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Unformatted text preview: Experimental siRNA Targeting the 469-624bp Region of Renilla Luciferase is Ineffective in Reducing Expression in Mouse Embryonic Stem Cells Jessica Keenan and Augusto Tentori Abstract dsRNA is an increasingly well studied and widely used tool for the regulation of gene expression at the post-transcriptional level. In this experiment, we designed an siRNA intended to specifically target the Renilla reniformis luciferase gene in order to reduce the amount of luciferase produced by the gene. However, Luciferase assays of the lysates from mouse embryonic stem cells transfected with this siRNA and a plasmid containing the Renilla luciferase gene suggest that the designed siRNA is not able to reduce Renilla luciferase expression. Moreover, microarray analysis suggests that the siRNA has widespread off-target effects. Over 8,000 genes were differentially expressed between cells transfected with the luciferase-containing plasmid only, and those transfected with the experimental siRNA and the plasmid. There is no clear pattern between the genes that showed the greatest differential expression between the two samples. Thus, we have determined the experimental siRNA is not able to specifically reduce Renilla luciferase expression, and instead has many off-target effects. To make RNAi a more useful tool than traditional techniques like homologous recombination, further research must be done to improve siRNA design algorithms. Introduction In order to perform their specified functions, cells must constantly regulate the amount and types of proteins they produce. When a cell is no longer able to properly regulate protein production, cell death or defects in the tissue or organism often result. For example, cancer is often caused by decreased production of proteins that inhibit cell growth, or by over production of proteins that cause the cell to continuously divide. Healthy cells, however, are able to regulate their protein expression at many levels, including transcription, translation, and after transcription and translation. One way in which cells regulate protein expression at the post-transcriptional level is by preventing the translation of the mRNA transcript in a process called RNA interference (RNAi). RNAi, which occurs in organisms as diverse as C. elegans (Fire 1998), planarians (Snchez-Alvarado 1999), and humans (Elbashir 2001), uses a double- stranded piece of RNA (dsRNA) to prevent the translation of a target mRNA transcript. One strand of the dsRNA is complementary to a region of the target mRNA. This strand is incorporated into the RNA-induced silencing complex (RISC) by a process that is not well understood, but is believed to involve the Argonaute family of proteins (Song 2003)....
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- Spring '06