RNAi Products

siIMPORTER siRNA TRANSFECTION REAGENT

siRNA/siAB ASSAY KITS

siIMPORTER DELIVERY OF siRNA

AdenoSilence Library

The AdenoSilence library was designed and optimized specifically for RNA interference (RNAi) mediated gene-silencing. RNAi is a sequence specific post-transcriptional gene knockdown event that has become the standard format for studying gene function and elucidating drug target discovery. The viruses of the AdenoSilence library mediate RNAi through the intracellular expression of a short hairpin RNA (shRNA) that is driven by the strong and ubiquitous U6 promoter. The shRNA is then processed into small interfering RNA (siRNA), which is ultimately responsible for the degradation of target messenger RNA (mRNA) that contains complementary sequences. Every gene in the library is targeted by three separate viruses, each harboring a unique shRNA sequence that is specific to that gene. The sequence of each shRNA was selected by a proprietary search algorithm that identified putative shRNA sequences that contained no homology to other sequences within the mammalian genome. Numerous other selection criteria deemed vital for RNAi mediated gene-silencing were also met.

The AdenoSilence library is based on the adenovirus serotype 5 genome. The viruses are produced by a vector system in which two essential viral genes have been deleted and are provided by the proprietary packaging cell line, PerC6/E2A (Michiels et al., 2002). The flanking sequences of both genes were replaced such that no sequence overlap with the wild type genome exists. This sequence modification eliminates the possibility of homologous recombination, thus all of the viruses produced are replication incompetent. The viruses are capable of infecting cells but they cannot replicate after infection. Thus, the AdenoSilence viruses do not induce an infected cell to manufacture additional viruses and therefore they exert a very limited disruption to normal cellular metabolism.

Adenoviral infection is dependent upon the interaction between the capsid fiber variant and distinct cellular receptors. The AdenoSilence viral library was constructed using the C01 fiber variant which binds to the human coxsackievirus and adenovirus receptor (hCAR) protein (Bergelson et al., 1997). The hCAR protein is commonly expressed in numerous mammalian cell types, allowing the AdenoSilence viruses to infect an extensive number of different host cells, including primary cells. This broad tropism provides an important advantage for adenoviral based delivery systems over conventional transfection technologies (such as those used for plasmids or siRNA synthetics), which are hampered by toxicity issues and poor efficiencies in many different cell types, especially primary cells. Adenoviruses not only infect a wide variety of cell lines, they also typically display a high transduction efficiency in the cells they do infect. Additionally, the viruses mediate long term gene knockdown (often greater than 10 days), allowing efficient and extended silencing of the gene of interest (Arts et al., 2003). Unlike synthetic and plasmid based assays, which typically demonstrate shorter, more transient gene-silencing effects, the extended duration of gene knockdown provided by AdenoSilence viruses affords the researcher an opportunity to perform phenotypic assays that require longer term gene silencing such as cellular differentiation, cellular proliferation, and apoptosis analysis.

Adenoviruses also provide many other advantageous attributes. They are capable of infecting both dividing and non-dividing cells. This is in contrast to alternative viruses which can only infect actively dividing cells hence limiting their usefulness. Adenoviruses do not undergo chromosomal insertion of their genetic material into the host cells genome following infection. Therefore, their shRNA expression is not affected by the location of insertion. Finally, the adenoviruses consistently produce high titers of virus from the PerC6/E2A cell line. The high titers obtained aid in experimental setup since only a small volume of material is typically needed for testing.

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Product Offering

• Druggable/Secreted
• Enzymes
• GPCRs
• poly(ADP-ribosyl)transferases
• methyltransferases
• choline acetyltransferases
• dehydrogenases
• deacetylases
• phospholipases
• proteases
• Ion Channels
• Kinases
• Nuclear Hormone Receptors
• Receptors
• Transporters

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pKD MAMMALIAN siRNA EXPRESSION PLASMIDS

siRNA – Mediated mRNA Degradation Pathway

Mammalian cells express endogenous, short genes using RNA polymerase III enzymes and promoters. Millipore’s Upstate plasmid-based siRNA constructs utilize this endogenous system to express short-hairpin RNA (shRNA) from pKD plasmids. The shRNA is ultimately processed by the cell to form a functional siRNA.

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Advantages of pkD Mammalian siRNA Expression Plasmids

• Pre-made and gene target-specific
• Extensively validated in cell culture through either quantitative, real-time PCR or Western blotting
• Expressed siRNA in vivo persists longer in the cell because as some is degraded, more is made to replace the loss
• The plasmids can be stably integrated into the host genome
• The sequence of the cloned siRNA is included at no additional charge
• When more is needed, an inexpensive plasmid prep will generate more

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Do you want a pKD for a gene target that we don't have?

Extensive Validation of pKD Mammalian siRNA Expression Plasmids in Cell Culture

Multiple Versions of Gene-Specific pKD siRNA Plasmids

For most of Millipore pKD siRNA plasmids, multiple versions are available for use as specificity controls. Each version is specific for the same gene but is designed against different regions of the target’s mRNA.

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pKD Series of Mammalian siRNA Expression Plasmids

Cloning into the pKD mammalian siRNA expression plasmid. In the pKD plasmid (cat. no.62-001), use the Smal site immediately downstream of the H1 promoter and any of the other sites to clone in the double stranded DNA oligos that will be expressed as a shRNA (short-hairpin RNA) which will get processed into a siRNA. By using the blunt Smal site, less non-specific sequence will be contained in the shRNA transcript. Make sure to include in the cloned DNA oligos a stretch of 5 thymidine residues that will serve as a transcription terminator. All of the pKD siRNA plasmids have a similar structure. A human H1 promoter drives the expression of a cloned sequence that when transcribed forms a short-hairpin RNA (shRNA). The transcription is terminated by a dT sequence, and the shRNA gets processed by the cell into an siRNA. pKD (cat. no. 62-001) is available to clone your own double stranded DNA oligos into, or you can purchase any of the validated, gene target specific pKD-siRNA plasmids.

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