Site Tools
Account Information
Guest Alerts | Access Rights | My Account | Sign In
In This Section
Related Information |
This Special Advertising Feature is brought to you by AAAS OPMS
Ten years ago, RNA interference was an interesting but arcane phenomenon that excited a small group of researchers working on Caenorhabditis elegans. At the same time, embryonic stem cells were a useful but cumbersome system for engineering targeted gene deletions in mice. Neither tool seemed to have much use beyond its narrowly defined specialty. What a difference a decade makes. By Alan Dove Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies. Today, RNA interference, or RNAi, is simultaneously one of the hottest techniques in molecular biology and a bold new frontier in the study of gene regulation. Stem cells, meanwhile, are the subjects of loud protests, presidential campaign discussions, and multibillion-dollar funding initiatives. Besides their simultaneous climbs to fame, RNAi and stem cells have something else in common: researchers are increasingly combining the two. Indeed, RNAi has quickly become an important tool for probing the nature of stem cells, as well as engineering "knockdowns" of animal genes. Stem cells modified with RNAi techniques may also enable the slowly developing field of tissue engineering to break into the clinic. Equipment and reagent manufacturers have been quick to cater to scientists using both stem cells and RNAi, offering everything from nucleic acids with special modifications for RNAi work to complete kits for popular types of stem cell studies. Both technologies are still in their infancy, though, and both present numerous pitfalls for researchers who combine them. Searching for Self-renewalWhen molecular biologists refer to RNAi, they often mean using synthetic small interfering RNA (siRNA) molecules to silence specific genes in a cell. As cultured cells take up the siRNA, the molecule binds complementary messenger RNA, targeting it for destruction by a highly conserved—and very efficient—pathway. In a mouse embryonic stem cell line, this enables investigators to generate targeted disruptions of nearly any gene product much faster than with traditional recombinant DNA techniques. "The biggest advantage of using RNAi, not only in embryonic stem cells but also in other cells, is the speed at which you can deplete the protein product," says Frank Buchholz, a group leader at the Max Planck Institute of Molecular Cell Biology and Genetics in Dresden, Germany. The catch is that siRNA-based gene silencing is often not as precise or thorough as a traditional gene deletion. Rather than targeting a particular genetic locus, an siRNA floats freely in the cell, often binding not only its primary target, but other messenger RNAs as well. These off-target effects often produce spurious results. In addition, an siRNA is unlikely to shut down its primary target completely. "With RNAi technology, it's always a knockdown, it's probably never really a 100 percent knockout, so there may be differences in phenotypes that one may observe," says Buchholz. That drawback can be an advantage for some studies. Many gene products exhibit dose-dependent effects, and Buchholz says the ability to generate different degrees of knockdowns with siRNA has helped researchers study that phenomenon. Those hoping to use siRNA as a shortcut to making a complete knockout animal, however, need to interpret their results cautiously. Nonetheless, Buchholz and his colleagues have already used siRNA for an initial survey of the genes involved in maintaining "stemness," or stem cells' ability to self-renew and form all tissue types. "RNAi of course offers an ideal technique to find the molecules or to find the genes that are involved in this differentiation process," says Buchholz. Taking advantage of the speed of siRNA compared to traditional gene knockouts, the researchers knocked down each mouse gene in the genome. They then monitored the cells' subsequent expression of Oct-4, a marker for the stem cell state. The result was a list of candidate genes involved in maintaining self-renewal. Despite combining two relatively new technologies, the experiment was surprisingly straightforward. "Of course working with stem cells requires certain skills, because you always have to treat them nicely," says Buchholz. But he adds that "we actually were able to very rapidly establish a transfection protocol, which established very good knockdown levels in mouse embryonic stem cells by lipofection." Indeed, stem cells seem particularly suited for siRNA-based studies. "Stem cells are fairly amenable to lipid-mediated delivery. As the cell becomes more differentiated, it becomes harder to transfect," says Devin Leake, director of research and development for the life science research group at Thermo Fisher Scientific in Milford, Massachusetts. It's the NetworkThings get trickier when researchers try to probe the cells' own RNAi system, in which endogenous microRNA, or miRNA, molecules silence specific gene targets. In recent years, the miRNA system has emerged as a fundamental and evolutionarily conserved component of gene regulation, at least as important as transcriptional silencing. Each endogenous miRNA can regulate multiple genes. The silencing pattern often targets multiple components of a single metabolic pathway, raising hopes that miRNAs will both illuminate new biology and point the way to promising new drugs. In stem cells, miRNA-mediated gene regulation is clearly one of the major mechanisms controlling differentiation, but scientists have only recently gained access to the tools to probe this system. "Although miRNAs have been known for many years, tools to manipulate miRNAs in cells have been around only for the last couple of years," says Eric Lader, director of research and development at Qiagen in Germantown, Maryland. The first step of a typical miRNA experiment—exposing cells to a particular set of conditions and then isolating their expressed miRNAs—is usually the easiest. Because miRNAs are just short RNA molecules, researchers can simply modify traditional RNA isolation methods or, for even greater convenience, buy an appropriate kit. "Qiagen has adapted our RNA isolation techniques to quantitatively recover small RNAs in the total RNA prep, and to also allow further enrichment," says Lader. Qiagen is just one of many kit makers that offer reagents for miRNA isolation. Characterizing and quantifying the isolated miRNAs can be more challenging, but several companies offer validated assays for all known miRNAs in popular model systems. Thermo Fisher, for example, offers a microarray for profiling all of the miRNAs in human cells, which could be particularly useful for stem cell researchers. "With this microarray, you can detect the endogenous levels of miRNAs in an undifferentiated cell or a differentiated cell, and look at the differences between those two," says Leake. Not to be outdone, Qiagen targets the same market with a completely different technique: a quantitative miRNA RT-PCR kit. After identifying some miRNAs that might regulate a particular process, such as stem cell differentiation, researchers typically try to perturb those miRNAs to confirm their functions. Often, that brings them back to siRNAs, which can be custom-built to either inhibit or mimic specific miRNAs. Unfortunately, the process is not as simple as noting the miRNA sequence and targeting it with a complementary siRNA. Leake explains that to mimic or inhibit an endogenous miRNA, the artificial siRNA must be highly specific, capable of directing its target to the cell's RNA degradation machinery, and able to survive in the cell without being broken down itself. The final challenge is that, even if investigators generate a perfect mimic or inhibitor for a given miRNA, the results can be difficult to interpret. "The difficulty is figuring out what's going on, because a single microRNA can affect the expression of any number of genes, and sometimes it exerts that effect coordinately with other microRNAs," says Lader. Target PracticeBecause of the miRNA system's enormous complexity, researchers inevitably turn to computers for help dissecting these tangled regulatory networks. In theory, one should be able to predict any miRNA's targets given nothing but the miRNA's sequence and the genome sequence of the organism. In practice, bioinformatics researchers are still struggling with the problem. "Target prediction is one of the most unresolved issues in the field of miRNAs," says Søren Møller, chief scientific officer at Exiqon in Vedbaek, Denmark. While Exiqon focuses on improving the chemical specificity of synthetic siRNAs, Møller has also followed the problem of miRNA target prediction closely. "You can analyze miRNAs in a lot of different ways, but the next step, moving from miRNA differential expression to what signaling pathways are affected, is a key focus of many of our customers' research," he says. The good news about miRNA target prediction is that there are numerous computer algorithms available to tackle the problem, and because most of them have been developed by basic researchers in academic labs, they are generally free, with pub-lished source code. The bad news is that the different algorithms often disagree with each other. "We advise [researchers] to use as many different target prediction algorithms as are out there, and look at the output and correlate that to the biology that they are familiar with," says Møller. In stem cell research, where both the basic biology and the functions of individual miRNAs are unclear, researchers should be especially skeptical of the computer's predictions. Weaknesses in the software stem mainly from gaps in scientists' current understanding of the miRNAs themselves. Some algorithms, for example, require perfect complementarity between specific miRNA bases and a potential target messenger RNA, while others allow more room for noncanonical targets. The former strategy can miss targets that are biologically important, while the latter can yield spurious leads. As molecular biologists develop a clearer understanding of the underlying rules of miRNA targeting, bioinformaticians will be able to find a better balance in the algorithms. A simpler version of the target prediction problem comes up when investigators design artificial siRNAs, and most companies offering siRNA-production services now provide free access to their proprietary siRNA prediction algorithms online. Typically, these programs take several sequence parameters of a target messenger RNA and build an siRNA or a set of siRNAs that should bind it. While relatively basic siRNA algorithms are adequate for many uses, they still have room to improve. For example, using a mathematical technique called support vector machines (SVMs), software developers in Applied Biosystems' Ambion division in Austin, Texas, recently released a new application, which they claim produces siRNAs with substantially better efficacy. "I think one of the important differences is that we do take into account the local folding structure of the target messenger RNA, as well as looking at the sequence parameters of siRNAs themselves," says Kathy Latham director of the Ambion RNAi product line. The algorithm itself is proprietary, but researchers can access it for free through the company's website. Better Binding through ChemistryAmbion's new SVM-based algorithm is aimed at addressing an underappre-ciated problem in siRNA research: inconsistent silencing. While the field has focused extensively on reducing off-target effects, siRNAs can also confuse experimenters by failing to silence their primary targets. "A really good siRNA might give you 90–95 percent silencing, but an okay siRNA might only give you 80 percent silencing," says Latham. That difference may be irrelevant for some experiments, but in differentiating stem cells, many regulatory gene products work like switches, moving the developmental process forward at a particular threshold of gene expression. For these genes, a partially active siRNA may be the same as no siRNA at all. "Differences in phenotypes are often attributed to off-target effects; however, differences in phenotypes can also be due to inconsistent silencing, which is why we combined chemical modifications to improve specificity with the new algorithmic approaches," says Ambion's Latham. The specific siRNA chemical modification Ambion is using is the Locked Nucleic Acid design, which the company licensed recently from Exiqon. While Ambion now markets Locked Nucleic Acid–based siRNAs, Exiqon still develops and sells a variety of other products that use the same chemistry. "Based on our technology, we have developed a set of detection techniques for analysis of miRNAs in cells, but also some functional products that can down-regulate or knock down specific miRNAs in cells," says Exiqon's Møller. For miRNA detection, Exiqon offers different kits based on quantitative PCR, microarrays, and Northern blots, and some of these may offer distinct advantages for stem cell researchers. Investigators looking at later stages of development may find Locked Nucleic Acids particularly attractive as probes for miRNA expression in tissues. Regular DNA or RNA oligonucleotides cannot detect miRNAs by in situ hybridization, but the more specific Locked Nucleic Acid oligonucleotides can. "A tissue is not a homogeneous set of cells. It's very diverse, and we've seen when we look in situ that different cell types express different miRNAs," says Møller. Exiqon and Ambion are not the only companies offering chemically modified siRNAs, and investigators should shop around for the specific characteristics they need. Those hoping for easy delivery of siRNAs to target cells may find Thermo Fisher's new Accell siRNAs particularly useful. "Accell siRNAs are chemically modified to promote cellular uptake, without any kind of formulation, any kind of instrumentation," says Leake. Rather than encapsulating siRNAs in lipid-based delivery compounds, researchers simply add their custom-built siRNAs to a proprietary cell culture medium, then bathe the cells in it. Regardless of the specific techniques they use, scientists combining stem cells with RNAi have good reason to be hopeful. Looking ahead to the next few years, Buchholz sees both techniques yielding major breakthroughs. "In the lab, I think we will learn a lot more about stem cells and the pathways that are driving [development]," he says, adding that "this is a prerequisite to really move the knowledge that we gain there closer to the clinic."
Alan Dove is a science writer and editor based in Massachusetts. DOI: 10.1126/science.opms.p0800026
Gene SilencingThermo Scientific Dharmacon Accell siRNA is a novel form of short-interfering RNA (siRNA) that is absorbed directly by cells without the use of conventional delivery methods such as transfection reagents, viruses, or electroporation. It effectively silences genes in all cell types tested to date. Previous technologies for delivering siRNA into difficult cell types, such as primary cells, suspension cells, stem cells, and neurons, were often ineffective and resulted in high levels of cell death. Dharmacon Accell siRNA has been able to penetrate every cell type tested, without the need for delivery reagents that can skew experimental results. The siRNA is simply mixed with the optimized Accell delivery medium then added to cultured cells. The easy, two-step process avoids the experimental variability caused by toxicity and off-target effects of conventional siRNA delivery methods.Thermo Fisher Automated Stem Cell CultureVelocity11 has released a data sheet that describes a high throughput, automated stem cell culture and screening system using its Filtered Plus Biocel platform. The automated platform was developed to use stem cells as the basis of high throughput assays and drug discovery programs to increase throughput and find more promising lead compounds. The system maintains a temperature of 37°C inside the Biocel, with air filtered to a class 100 specification, creating an optimized environment for plate-based cell maintenance and cell-based assays. With a novel method for controlling air velocity and ultralow penetration air filters, the system achieves increased turnover in filtered air, reducing the number of foreign particles and cell contamination. The automated procedure involves seeding plates from a bulk culture, adding compounds to cell plates, and transferring supernatant or cell lysate to an assay plate.Velocity11 Synthetic miRNA Mimics and InhibitorsNew synthetic microRNAs (miRNAs) from Qiagen, including the miScript miRNA Mimics and the miScript miRNA Inhibitors, are ready-to-transfect to carry out gene regulation and cellular pathway analysis for applications from in vitro to animal models. These molecules are based on all known human, mouse, and rat miRNAs in the latest version of miRBase, the Sanger Institute’s database for miRNA. These mimics and inhibitors are available in flexible formats, from single tubes to 96-well or 384-well plates. They are available in scales of 1 nmol and 5 nmol for in vitro use and 20 nmol for animal studies.Qiagen RNAi VectorsTwo new vectors are available from Invitrogen for RNA interference (RNAi) research. The Block-iT HiPerform Lentiviral PolII miR RNAi Expression System with Emerald Green Fluorescent Protein offers the ability to deliver to nondividing, primary, and hard-to-transfect cells with lentiviral technology coupled to multisite technology for promoter flexibility, making it suitable for difficult applications, including in vivo RNAi. It contains a messenger RNA stabilizing sequence and a nuclear import sequence that generate up to five-fold higher virus titers and fluorescent protein expression than previous versions. The Block-iT Inducible PolII miR RNAi Expression System makes use of a tetracycline-inducible promoter that allows researchers to knock down even genes that are essential to the function of a cell.Invitrogen Media Preparation SystemThe Mediajet vario is a versatile petri dish filler that can be used to fill 35 mm, 55/60 mm, and 90/100 mm dishes. With a compact footprint of only 70 cm by 70 cm, it fits conveniently on the laboratory bench and can fill 900 petri dishes per hour. It features walk-away automation with novel feed-in and stack-out technology. When working with 35-mm petri dishes, it can perform automated production of thousands of agar plates on which to cultivate the nematode C. elegans. This nematode is a popular geneticists’ tool because the simplicity, transparency, and speed of its biological functions facilitate studying genes and their functions.Integra Biosciences
Note: Readers can find out more about the companies and organizations listed by accessing their sites on the World Wide Web (WWW). If the listed organization does not have a site on the WWW or if it is under construction, we have substituted its main telephone number. Every effort has been made to ensure the accuracy of this information. Inclusion of companies in this article does not indicate endorsement by either AAAS or Science, nor is it meant to imply that their products or services are superior to those of other companies.
|
|||||||||||||
For PDF
version
Science. ISSN 0036-8075 (print), 1095-9203 (online)
Magazine | News | Signaling | Careers | Multimedia | Collections | Help | Site Map | RSS
Subscribe | Feedback | Privacy / Legal | About Us | Advertise With Us | Contact Us
© 2008 American Association for the Advancement of Science. All Rights Reserved.
AAAS is a partner of HINARI, AGORA, PatientInform, CrossRef, and COUNTER.
You have reached the bottom of the page. Back to top