Transcript RNAi in C.remanei - SPUR

The examination of the
insulin-like pathway
Caenorhabditis remanei
Natasha Naidoo
SPUR Project 2009
Why study aging?
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It is a phenomena that effects every organism on the planet
It is variable between individuals
Find the causes of aging
Why do individuals age differently?
Identify the pathways to life extension
Life-extending mutants
• The Insulin receptor triggers a
chemical cascade (daf-2)
• Down-regulates the transcription
factor. (daf-16)
• The transcription factor activates
the “stress response machinery”
which extends lifespan
• daf-2 mutants are longer lived
• Extends lifespan by about 2x in
C.elegans
• daf-16 mutants are shorter lived
• age-1 mutants longer lived
• Do individuals have natural
variation in these pathways?
Nemoto, Shino, and Toren Finkel, "ageing and the mystery
at Arles." Nature, no.429 (2004): 149-152.
What are the benefits of C.elegans
Short generation time
• Short lifespan
•
•Well characterized
genome
•Transparent
• Freeze-able
• Self-fertilization
Double-edged sword of selfing
• Self-fertilization
• Conserves genes of interest
• Little genetic variation
• Outcrossing
• Lots of natural genetic variation
• C. remanei as a model organism
-3
t (10 )
nucleotide diversity
p
20
15
10
5
0
Ce Cb Cr
Ce Cb Cr
odr-7
odr-10
Ce Cb Cr
odr-3
Ce Cb Cr
tax-2
Richard Jovelin ‘09
SPUR project goals
Elegans and lifespan
• Getting comfortable with C.elegans and their genetic
toolbox
• Performing lifespan Assays
RNAi in C.remanei
• Making parts of a new genetic toolbox for a
novel model organism
Oxidative Stress
• Testing the RNAi method
• Examining daf-2, and age-1 pathways in
C.remanei
RNA interference (RNAi)
RNA interference (RNAi)
What this means for the
field
• We can do RNAi on C. remanei for
any gene we want and look for
phenotype expression
• Candidate genes can be identified
• We can look at the variance between
individuals
Nemoto, Shino, and Finkel, ‘04
Oxidative stress testing
The benefits of Oxidative
stress tests
• Short timeframe
•Early indicator of success/failure
• Positive correlation with lifespan
Larsen, ’92
Reynolds ’09
Oxidative stress testing—
Paraquat Paraquat=Methyl viologen dichloride hydrate
Methods and Materials
• 96 well plates
• Various concentrations of paraquat
120
Paraquat Treatment
100
[250mM]
[125mM]
% Alive
80
[62.5mM]
60
[31.25mM]
40
[15.625mM]
20
S. basal
0
0
2
4
6
Time (hours)
8
10
12
Naidoo ‘09 (unpublished)
Paraquat results
General Trends
• daf-2 worms die first
• age-1 and the empty vector (PL4440) are very similar
Arantes-Oliveira et al. ’03
Naidoo ‘09 (unpublished)
Summary
RNAi
• Constructed a way to do RNAi in a novel organism
• Now we can see if specific genes are functionally
conserved between species
• C. remanei can help answer the question of natural
variation between individuals
• Tested RNAi with the paraquat assay
• The process worked — daf-2 is different then
the empty vector
• It is possible that the daf-2 pathway is not
functionally conserved across species
• Reinforces the usefulness/speed of RNAi
Future Directions
• Do more replicates to solidify the paraquat results
• Sequence the RNAi to make 100% certain the
results were accurate
•Use the technique to further investigate the
aging pathway in C. remanei
•Do lifespan assays on C. remanei that have been
affected by RNAi
Thank You’s
John Willis
Patrick Phillips
The Phenomenal Phillips Lab
Peter O’Day and SPUR
University of Oregon
Family and Friends
Special thanks to Bryn Gaertner
Special thanks to Bryn Gaertner
Special thanks to Bryn Gaertner