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Silencing UNC-50 in Caenorhabditis Elegans
Dylan Roper, Department of Biology, York College of Pennsylvania
Methods
http://web.science.uu.nl/developmentalbiology/boxem/images/adult_worm.jpg
Introduction
• Small interfering ribonucleic acids (siRNA’s)
are double stranded RNA molecules used to
post transcriptionally silence genes by binding
to specific mRNA sequences. Binding to the
mRNA prevents translation and gene
expression (Guo et. al. 2010).
• The nematode, Caenorhabditis Elegans (C.
elegans), is a model for gene silencing. The C.
elegans genome has been sequenced and it
was determined that many human disease
pathways in humans are also present in C.
elegans (Kaletta and Hengartner 2006).
• UNC-50 is a genetic region that is homologous
between humans and C. elegans. In C. elegans
the region encodes an integral membrane
protein that is used to traffic specific nicotinic
acetylcholine receptors (Eimer et. al 2007).
• When the receptors get degraded after the
gene is silenced the C. elegans experiences
slower movement as well as experiencing
kinks in their general movement (Eimer et. al
2007).
Design a primer
sequence for a dsDNA
that codes for a UNC-50
specific siRNA
Create the dsDNA using
PCR and ligate into a
plasmid with a T7
promoter
Extract plasmid, and
transform into HT115 E.
coli cells (strain of E.coli
the C. elegans prefers)
Transformation into
OmniMAX 2 T1 E. coli
cells (strain of E.coli is
heartier)
Add transformed HT115
E. coli to agar plates (2
per primer sequence) 1
with treatment (IPTG),
and 1 without
Add 4 C. elegans per
plate, and observe the
second generation for
72 hours
Figure 3: C. elegans on a treatment plate of HT115 with plasmid
1. The plate was imaged 48 hours after initial transfer of C.
elegans. There are few visual track marks, and the worms
circled are demonstrating abnormal bending.
Results
1
2
3
Primer 1
4
5
Primer 2
Objectives
• Transform HT115 E. coli cells so they code for
a specific siRNA that will post-transcriptionally
silence the UNC-50 protein
Conclusions
• Successful transformation of HT115 E.
coli shown by C. elegans
• The C.elegans expressed phenotypical
changes that correlate with silencing
the unc-50 gene region
Future Studies
100 bp
• Using specific siRNA sequences to
silence proteins involved in cancer cell
proliferation
References
• Use the transformed HT115 E.coli cells as a
food source for C. elegans so that they will
uptake the siRNA
• Observe phenotypical changes in the first
generation of C. elegans with silenced UNC-50
Figure 2: C. elegans on a control plate containing HT115
bacterial cells with plasmid 1. Plate was imaged 48 hours
after initial transfer of C. elegans. The area circled indicates
the disturbed areas of bacteria in which the C. elegans has
already traveled over. The fact that there are increased
amount of movement markers shows that the C. elegans
were active.
Guo, Peixuan., Coban, Oana., Snead, Nicholas M., Trebley, Joe., Hoeprich, Steve., Guo, Songchuan., and Shu,
Yi. 2010. Engineering RNA for Targeted siRNA Delivery and Medical Application. Advanced Drug Delivery
Reviews 62: 650-666.
Kaletta, T. and Hengartner, M.O. 2006. Finding function in novel targets: C. elegans as a model organism.
Nature Reviews Drug Discovery: 1-12.
Figure 1: A 2% agarose gel ran at 300 volts for ten minutes. In lane 1 there is
a 100 base pair ladder. The lowest band in lane 1 represents 100 base pairs.
In lanes 2-5 the PCR products are shown at similar migration distances as
the 100 base pair ladder.
1: 100bp ladder, 2&3: Primer 1, and 4&5: Primer 2
Eimer, S., Gottschalk, A., Hengartner, M., Horvitz, R.H., Richmond, J., Schafer, W.R., and Bessereau, J. 2007.
Regulation of nicotinic receptor trafficking by the transmembrane Golgi protein UNC-50. The EMBO Journal
26: 4313-4323.
Acknowledgements
I would like to thank Dr. Kaltreider and Dr. Thompson for their help and guidance in completing
this project. I would also like to thank the York College Biology department for their support.