RNAi: Silencing never sounded better - siRNA

It also offers the flexibility to shuttle hairpin inserts from one vector to another depending on the researcher's needs.
"There are different variations of plasmid vectors in terms of promoters and selection markers, but all of them work on the same principle," says Sujay Singh, a researcher with Imgenex Corporation in San Diego, California. Imgenex sells GeneSuppressor Construction Kits that use both plasmid and viral vectors to knock down any gene. In addition, the company sells kits containing prevalidated cloned shRNA inserts in GeneSuppressor vectors together with gene-specific antibodies. Similarly, Upstate, in Charlottesville, Virginia, sells predesigned shRNA mammalian plasmids for about 100 kinase genes. The company guarantees that their plasmids will silence a gene by more than 75%. Galapagos Genomics offers adenoviral shRNA reagents and guarantees a 75% silencing efficiency at the mRNA level.

Monitoring transfection efficiency with fluorescently labeled siRNA. (Courtesy of Upstate.)

Another advantage of vector systems is that they can be engineered for inducible expression. Invitrogen sells the BLOCK-iT inducible RNAi plasmid and lentivirus vectors that can be induced to express shRNA in the presence of tetracycline. Upstate is also developing a ligand-inducible plasmid expression vector, based on the synthetic ecdysone system, using technology licensed from RheoGene in Norristown, Pennsylvania.
Getting over delivery
Many researchers say that delivery is one of the biggest hurdles on the way to a successful RNAi experiment. Polyamine-based reagents or cationic lipid mixtures have been available for many years to transfect nucleic acids into cells. "But what is good for DNA and mRNA delivery is not ideal for delivering siRNA," says James E. Hagstrom, vice president of scientific operations at Mirus Corporation in Madison, Wisconsin.
One of the more popular siRNA-specific transfection reagents is Invitrogen's Lipofectamine 2000, but a slew of others are available. Mirus sells TransIT-TKO, and Upstate has recently released sIMPORTER. Novagen has amine- and lipid-based reagents in a single formulation called RiboJuice siRNA Transfection Reagent, which is meant to target a wide range of mammalian cell lines. Dharmacon will soon be releasing a whole range of transfection reagents suitable for different cells and experiments. "The efficiency of transfection may depend on cell type but also on the passage number and the confluency of the cells. The time and manner of formation of siRNA-liposome complexes are also critical," says Deines.
Transfection reagents, however, do not work in vivo. Viral vectors, such as adenovirus or lentivirus, carrying shRNAs inserts can be used to induce RNAi in different tissues of an animal. Mirus has developed an alternate mode of in vivo delivery using high-pressure tail-vein injection of siRNA in physiological solution, such as saline, to deliver siRNAs to highly vascularized mouse tissues such as the liver and muscle. The technique works with both naked siRNA and plasmid vectors. "Silencing is transient, but in some areas it lasts more than a week. You can get 30-60% reduction of gene expression in liver hepatocytes in vivo," says Hagstrom. Judy Lieberman's group at Harvard Medical School showed that intravenous injection of siRNAs targeting the gene Fas blocked the development of fulminant hepatitis in mice1, providing the first in vivo evidence that infusion of siRNAs can alleviate disease in an animal model.
According to Hagstrom, the delivery of naked siRNAs has the advantage that no other proteins are delivered or expressed. "Nucleic acids are not immunogenic," he says. But one of the limitations of the technology is that although the liver seems to be particularly receptive to exogenous RNA, it is difficult to target other tissues. Mirus recently obtained evidence of siRNA delivery to skeletal muscle.
Mirus is now focusing on identifying siRNAs that are more stable for in vivo work. "We have been able to significantly extend gene knockdown times by chemically modifying siRNAs. Normally the half-life of the knockdown effect in vivo is 2-4 days. With chemically modified siRNA, we can extend knockdown times from days to weeks," says Hagstrom.
Controls and validation
As with any experimental protocol, having proper controls is essential. "People are typically not well informed about what controls to use for their siRNA experiments," says Deines. "We invested a lot of time and have collaborated with Agilent Technologies [in Palo Alto, California] to develop an siRNA that does not have any effects on cells as determined using microarrays." Such inert siRNAs make suitable negative controls in an RNAi experiment; fluorescently labeled versions are also available to monitor the efficiency of transfections. In addition, many companies sell prevalidated siRNAs that target housekeeping genes to be used as positive controls.
Upstate sells recombinant proteins and antibodies that can be used to validate that siRNA-mediated gene silencing worked. In partnership with Dharmacon, Upstate also offers validated SMARTpool siRNA products as kits with corresponding antibodies. In a similar vein, Dharmacon and Genospectra, Inc. have joint forces to sell SMARTpool siRNA reagents along with QuantiGene bDNA assays?which measure the amount of messenger RNA produced by a target gene from crude cell lysates or tissue homogenates directly, without the need for PCR.
Experts suggest monitoring both mRNA and protein levels. "Sometimes researchers will get stable knockdown of mRNA but will not see this effect because they are looking at protein levels. But for proteins with a long half-life, you may not see levels decreasing," says Imgenex director of business development Lisa Stein.
One of the worries with RNAi experiments is that siRNAs might knock down or induce genes other than the intended target. For example, an siRNAs "may be perfectly complementary to a gene but also have some complementarity to another gene and may act to silence the gene at the protein level," says Carthew. Another concern is that using high concentrations of siRNAs may trigger an interferon or other response in cells.
Helmut H.G. van Es, director of research at Galapagos Genomics in Leiden, The Netherlands, says that he has not observed off-target effects in his work but "it is important to validate results with other independent siRNAs targeting the same gene and get the same readout." The ultimate test of specificity is to insert a gene construct with a point mutation that cannot be knocked down by the siRNA and show that it rescues the phenotype induced by the siRNA." You need to build confidence biologically and molecularly," says van Es. "It is just like classical genetics," says Zamore. "RNAi has changed the way in which mutations are created. Instead of mutating the DNA, the message is cleaved, but all the same caveats remain."
Libraries of siRNAs
Until now, genome-wide screens to identify genes involved in different cellular pathways were mostly limited to nonmammalian model organisms. The advent of RNAi has made feasible to conduct similar screens in mammalian cells. Two groups?one at Cold Spring Harbor Laboratory in New York and the other at The Netherlands Cancer Institute?created human shRNA libraries that target between 8,000 and 9,000 genes and used them to identify new genes involved in two distinct signaling pathways2, 3
Using a retrovirus-based siRNA library, the group led by Ren? Bernards in The Netherlands identified five new genes required for p53-dependent arrest of cell proliferation. To increase the speed of RNAi screening, the researchers took advantage of the fact that each shRNA vector harbors a unique sequence identifier (bar code). The abundance of each shRNA construct in a pool of constructs could be assessed by monitoring the relative levels of each bar code using a microarray. "How long have people been looking for genes in the p53 pathway, 20 years? With one screen we get five more," says Bernards.
According to Bernards, the key to success is having a good "readout" for the screen. van Es, who is using an arrayed adenovirus-based library to target the 5,000 or so genes representing the "druggable genome," agrees. "What is important is understanding a disease so that you can model it in vitro in a cellular system, preferably in human cells," says van Es. His group has used its library to identify novel drug targets for diseases such as osteoarthritis and rheumatoid arthritis.
"In the end there will be very little difference among libraries. They may differ in probe selection or vector choice, or they may target different gene sets, but the results will depend on the strength of the screens," says Bernards, whose group has conducted a dozen or so additional screens since the Nature publication. "In 6-10 months everyone will have all the tools to perform genome-wide screens [in human cells], but the strength of the screen rather than the strength of the library will give the advantage to one group over another."
Bernards' library is not yet available for distribution, although discussions with potential vendors are at an advanced stage. In the interim period CTR, the technology transfer arm of Cancer Research UK, has handled requests for access to the library. Hannon's library is being distributed by Open Biosystems in Huntsville, Alabama. MRC Geneservice, in the UK, recently acquired distribution rights for the United Kingdom. In addition, a number of companies have siRNA libraries targeted to hundreds of genes in specific families. Examples include the kinase libraries sold by Dharmacon and Ambion and Qiagen's druggable genome library.
The design and delivery of siRNAs have come a long way since Thomas Tuschl's group (at the time at the Max Planck Institute for Biophysical Chemistry in Gottingen, Germany) first demonstrated that siRNAs could knock down tar