Knocking Down Genes for Fun and Function

Reprinted with permission from Nature PROTEIN SILENCING: Silencing of nuclear envelope proteins lamin A/C siRNA duplex (a,d) with GL2 luciferase siRNA duplex (nonspecific siRNA control) (b), and with buffer only (c). Staining with lamin A/C specific antibody (a,c); staining with NuMA-specific antibody (d). Data derived from the Science Watch/Hot Papers database and the Web of Science (ISI, Philadelphia) show that Hot Papers are cited 50 to 100 times more often than the average paper of the same type and age. S.M. Elbashir et al., "RNA interference is mediated by 21- and 22-nucleotide RNAs," Genes Devel, 15:188-200, Jan. 15, 2001. (Cited in 158 papers) S.M. Elbashir et al., "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells," Nature, 411:494-8, May 24, 2001. (Cited in 276 papers) Scientists once thought of eukaryotic RNA as solely active in transcription as messenger RNA (mRNA), and in translation as transfer RNA (tRNA). After Fire and colleagues first reported that double-stranded RNA (dsRNA) can interfere with gene expression,1 the conception of how gene transcription works was knocked on its head. Suddenly, these pieces of RNA, which appeared to be cellular detritus sliced out of mRNA, took on importance: They could have a role in gene suppression. Subsequent papers by many groups, including these two Hot Papers by Thomas Tuschl and colleagues,2,3 formerly of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, are clarifying how the naturally occurring method of RNA inhibition works and how engineered small inhibitory RNAs (siRNAs) can be introduced into a cell to knock down, or suppress, specific gene expression. Barely five years since the concept was first published, researchers are looking to use siRNAs clinically. The first paper, Tuschl explains, "is the formal proof that siRNA can do gene silencing." The second paper, he says, shows that siRNAs can direct gene silencing in mammalian cells without interference that kills the cells. CRUCIAL INFORMATION In a naturally occurring system, posttranscriptional gene silencing (PTGS) may be a means of protecting cells from transposons or viruses that can produce dsRNA.2 Within the cell, these dsRNAs are enzymatically cut into smaller RNA pieces, which then bind to homologous mRNA, preventing translation of certain gene products. RNA fragments longer than about 30 nucleotides may destroy cellular mRNA, resulting in cytotoxicity and apoptosis. But Tuschl's group, using a Drosophila lysate system, showed that RNAi--the suppression of gene expression by RNA fragments--occurs when the RNA fragments are 21- or 22-nucleotide duplexes of sense and antisense RNA.2 These fragments are broken down from the dsRNA by the Dicer enzyme.4 Tuschl and colleagues identified efficacious segment lengths for PTGS using synthesized siRNA artificially introduced into cells.2 Identifying the siRNA lengths that best suppressed gene expression in an in vitro Drosophila system was helpful in studies of invertebrate and plant systems, but methods for successfully using these extrinsic siRNAs in mammalian systems were elusive. Tuschl says that geneticists discovered the RNA silencing system, but "there was no biochemical system that showed [how] a specific long RNA is degraded" to siRNA. Brenda Bass, a Howard Hughes Medical Institute investigator at University of Utah, Salt Lake City, notes that "conventional methods of initiating RNAi with long double-stranded RNA do not work in most mammalian cells." The key to inhibition of mammalian gene expression was found in the shorter siRNA segments. Nearly two years ago, Tuschl's team reported3 on the successful use of extrinsic siRNAs in a mammalian cell culture system. "Once we showed how it worked in flies ... we could go into mammalian cells and see if we can silence a gene," says Tuschl. They could. "This paper provided the crucial information that allowed researchers to use RNA interference in mammalian cells," explains Bass, who wrote a commentary on the Nature paper.5 "This paper showed that ... an intermediate in the RNAi pathway [siRNA], could be successfully used to initiate RNAi in mammalian cells." In a recent paper,4 Tuschl wrote, "[siRNAs] are powerful sequence-specific reagents designed to suppress the expression of genes in cultured mammalian cells through a process known as [RNAi]." On his Web site, Tuschl said the group had "shown that dsRNA is processed to duplexes of 21-nt [nucleotide] small interfering RNAs, which guide sequence-specific degradation of the homologous mRNA." These siRNA duplexes then form complexes with proteins that have endonuclease activity. They "cleave target RNA in the middle of the region covered by the siRNA duplex," the Web site notes. "Delivery of siRNA duplexes into cultured mammalian cells is followed by degradation of targeted mRNAs, thus producing 'knock-down' cells with characteristic 'knock-down' phenotypes." Tuschl explains that any protein-encoding gene can be targeted by siRNAs, which can be introduced through a genetically engineered vector or via microinjection. Courtesy of Max Planck Institute for Biophysical Chemistry Göttingen Klaus Weber, Jens Harborth, Thomas Tuschl, Winfried Lendeckel, Sayda M. Elbashir and Abdullah Yalcin. A SIMPLE TECHNIQUE Finding the way to use this technique in mammalian cells was the next step. "There had been a lot of elegant work in plants, worms, and other animals to investigate the mechanisms of RNA interference, but the grail was being able to use it in mammalian cells," says David R. Engelke, professor of biological chemistry, University of Michigan, Ann Arbor. Engelke, who cited both Tuschl papers in his recent paper on siRNAs,6 explains the work's importance: "This not only would allow rapid pseudogenetics in mammals, including human cultured cells, but would present a number of obvious therapeutic possibilities." Now, through a relatively simple technique, siRNAs that can probe particular genetic sequences can be used in functional genomics studies. Rather than producing a full knockout organism that lacks a specific gene entirely, siRNAs bind to specific sites on mRNA and prevent protein synthesis (or gene expression). Thus, siRNAs are a powerful tool for studying gene function. Using homologous siRNAs to knock down mRNA's ability to produce protein effectively removes gene activity without removing or otherwise altering the gene. Engelke says that the paper on the mammalian system was greeted "with great enthusiasm" by the research community, but also with a bit of caution. He explains: "Previous results with antisense nucleic acids, ribozymes, and artificial RNA inhibitors ('aptamers') had not been as broadly applicable in vivo as had been hoped." Tuschl adds that a method to use siRNAs against infection awaits development: "If you want to cure a patient, you have to somehow get these siRNAs into every cell of that organism that has been infected." Nonetheless, researchers tried the technique, and it worked. "There was a spreading wave of people who were successful with siRNA after trying relatively few sites, creating a great buzz on the street for the method," notes Engelke. "There was a huge surge of enthusiasm from a broad range of scientists, whose experiments had been limited by the inability to selectively knock out single gene products in mammals and examine the consequences." Tuschl remarks that researchers working on human cell cultures, until now, have been unable to turn off genes. Now, using siRNAs, they can. He adds that since the group's work was published, "synthetic RNA is as important as synthetic DNA." RAPID PROGRESS The researchers, in light of their success, have since left Max Planck for their own biotech firm, Alnylam Pharmaceuticals, in Cambridge, Mass. (Tuschl, a company founder, is now at Rockefeller University in New York.) And they are sharing their successes. The unique illustration of a method in mammalian cell culture has evolved into a series of online instructions--The siRNA User Guide--that explains design and synthesis of siRNA duplexes. The guide lists: sources for synthesized siRNAs and reagents; companies offering siRNA synthesis or off-the-shelf siRNAs; instructions for obtaining a site license for certain knockdown experiments; and reagents and methods for cell transfection. Readers are advised when to expect to find a knockdown phenotype, and what to do if no knockdown phenotype is expressed. Also, the siRNA sequences the researchers used in the Nature paper3 are included. In the booklet, Tuschl and colleagues leave no doubt that replicating their work should be elementary: "Good luck, and we are sure you will be amazed how easy it will be to knock down your favorite mammalian gene." Myrna E. Watanabe is a freelance writer in Patterson, NY. References 1. A. Fire et al., "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans," Nature, 391:806-11, 1998. 2. S.M. Elbashir et al., "RNA interference is mediated by 21- and 22-nucleotide RNAs," Genes Devel, 15:188-200, Jan. 15, 2001. (Cited in 158 papers) 3. S.M. Elbashir et al., "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells," Nature, 411:494-8, May 24, 2001. (Cited in 276 papers) 4. T. Tuschl, "Expanding small RNA interference," Nat Biotechnol, 20:446-8, 2002. 5. B.L. Bass, "RNA interference: the short answer," Nature, 411:428-9, 2001. 6. C.P. Paul et al., "Effective expression of small interfering RNA in human cells," Nat Biotechnol, 20:505-8, 2002.

Knocking Down Genes for Fun and Function

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