PerspectivesAre you interested in submitting a Perspective Article? Be sure to read The Science Advisory Board's Editorial Guides for Perspective Articles. Click here. Special Topics Focus: RNAi Technology Tools and Techniques of RNA Interference by Jean-Claude Zenklusen, M.S., Ph.D. Disruptive (also known as repressive or interference) techniques have been very useful in the field of reverse genetics to help in the functional elucidation of newly discovered genes. This approach is based on the idea that by producing cells or whole organisms lacking the gene of interest, one can determine its function and co-effectors by studying the metabolic defects occurred on the test subjects. Although this approach has been successfully utilized in the creation of Knockout animals for several different genes, the technique has serious limitations:
Even if these approaches have helped in circumventing some of the problem aforementioned, they do have difficulties on their own (such as leakyness of the tet promoter, and cost of double transgenics in the cre-lox) that make them unusable for cell based assays. With the discovery of small interference RNA (siRNA), a new horizon has opened in the reverse genetics field using cell based experimental systems. This new technology allows researchers to transfect a small double stranded duplex of RNA homologous to any target gene and accelerate its degradation through the enzymes of the RNAi Silencing complex. This approach effectively negates the expression of the targeted gene at a fraction of the cost of Knock-out production, and more importantly in a way which does not allow the cells to adapt, mimicking more closely the pathological state of disease. siRNA can be used as a standard reagent in which double strand RNA duplexes are transiently transfected into the cell type to be studied, or as an engineered plasmid containing a hairpin loop structure that will give rise to the dsRNA duplexes in vivo after stable transfection. This last approach has the potential for producing transgenic animals that will produce the siRNA in vivo in certain tissues according to the promoter with which they are outfitted; effectively producing a regulated, conditional, knock-out organism. ****************************** Dr. Zenklusen has been a member of The Science Advisory Board since 1997. ****************************** To discuss this perspective or RNAi technology, visit the Discussion Forum. ### << Previous Next >> [ View All Perspectives ] |
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