biochemistry poster
TRANSCRIPT
Utilizing E. coli to Express dsRNA to Inhibit Molecular Pathways in C. elegans
ABSTRACT
Alpha-1 Antitrypsin (α-1 AT) deficiency is a common genetic disorder that affects 1
in 2,000 individuals in the USA. Additionally, over 20 million people have been identified as
carriers for this genetic disorder. In severe cases, α-1 AT deficiency can cause substantial
lung and liver damage, which if left untreated could result in death and there are no current
available treatments. Alpha-1 protein is produced in the liver, travels in the bloodstream
and utilized in the lungs to protect healthy lung tissue from harmful destruction by elastase.
A common single amino acid substitution, located at E342K (ATZ) was identified in α-1 AT
deficient humans. When this specific mutation occurs two phenotypes can result: 1) ATZ
can polymerize in the liver causing cellular toxicity 2) inhibits alpha-1 antitrypsin from
inhibiting elastase which can result in lung disease. Currently; little is known about the
cellular mechanisms that clear the accumulated proteins in the liver. Therefore, an
investigative study utilizing C. elegans model of ATZ was performed in order to help
determine the cellular mechanisms that dispose of accumulated proteins. Specifically RNA
interference was utilized to knockdown expression of specific genes. This investigation
examined genes involved in the heat-shock pathway (HSP), unfolded protein response
(UPR), and insulin signaling pathway (IS). Phenotypic analysis including: embryonic
lethality, protein aggregation expression, and longevity, was completed after knockdown of
genes to determine effect on ATZ accumulation. Currently with our preliminary data
suggests that the heat-shack pathway may play a role in ATZ accumulation. Determining
the mechanism of protein accumulation in the investigation of C. elegans may lead to
possible drug targets and therefore the development of a treatment which may alleviate
those diagnosed with this disorder.
Zachary M. Weisner and Oliva S. Long*,
* Corresponding author, [email protected], (724) 836-9891
Biological Sciences Department, Division of Natural Sciences, Mathematics, and Engineering, University of Pittsburgh at Greensburg, Greensburg, PA 15601
EXPERIMENTAL PLAN
DISCUSSION
FUTURE WORK
INTRODUCTION
ACKNOWLEDGEMENTS
CITED LITERATURE
[1] Altun, Z. F., and D. H Hall. "INTRODUCTION TO C. elegans ANATOMY."
Wormatlas Homepage. Worm atlas , 1 Jan. 2002. Web. 9 Nov. 2012.
<http://www.wormatlas.org/ver1/handbook/anatomyintro/anatomyintro.htm.>
[2] M, zova, Verduyn C, De Brouwer D, and De Block M. Transforming petals into
sepaloid organs in Arabidopsis. "RNA Interference and Hairpin Mechanism."
RNAi: Hairpain Mechanism. N.p., 8 Oct. 2003. Web. 2 Oct. 2012.
<http://www.bioon.com/biology/Class422/1781.shtml>
[3] "Alpha-1 Antitrypsin Deficiency." Learn.Genetics™. N.p., n.d. Web. 28 Sept.
2012. <http://learn.genetics.utah.edu/content/disorders/whataregd/a1ad/>
[4] "Alpha-1 Antitrypsin Deficiency." Learn.Genetics™. N.p., n.d. Web. 28 Sept.
2012. <http://learn.genetics.utah.edu/content/disorders/whataregd/a1ad/>
[5] Altun, Z. F., and D. H Hall. "INTRODUCTION TO C. elegans ANATOMY."
Wormatlas Homepage. Worm atlas , 1 Jan. 2002. Web. 9 Nov. 2012.
<http://www.wormatlas.org/ver1/handbook/anatomyintro/anatomyintro.htm.>
Division of Natural Sciences, Mathematics, and Engineering
• Fast reproduction rate
• Fast life cycle
• Genome has a 40% homology to humans
• Economically inexpensive
• Large # of offspring
• Hermaphrodites
Why C. elegans?
RNA Interference
• Helps moderate the activity of
genes
• Utilize this mechanism to silence
gene expression through post-
translational gene silencing.
• This structure is pathway specific,
and produces accurate results
• In C. elegans RNAi is synthesized
and expressed by modified E. Coli,
which the worms ingest resulting in
specific gene knockdown
RNAi
What is Alpha-1
Antitrypsin Deficiency ?
• Disease affects 1 in 2,000 individuals
• Alpha-1 Antitrypsin Protein responds to
neutralize elastase in the lungs
• Z mutation can cause polymerization of
ATZ protein in the lung
• ATZ prevents α-1-AT from binding with
elastase and can result in damage to the
lung tissues
RNAi Clones
Transgene Expression Experiment
I would like to take the time to show my sincere gratitude to the University of
Pittsburgh at Greensburg Department of Biology and Chemistry for the use of their
laboratories and equipment. Additionally, I would like to take the time to thank my
mentor, Dr. Olivia Long for her time and assistance with this project. Also a thank
you to Dr. Mark Stauffer, Dr. Matthew Luderer, Dr. Kerry Holzworth, Professor
Diane Cheek, and especially Professor Barbra Barnhart for the support through out
this project.
Figure 5 : A) Large Scale Cell Culture Protocol B) Experiments executed on C. elegans
Figure 4: Mechanism of RNAi [2]
Figure 1: Anatomy of C. elegans [1]
Figure 3: A) Normal Pathology of Lungs
B) α-1 AT Deficiency Pathology [4]
Table 1: List of selected RNAi’s used in Research
This experiment utilized E. coli strain HT115 containing the L4440 vector. The L4440
vector has been modified to express the dsRNA of interest. Figure 5A explains the large scale cell
culture protocol used to make the RNAi plate that were used to selectivity knockdown the gene of
interest. Figure 5B elucidates the experimental protocols utilized during the phenotypic analysis,
specifically embryonic lethal, longevity and transgene expression.
Figure 7: Results of Embryonic Lethal
Results of the embryonic lethal experiment indicate that hsf-1 has significant increase in
embryonic death of C. elegans. Additionally, “bagging” was noted in the stc-1 knockdown.
Figure 6: Stages of C. elegans Life Cycle [6]
Figure 8: Kaplan-Meier Survival Curve
ATZ mutated C. elegans have decreased lifespan. We predict enhancers of the removal
of the aggregated protein will result in a corresponding increase in lifespan while conversely
inhibitors of disposal of aggregated protein will result in a shorter lifespan.
Figure 9: Results of the transgene expression experiment on selected RNAi’s. The GFP and
vec are the controls. This examines the protein aggregations formed in the C. elegans.
The transgene expression experiment observed the effects of selected RNAi’s on ATZ
expression. This experiment revealed that the hsf-1 had a significant increase in accumulations of
ATZ compared to the control, vec. Additionally, daf-16, ZC395.10, stc-1, F08h9.4, and Y41e3.11
all showed a significant increase in expression.
Figure 10: Quantification of GFP Expression
• Complete more trial of longevity experiment
• This investigation will primarily examine how the RNAis can either benefit
or harm the C. elegans.
• Repetition of the embryonic lethal experiment to ensure accurate and precise
data.
• This will minimize human error and promote better results.
• In years to come, the objective will become to examine more gene pathways
in regards to how it will react with a ATZ worm.
• The goal is to discover a new pathway that may lead to a cure in this type of
disease.
• Drug targets for these pathways will additionally be investigated.
B.A.
Gene Pathway Associated with Gene
daf-16 Insulin Signaling Pathway
dve-1 Defective Proventiculus in Drosophilia
hsf-1 Heat Shock Factor
ire-1 UPR
F08H9.4 Heat Shock Factor
stc-1 Heat Shock Factor
zc395.10 Arachidonic Acid Metabolism
Y41E3.11 Isoform b
• The embryonic lethal experiment showed a significant lethality with the hsf-1
RNAi.
• This indicates that the hsf-1 gene must play a significant role in the
development of the animals.
• The longevity experiment revealed that there was a significant difference
between the knockdown of vec and GFP.
• GFP removed the accumulations of ATZ resulting in longer lifespans
• Further studies will look into how different RNAi’s affect the longevity of
the C. elegans.
• With the exception of the ire-1, all the RNAi’s tested showed an increase of
expression after 48 hour knockdown.
• This indicates that these pathways may play a role in the disposal of ATZ
accumulations.
• The stc-1 pathway exhibited “bagging” in multiple experiments. This
indicates that the stc-1 RNAi affects the reproductive system.
Expression in C. elegans
Figure 2: nhx-2::GFP::ATZ
Transgenic expression of ATZ
protein accumulation in the
worm
A. B.
RESULTS