA2
PLA2
PL-HETE
GPx4
HETE
Proposed pathway for lipoxygenase activity
Gluatathione peroxidase 4 (GPx4)Catalyzes the reduction of the
hydroperoxides (HpETE) created by lipoxygenase
Produces hydroxy products (HETE)Important signaling molecule for normal
biological functionPhospholipid antioxidant that utilizes
glutathione and selenium
OOH12-HpETE
OH12-HETE
Phospholipase A2 (PLA2)Cleaves molecules from the phospholipid at
the sn-2 position
Produces HpETE and HETE from PL-HpETE and PL-HETE
HPETE
PL-Arachidonic
LOX
PL-HPETE
GPx4 PLA2
PLA2
PL-HETE
GPx4
HETE
Proposed pathway for lipoxygenase activity
ConclusionKnock 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
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
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