Selection and validation of optimal siRNA target sites for RNAi-mediated gene silencing
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Transcript of 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
Gene 395(2007) 160-169
Chongqing University of Medical Sciences
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Content
1. Introduction
2. Materials and methods
3. Conclusion
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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.
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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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