Three Boston University biomedical engineers have created a genetic dimmer switch that can be used to turn on, shut off, or partially activate a gene's function. Professor James Collins, Professor Charles Cantor and doctoral candidate Tara Deans invented the switch, which can be tuned to produce large or small quantities of protein, or none at all.
This switch helps advance the field of synthetic biology, which rests on the premise that complex biological systems can be built by arranging components or standard parts, as an electrician would to build an electric light switch. Much work in the field to date uses bacteria or yeast, but the Boston University team used more complex mammalian cells, from hamsters and mice. The switch has several new design features that extend possible applications into areas from basic research to gene therapy.
"There are a number of technologies available to regulate gene expression, but they each come with limitations," said Collins. "One of the central problems is you can't get a really tight 'off' state."
Even when genetic switches are turned off, a trickle of the protein that is meant to be repressed still gets made. Some genetic switches get around this by entirely snipping out a gene to stop production of a specific protein, but this approach is irreversible.
To overcome these challenges, "Tara came up with a design that really combined two different technologies to repress or shut down gene expression," Collins added. "We said, okay, we've got these two technologies, both that give a pretty good 'off,' why not try to combine them together to get a really clear and strong 'off,'" said Collins.
The first strategy, a repressor protein, sits on DNA like a roadblock, preventing any gene product -- messenger RNA (mRNA) -- from being made. If any mRNA gets past this repressor, the second technique, interfering RNA (RNAi) attaches to the functional mRNA, rendering it useless. The cell cannot turn it into protein.
"I was delighted to see that when the two systems are coupled, it is possible to completely turn a gene's function off," said Deans.
This switch is also reversible and tunable. By adding a chemical -- Isopropyl-â-thiogalactopyranoside -- the repressor components are blocked and the gene turns on again. The gene's activity can be tuned up or down by adjusting the amount of this chemical.
The researchers demonstrated the strength of their "off" switch by hooking it up to the gene for diphtheria toxin, then inserting it into cells. One molecule of diphtheria toxin can kill a cell, but with the genetic switch turned off, the cells survived for weeks. When the researchers flipped the switch, toxin production was triggered and the cell died.They also showcased the switch's capability for delicately tuning gene expression, by installing it alongside a gene that leads to apoptosis, programmed cell death, once a certain threshold concentration of the gene's product is reached. They gradually increased the gene's activity until they met and passed this threshold.
This tuning feature is important, said Deans, because "many diseases are not a result of missing a gene, but rather a result of too much or too little expression. With the ability to tune the level of gene expression in our switch, we could explore threshold responses and how these result in disease phenotypes."
The switch may also hold promise for therapeutic applications. "It gives a really nice regulator scheme for cell and gene therapy," said Collins. "I think in the coming decades we'll increasingly see these therapies being introduced as part of routine medical practice."
The research detailing their new switch, "A Tunable Genetic Switch Based on RNAi and Repressor Proteins for Regulating Gene Expression in Mammalian Cells," appears in the July 27 issue of Cell.
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