- Efficient delivery of siRNA expression construct into a broad range of mammalian cells and organisms
- Ideal for difficult-to-transfect cells, including many primary and terminally differentiated cells
- Proven system demonstrated to specifically silence target genes in both cultured cells and in animals (1)
- Does not integrate into host genome so little chance of insertional mutagenesis
Important Licensing Information
Ambion's pSilencer adeno 1.0-CMV System combines the advantages of adenoviral vectors with an efficient siRNA expression system. With pSilencer adeno, you can easily deliver an siRNA expressing construct into a variety of mammalian cells and organisms. The modified CMV promoter in the vector efficiently expresses hairpin siRNAs, which are effective at gene silencing through the RNAi pathway. The figure at right shows the silencing of GAPDH in HeLa cells after infection with pSilencer adeno 1.0-CMV engineered to express the corresponding siRNA. Protein levels were reduced ~90% compared to noninfected controls. The vector and promoter featured in the pSilencer adeno 1.0-CMV System were developed by Beverly Davidson and colleagues and have been used to silence GFP and endogenous ß-glucuronidase in HeLa cells and adult mice (1).
Efficient Delivery via Viral Infection
Adenoviruses are popular gene delivery vehicles because they efficiently transduce many different cell types, including terminally differentiated cells, and typically express high levels of the desired RNA. Infection is independent of cell cycle, so adenoviruses can be used to express RNA in both dividing and non-dividing cells. Integration of the adenoviral DNA into the host genome is rare, which means that there is little chance of insertional mutagenesis. Because of this feature and the fact that most recombinant adenoviruses elicit an immune response in animal systems, these viral vectors are appropriate only for transient RNA expression.
How to Use pSilencer adeno 1.0-CMV
Producing recombinant adenovirus that expresses a desired siRNA sequence is a relatively straightforward process. First an oligonucleotide insert encoding the desired siRNA is cloned into the provided, pre-linearized shuttle vector. This shuttle vector includes a modified CMV promoter for efficient expression of the siRNA sequence. The shuttle vector containing the desired sequence is then linearized and introduced into HEK 293 packaging cells (not included) along with the linearized adenovirus backbone (see schematic at right). Inside the HEK 293 cells, the shuttle vector and the adenoviral backbone recombine, and recombinant adenoviral particles are produced. These adenoviral particles are then harvested and used to infect cells or animals. Adenoviral particles can be stored for long periods of time, so that experiments can be performed long after adenovirus production.
The Complete Adenoviral Production System
The pSilencer adeno 1.0-CMV System includes everything needed to produce five preparations of recombinant adenovirus, except the specific siRNA encoding oligonucleotides to be cloned into the vector and the HEK 293 packaging cells (HEK 293 cells are available from several sources, including ATCC). The kit includes linearized Shuttle Vector 1.0-CMV (20 rxns), negative control shuttle vector that encodes a scrambled siRNA sequence, a positive control oligonucleotide insert that encodes an siRNA targeting GAPDH, and an adenoviral backbone that includes a LacZ sequence for ready monitoring of transfection efficiency. Also included are reagents for transfecting the HEK 293 cells with the shuttle and backbone vectors, forward and reverse sequencing primers to verify clones, and 5X Annealing Buffer for preparing the siRNA encoding oligonucleotides for ligation.
REFERENCES
1.Xia H, Mao Q, Paulson HL, and Davidson BL (2002) siRNA-mediated gene silencing in vitro and in vivo. Nat Biotech 20: 1006-1010.
The recombinant adenovirus produced with this kit are classified as a
Biosafety Level 2 hazard. We strongly recommend that you read the
information on safe handling of Biosafety Level 2 hazards in the following
CDC/NIH publication: Biosafety in Microbiological and Biomedical
Laboratories, 4th Edition May 1999.
This publication is available on the internet at this address:
http://bmbl.od.nih.gov.
It is also available for sale by the Superintendent of Documents, US
Government Printing Office (GPO), stock #017-040-00547-4.
Contact the GPO by telephone (7:30 AM to 4:30 PM EST) at 1-202-512-1800, by
fax at 1-202-512-2250, or write to: Superintendent of Documents, US GPO,
Washington D.C. 20402.
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