selection and validation of optimal sirna target sites for rnai-mediated gene silencing
DESCRIPTION
Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing. Gene 395(2007) 160-169 Chongqing University of Medical Sciences. Content. 1. Introduction 2. Materials and methods 3. Conclusion. www.themegallery.com. Company Logo. 1. Introduction. - PowerPoint PPT PresentationTRANSCRIPT
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Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing
Gene 395(2007) 160-169
Chongqing University of Medical Sciences
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Content
1. Introduction
2. Materials and methods
3. Conclusion
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.
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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.
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1. Introduction
Objective: Developing a simplified and
efficient fluorescence-based screening and
validation system, namely pSOS, to assess
gene-silencing efficacy of siRNAs.
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1. Introduction
Introducing pSOS-based siRNA plasmids
into mammalian cells, the reduction in GFP
signal would reflect the silencing efficiency
of siRNAs.
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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 ） .
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Detailed steps for subcloning siRNA oligonucleotide cassettesinto pSOS vector
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A proposed model of pSOS
action
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2. Materials and methods
HEK-293 were purchased from ATCC.
2.1 Materialshuman
embryonic kidney(cells)
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2. Materials and methods
1
silencing GFP
expression by using
GFP specific siRNA.
2
silencing GFP
expression by using
human β-catenin
specific siRNA.
2.2 Methods
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RNAi-mediated inhibition of GFP expression
Candidate siRNA sites of the GFP coding region
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Fluorescence measurement of silencing efficiency of the three
GFP target sites
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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.
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Candidate target sites of humanβ-catenin coding region(267nt–2266nt)
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Inhibition of GFP expression by siRNAs
targeting human β- catenin
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Quantitative comparisonof the knockdown efficiency of GFP signal by pSOS-based siRNA vectors targeting human β-catenin.
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These results demonstrate that β-catenin siRNAs can effectively knockdown the chimeric transcript between GFP and human β-catenin.
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the pSOS-siBC vectors-mediateddecrease in GFP signal correlates with inhibition of β-cateninsignaling
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Taken together, our results have proven the principle of the pSOS-based system for selection and validation of siRNA target sites.
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Schematic representation of the retroviral vector pSOS
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adenoviral shuttle vector
pSES
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GFP expression of the retroviral vector pSOS or pSOS-siBC5U6-mediated
293 stable cells.
Quantitative real-time PCR analysis of
β-catenin expression
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Effective transduction of human osteosarcoma MG63 cells by adenoviruses expressing siBC5U6 and siBC6U6 target sites.
Adenovirus-mediated knockdown of β-catenin
in MG63 cells
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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.
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.
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