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

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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

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

Gene 395(2007) 160-169

Chongqing University of Medical Sciences


Content

Content

1. Introduction

2. Materials and methods

3. Conclusion

www.themegallery.com

Company Logo


1 introduction

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 introduction1

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 introduction2

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 introduction3

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 introduction4

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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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

www.themegallery.com

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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

www.themegallery.com

Detailed steps for subcloning siRNA oligonucleotide cassettes

into pSOS vector

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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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A proposed model of pSOS action

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2 materials and methods

2. Materials and methods

HEK-293 were purchased from ATCC.

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2.1Materials

human embryonic kidney(cells)

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2 materials and methods1

2. Materials and methods

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1

2

silencingGFPexpression by using GFP specific siRNA.

silencingGFPexpression by usinghumanβ-catenin specific siRNA.

2.2 Methods

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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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Candidate siRNA sites of the GFP coding region

RNAi-mediated inhibition of GFP expression

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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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Fluorescence measurement of silencing efficiency of the three GFP target sites

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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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Candidate target sites of humanβ-catenin coding region

(267nt–2266nt)

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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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Inhibition of GFP expression by siRNAs targeting human β- catenin

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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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Quantitative comparison

of the knockdown efficiency of GFP signal by pSOS-based siRNA vectors targeting human β-catenin.

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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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

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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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the pSOS-siBC vectors-mediated

decrease in GFP signal correlates with inhibition of β-catenin

signaling

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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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Schematic representation of the retroviral vector pSOS

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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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adenoviral shuttle vector pSES

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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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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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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

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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

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 conclusion1

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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Selection and validation of optimal sirna target sites for rnai mediated gene silencing

Thank You !


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