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Delivering siRNA Using Adenoviral Vectors

Efficiently deliver an siRNA expression construct into a variety of mammalian cells and organisms

Ideal for many primary and other difficult to transfect cells

Modified CMV promoter efficiently expresses hairpin siRNAs

Positive Control Oligonucleotide Insert and Negative Control Shuttle Vector included

Efficient delivery of siRNA or an siRNA expression construct into cells represents a critical step in most RNA interference (RNAi) based gene silencing experiments. While traditional lipid and amine based reagents work well for transfecting some cultured cells, they can be inefficient for transfecting many primary cells, neural cells, etc. Viral delivery is one way to overcome this limitation. In addition, viral vectors permit the delivery of siRNA expressing constructs into a variety of animal species.

Adenoviruses are popular gene delivery vehicles because they efficiently transduce many different cell types, including hard-to-transfect terminally differentiated cells. Also, adenovirus gene delivery typically results in higher levels of RNA expression. 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 ani mal systems, these viral vectors are appropriate only for transient RNA expression but are well suited for gene-therapy.

Ambion's new pSilencer adeno 1.0-CMV System combines the advantages of adenoviral vectors with an efficient siRNA expression system (see A Bit About Adenoviral Biology, at right). With pSilencer adeno, you can easily deliver an siRNA expression construct into a variety of mammalian cells and organisms. The modified CMV promoter in the vector efficiently expresses hairpin siRNAs, which elicit gene silencing through the RNAi pathway. Figures 1 and 2 show silencing of GAPDH and GFP in HeLa cells after infection with pSilencer adeno 1.0-CMV engineered to express the corresponding siRNAs. In both examples, 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 at the University of Iowa. This vector has been used by that lab to silence a number of genes in HeLa cells and adult mice (1).

Figure 1. Silencing of GAPDH Induced by pSilencer adeno. HeLa cells were infected at an MOI of 80 with adenovirus derived from the pSilencer adeno 1.0-CMV System. The virus was designed to express a GAPDH siRNA or a scrambled negative control siRNA. The media was changed 4 hours after infection and cells were analyzed after 72 hours. Immunofluorescence was performed using mouse anti-GAPDH antibody. Green: GAPDH protein. Blue: DAPI stained nuclei.

Figure 2. Silencing of GFP with pSilencer adeno. HeLa cells were infected (MOI = 160) with recombinant adenovirus derived from pSilencer adeno 1.0-CMV engineered to express an siRNA targeting GFP. Cells were analyzed 48 hours after infection by fluorescence microscopy.

How to Use pSilencer adeno 1.0-CMV
First an oligonucleotide insert encoding the desired siRNA is cloned into the pre-linearized shuttle vector (provided), which 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 (Figure 3). Inside the HEK 293 cells, the shuttle vector and the adenoviral backbone plasmid recombine to produce an adenoviral genome including the siRNA expression cassette. The adenoviral genome is packaged into a protective coat and released into the culture medium. These adenoviral particles are then harvested and used to infect cells or animals. Adenoviral particles can be stored for long periods of time, so experiments can be performed long after adenovirus production.

Figure 3. Schematic of pSilencer adeno Recombination and Packaging in HEK 293 Cells. The shuttle vector and backbone plasmid are linearized and then transfected into HEK 293 cells. Inside the cell, the plasmids recombine and the recombined genome is replicated and packaged into adenoviral particles. mu=map units.

The Complete Adenoviral Production System
The pSilencer adeno 1.0-CMV System includes everything needed to produce f ive preparations of recombinant adenovirus, except the siRNA template oligonucleotide 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 the 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 Vector and Backbone, forward and reverse sequencing primers to verify clones, and 5X Annealing Buffer for preparing the siRNA-encoding oligonucleotides for ligation.

The use of these materials is permitted for research purposes only. Any other use requires a license from the University of Iowa Research Foundation, 214 Technology Innovation Center, Iowa City, IA 52242.

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Ordering Information
Cat# Product Name Size 5790 pSilencer adeno 1.0-CMV System 5 virus preparations


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