Browsing by Subject "Small interfering RNA."
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Item Anti-tumor activity of an oncolytic adenoviral construct expressing a small interfering RNA transgene.(Philadelphia, PA : American Association for Cancer Research., 2006-10-01) Samuel, Shirley Kulangara.; Tong, Alex W.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.Cancer is a leading cause of mortality in the world today. Mutation in the K-ras oncogene is common in most human cancers. K-ras oncogene expression was specifically downregulated by 58.7% by K-ras silencing siRNA, and this was accompanied by 66% growth inhibition of NCI-H441 lung cancer cells. To improve siRNA delivery and cause its stable expression in cancer cells, we used ONYX-411, an oncolytic adenovirus as a backbone to clone the K-ras silencing siRNA. This new adenoviral construct called Internavec, significantly downregulated mutant K-ras expression by 48.9% (p<0.05, n=3) as compared with 11.9% downregulation by parental ONYX-411 in HTB-79 pancreatic cancer cells. The anti-tumor activity of Internavec was examined in various cancer cell lines with and without the relevant K-ras mutation to observe the specificity of the siRNA transgene against the glycine to valine mutation on codon 12. Internavec showed enhanced anti-tumor activity in cell lines with the relevant mutation, compared with ONYX-411. Internavec (5 @ 1x108 pfu) significantly reduced the growth of subcutaneous HTB-79 pancreatic tumor xenografts in vivo by 85.5%, including complete growth suppression in 3 of 5 mice. Parental ONYX-411 or ONYX-411-siRNAGFP was markedly less effective (47.8% and 44.1% growth reduction, p<0.05, respectively). To characterize interferon-inducing activity of Internavec, landmark gene expression of the interferon pathway (OAS1, MX1) was examined following Internavec treatment, using HEK-293 cells as positive control. HEK-293 cells displayed an upregulation of OAS1 and MX1 following Poly (I:C) treatment. However, Internavec did not upregulate these interferon-pathway genes in HEK-293 or H79 lines, suggesting a lack of interferon activation by Internavec. To delineate underlying molecular events contributing to the enhanced growth inhibition, microarray experiments were performed on cells treated with Internavec. Internavec, but not ONYX-411, downregulated the expression of multiple Ras signaling-related genes (MAP4K5, PLCε1, IKBKB, FOXO3A and RAB28). These findings indicate that the knockdown of mutant K-ras serves to enhance oncolytic virus anti-tumor activity through the perturbation of additional cellular signaling events.Item Identification of phenotypes in Caenorabhditis elegans on the basis of sequence similarity.(2009-06-02T17:59:06Z) Batra, Sushil.; Baker, Erich J.; Lee, Myeongwoo.; Biomedical Studies.; Baylor University. Institute of Biomedical Studies.In biomedical research, Caenorabhditis elegans is an ideal choice as experimental organism due to striking similarity with human genome and its distinct features such as short life span, small reproductive cycle, simple body plan, easily observable mutant phenotypes and ease of cultivation in laboratory. The 97 megabase genomic sequence of C. elegans comprises approximately 19,920 genes, of which about 2807 genes (14% of total genome) are uniquely associated with one or more RNAi phenotypes. The challenge to assign phenotypes to remaining 86% genes has incited development of new rapid techniques and computational tools. Objective of this project was to identify phenotypes in C. elegans on the basis of sequence similarity using bioinformatics techniques. To find similarity in genes, we used BLAST as computational tool and predicted the phenotypes. Bi-directional pair wise BLAST was performed on 2,807 unique genes (associated with known phenotypes) against 19,920 genes. As a result, 141 new genes (with unknown phenotype) were obtained which share high sequence similarity with known RNAi phenotype genes of 16 categories. In the present work, putative genes associated with two phenotypes, Ste (37 genes) and Unc (29 genes), were studied by RNA interference (RNAi) in laboratory. The outcome of these experiments assigned sterility phenotype to 8 new genes and uncoordinated phenotype to 12 new genes which were not linked with any phenotype in previous studies. These observations were further verified by silencing the response using reverse transcriptase polymerase chain reaction (RT-PCR) for Ste genes. Thus, bioinformatics techniques were successfully utilized in identification of phenotypes on the basis of sequence similarity with a relatively high success rate of 22% and 41% for sterility and uncoordinated phenotypes respectively. High success rate of this bioinformatics technique will allow researchers to focus their efforts on identifying particular phenotypes of interest and understanding various biological processes and elucidating the pathogenesis of diseases.