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Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing

Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing

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Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing

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  1. Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing Gene 395(2007) 160-169 Chongqing University of Medical Sciences

  2. Content 1. Introduction 2. Materials and methods 3. Conclusion Company Logo

  3. 1. Introduction Double-stranded RNA (dsRNA) can induce gene-silencing processes in eukaryotes through the degradation of homologous mRNAs, a process known as RNA interference (RNAi) in animals and post-transcriptional gene silencing(PTGS)in plants. Company Logo

  4. 1. Introduction Practical challenges: selection of effective target sites,efficient transfer of siRNAs into cells or tissues,and achieving controllable long-term silencing of target genes.There is no reliable and efficient way to predict optimal siRNA sequences. Company Logo

  5. 1. Introduction Objective: Developing a simplified and efficient fluorescence-based screening and validation system, namely pSOS, to assess gene-silencing efficacy of siRNAs. Company Logo

  6. 1. Introduction Introducing pSOS-based siRNA plasmids into mammalian cells, the reduction in GFP signal would reflect the silencing efficiency of siRNAs. Company Logo

  7. 1. Introduction The pSOS system has two essential components: One of which expresses a chimeric transcript between GFP and the coding region of a target gene driven by hEF1α. The other expresses a siRNA duplex under the control of dual convergent Pol III promoters(H1 and U6). Company Logo

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  9. Detailed steps for subcloning siRNA oligonucleotide cassettes into pSOS vector Company Logo

  10. A proposed model of pSOS action Company Logo

  11. 2. Materials and methods HEK-293 were purchased from ATCC. 2.1Materials human embryonic kidney(cells) Company Logo

  12. 2. Materials and methods 1 2 silencingGFPexpression by using GFP specific siRNA. silencingGFPexpression by usinghumanβ-catenin specific siRNA. 2.2 Methods Company Logo

  13. Candidate siRNA sites of the GFP coding region RNAi-mediated inhibition of GFP expression Company Logo

  14. Fluorescence measurement of silencing efficiency of the three GFP target sites Company Logo

  15. Our results demonstrated that both 21nt and 27nt target sites can be equally efficient in gene knockdown . Our results also indicate that siRNAs whose sense-strand expression was driven by the U6 promoter were more effective than those driven by the H1 promoter. Company Logo

  16. Candidate target sites of humanβ-catenin coding region (267nt–2266nt) Company Logo

  17. Inhibition of GFP expression by siRNAs targeting human β- catenin Company Logo

  18. Quantitative comparison of the knockdown efficiency of GFP signal by pSOS-based siRNA vectors targeting human β-catenin. Company Logo

  19. These results demonstrate that β-catenin siRNAs can effectively knockdown the chimeric transcript between GFP and human β-catenin. Company Logo

  20. the pSOS-siBC vectors-mediated decrease in GFP signal correlates with inhibition of β-catenin signaling Company Logo

  21. Taken together, our results have proven the principle of the pSOS-based system for selection and validation of siRNA target sites. Company Logo

  22. Schematic representation of the retroviral vector pSOS Company Logo

  23. adenoviral shuttle vector pSES Company Logo

  24. GFP expression of the retroviral vector pSOS or pSOS-siBC5U6-mediated 293 stable cells. Quantitative real-time PCR analysis of β-catenin expression Company Logo

  25. Effective transduction of human osteosarcoma MG63 cells by adenoviruses expressing siBC5U6 and siBC6U6 target sites. Adenovirus-mediated knockdown of β-catenin in MG63 cells Company Logo

  26. 3. Conclusion In summary, we have developed a fluorescence-based siRNA screening method and demonstrated its utility for evaluating the gene-knockdown efficiency of candidate siRNA sites. The GFP-based assay for gene-silencing efficiency can be qualitative and quantitative. Company Logo

  27. 3. Conclusion Reduction of GFP signal is closely correlated to knockdown efficiency of the expression and functional activity of target genes.Thus,the pSOS system is an efficient,versatile,and yet user-friendly tool for selecting,validating,and delivering optimal siRNA sites for RNAi-mediated gene silencing. Company Logo

  28. Thank You !