Even with purebred mouse lines dating back centuries, geneticists needed tools for tinkering with mouse genes. The most powerful of those tools emerged only in the mid-1980s. While working in the lab of Martin Evans at Cambridge University, England, Elizabeth Robertson (now a professor at Harvard University) and Allan Bradley (now director of the Sanger Centre in Cambridge, England) showed that researchers could pluck special cells out of an early mouse embryo—known as embryonic stem, or ES, cells—alter them genetically, and reimplant them. These altered cells then resulted in genetically manipulated offspring.

This first mutation in an ES cell line, Bradley says now, "signaled the emergence of a new age in mouse genetics." The trick became figuring out how to alter these cells in a predictable way.

Robertson and Bradley went on to demonstrate in 1986 that retroviruses could be used to create mutations in ES cells. Retroviruses are viruses that have the unique ability to insert their genetic code into the DNA of the cells they infect; thus, genetically modified versions of retroviruses can be used to smuggle desirable information—a new gene, for example—into a cell or to knock out an existing gene by interrupting its coding sequence.

These early mutations were random—researchers couldn't dictate the exact location of the change they wanted to make. Nevertheless, Robertson and Bradley managed to create a mutation in the mouse Hprt gene that was similar to the human gene mutation that causes a devastating disorder called Lesch-Nyhan syndrome. It was a sign of things to come.

In 1987, Mario Capecchi, an Italian-born HHMI researcher at the University of Utah, developed a way to create specifically targeted mutations in mice. Researchers could essentially insert or erase a gene of their own choosing in mouse ES cells, using a technique called homologous recombination, and then implant the engineered cells in female mice, which would produce litters of genetically altered offspring.

At times nothing seemed to happen when researchers knocked out a single gene; this was surprising, though useful, information. But in many other cases researchers made spectacular connections between mouse genes and human diseases. The era of the knockout mouse had begun.

Capecchi, for example, discovered that by systematically knocking out genes in a family called Hox (originally discovered in fruit flies and often considered master switches that control the formation of the body plan during development), he could produce mice with dramatic developmental defects. When the Utah team mutated two Hox genes simultaneously, for example, the forelimbs of mice failed to develop, resulting in animals whose paws grew directly out of their elbows.

In recent years, such tools have become ever more powerful. Bradley is in the process of knocking out mouse genes one by one, as part of an international effort to create a vast number of mutant mouse lines that will be made available to all researchers. An increasingly popular technology known as the cre/loxP system has made it possible to control the place and time that a gene is turned on or off in the mouse. Researchers can thus target their mutations to any organ they choose or to any particular time in development.

— Stephen S. Hall

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Mario Capecchi devised a powerful method of gene targeting that enabled researchers to produce "knockout" mice.

Photo: Paul Fetters

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