1. 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). Silencing UNC-50 in Caenorhabditis Elegans Introduction Objectives Transform HT115 E. coli cells so they code for a specific siRNA that will post-transcriptionally silence the UNC-50 protein 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 http://web.science.uu.nl/developmentalbiology/boxem/images/adult_worm.jpg 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 Transformation into OmniMAX 2 T1 E. coli cells (strain of E.coli is heartier) Extract plasmid, and transform into HT115 E. coli cells (strain of E.coli the C. elegans prefers) 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 Methods Results 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. 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. Conclusions Dylan Roper, Department of Biology, York College of Pennsylvania 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 Using specific siRNA sequences to silence proteins involved in cancer cell proliferation References 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. 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. 1 2 3 4 5 100 bp 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 Primer 1 Primer 2