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