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A Faster Knockout
by Cassandra Willyard
With a virus, a needle, and an ultrasound machine, researchers have drastically cut the time it takes to disable a gene in mice.
There had to be a faster way to silence genes in skin stem cells. Elaine Fuchs managed to do it, carving years off the process, by using a well-timed injection of lentivirus—loaded with an RNA hairpin—into a mouse amniotic sac.
Elaine Fuchs was disappointed with the slow pace of conventional mouse genetics. To understand how skin stem cells develop and renew, she and her colleagues had been engineering strains of mice in which they could selectively turn off one or more genes. But the process, which requires breeding several generations of mice, was taking years.
Despite more than two decades of effort, Fuchs, an HHMI investigator at Rockefeller University, and her team managed to create only 30 engineered mouse strains—a small fraction of the genes they’d like to study. When a stem cell becomes active, for example, changes occur in the expression of roughly 200 genes. “There are still a myriad of genes up- or down-regulated whose role in stem cell biology is unknown,” says Fuchs. Making mutant mice to study all of them could take a lifetime.
Fuchs wanted a faster way. And last year, after several frustrating failed attempts, she found one. The technique bypasses breeding altogether. Using a virus and a tiny needle, she and her colleagues can now do in a few weeks what used to take a year or more. “It has opened up doors for us,” she says. “We can analyze genetic pathways [in mice] in ways previously possible only in flies and worms.”
The discovery has been years in the making. About a decade ago, scientists figured out how to alter lentiviruses—round, spiky viruses like HIV that cause chronic infection—to carry small, hairpin-shaped strands of RNA into mammalian cells. Scientists can engineer these RNA strands to block expression of particular genes, a technique called RNA interference. Fuchs thought the viruses might be a quick way to block gene expression in skin stem cells.
The technique worked well in skin cells growing in culture. But in an animal, skin tissue is made up of many layers. “When applied to the skin’s surface, the lentivirus went in, but only into the very top layer of dead cells,” Fuchs says. No matter what she and her colleagues tried—roughing up the skin, injecting the virus with a tattoo needle—they couldn’t get the lentivirus to go where they wanted: into the stem cells.
Then, around 2008, the team—Fuchs, postdoc Slobodan Beronja, postdoc Scott Williams, and graduate student Geulah Livshits—had a breakthrough idea. Why not try to infect mouse embryos rather than adult mice? Early in development, the embryos are covered by skin that is just a single cell layer thick. As development proceeds, these early skin stem cells generate the stratified epidermis and its hair follicles. If the researchers could inject the lentivirus into the amniotic sac, they speculated that the virus might infect this early cell layer and then pass on its genetic cargo to all those cells’ progeny. If so, the team would be able to selectively block expression of any gene in the skin in just a few days.
First, the team anesthetized a mother mouse. Then they carefully made an incision to access the embryos. The team relied on ultrasound to guide a glass needle—thinner than a human hair—into the pea-sized amniotic sacs. Once the needle was in place, they injected the virus. The virus dispersed throughout the amniotic fluid, infecting only the cells it touched—those covering the embryo’s surface.
The team had to time it just right, injecting the virus when the embryos were nine-and-a-half days old. Before then, the virus may infect cells that move inward to form the central nervous system. Wait too long, however, and the embryo develops a temporary protective covering, so the virus can’t reach the skin stem cells.
To test their technique, the researchers loaded the lentivirus with a gene that contained the blueprints for a fluorescent protein. After the pups were born, the team put them under a fluorescence microscope. Some of the pups’ skin glowed. The infection had worked. “That was really, really exciting,” Livshits says. And the effect was lasting—the mice still expressed the fluorescent protein as adults. The researchers also found that when they injected more virus, a larger proportion of skin cells became infected.
Image: Fuchs lab