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, osl5@pitt.edu, (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