New molecular switch for genes

Researchers design a regulator that can reversibly switch genes on and off and tune gene expression to a precise level

By | July 26, 2007

Researchers have created a molecular switch that can reversibly turn any mammalian gene on or off and control its level of expression. The results, published this week in Cell, provide a new level of precision in studying genes involved in biological processes and diseases, the authors say. The authors "promised a lot," with this research, said Perry Hackett of the University of Minnesota, who was not connected with the research. "And they lived up to their promise." Scientists have traditionally used three technologies to control gene expression, but all have limitations, explained James J. Collins of Boston University, who led the study. Genetic techniques to create "knockouts" are irreversible, making it difficult to study the function of genes at different points in development. Approaches that use small molecules such as tetracycline don't completely block expression of the target protein. RNA interference (RNAi), in which small RNAs block the function of messenger RNA, only partially block expression also, and often blocks not just the target gene but others with similar sequence. Collins and his colleagues took a synthetic biology approach, combining the main elements of these three techniques into a single gene regulation system. Although on its own, each only partially blocks a gene's expression, when combined they bring down expression to undetectable levels. When the switch is off, the system expresses a repressor protein that keeps the target gene turned off. A second brake is provided by a short hairpin RNA (shRNA), which blocks any mRNA that leaks through. The researchers designed the switch to be turned on by adding a chemical inducer. The inducer turns off production of the repressor protein, while turning on another repressor protein that blocks the shRNA gene. With both breaks off, the gene of interest is expressed and the mRNA translated. It took about three days to fully turn the switch on or off, but some effect was noticeable within hours. "Imagine having our switch control a gene that you want to see when it gets turned on later in development," Collins told The Scientist. "You want to be able to have it off during critical stages of development, and on at other stages. Present technologies don't allow you to keep it really tightly off without basically removing the gene." They tested the system in culture using several cell lines, and on several different genes. One test, which targeted the bacterial gene that expresses diptheria toxin (DTA), showed that expression was regulated extremely tightly: a single molecule of DTA can kill a cell, but cells in which DTA had not been turned on remained unaffected. Hackett noted that the key benefit of the switch was that expression of a target gene could be finely controlled by titrating the inducing molecule - expression can be tuned to a precise level, and reversibly be turned on and off. "Things are very reproducible in terms of levels of activity," Hackett said. Because of its modular nature, the switch can control any gene of interest, explained Collins. "For example, we can use tissue-specific promoters to create novel animal models." He also suggested that such a system could be used beyond the laboratory. The switch "might provide a fail-safe way" to deliver gene therapy, agreed Hackett. Linzhao Cheng of Johns Hopkins University in Baltimore, who was not a co-author of the study, wrote in an Email to The Scientist that an approach to regulate gene expression developed in his own lab showed "limitations months later in vigorous and long-term tests." As a result, he said, "I would like to see whether the new system is indeed better than other systems previously described." Josh P. Roberts Links within this article: T.L. Deans et al., "A tunable genetic switch based on RNAi and repressor proteins for regulating gene expression in mammalian cells," Cell, July 27, 2007. Perry Hackett James J. Collins L. A. Banaszynski et al., "A rapid, reversible, and tunable method to regulate protein function in living cells using synthetic small molecules," Cell, September 8, 2006. A. Katsnelson, "A Nuanced Knockout," The Scientist, July 1, 2007. J. Lucentini, "Is this life?," The Scientist, Jan. 1, 2006. A. Katsnelson, "An RNAi rogue's gallery," The Scientist, April 1, 2007. A. Fischer and M. Cavazzana-Calvo, "Whither gene therapy," The Scientist, Feb. 1, 2006. Linzhao Cheng B.Y. Zhou et. al., Inducible and reversible transgene expression in human stem cells after efficient and stable gene transfer," Stem Cells, March 2007.

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