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Nobel is a Knockout
POSTED 18 OCTOBER 2007

Nobel knockout in medicine: Round 2007
After 15 rounds (more like 30 years, actually), it's a knockout. The Nobel Prize for Medicine or Physiology went to Oliver Smithies, Mario Capecchi and Martin Evans for inventing a technique to precisely target and disable ("knock out") genes in mice, and then grow a strain of "knockout mice" that lacks the deleted gene.

Smiling man with wavy, silver hair smiles broadly at a mouse in his palmMario Capecchi of the University of Utah, with one of his favorite research subjects. Capecchi shared the 2007 Nobel Prize for developing a technique to de-activate ("knock out") genes in mice; the same technology can be used to insert or alter genes in mice. Photo by Tim Kelly, University of Utah

Operating largely in isolation from each other, this international trio opened the door to a vast number of basic studies in genetics, development, immunology, cancer and neurology. Courtesy of their efforts, more than 11,000 strains of mice now carry genetic alterations, and before long researchers will be able to buy mice off-the-shelf with an alteration in any given gene. For reasons that are not entirely clear, mice are far easier to knock-out than many other animals, although knockout rats and fruitflies also exist.

These knockout mice enhance the study of human disease -- and help satisfy the need to know what a particular gene does. In genetics research, mice matter, because their genes are so close to our own.

As the Nobel Committee said, "The toolbox of experimental genetic methods developed by Capecchi, Evans and Smithies, commonly called the knockout technology, has permitted scientists to determine the role of specific genes in development, physiology, and pathology. It has revolutionized life science and plays a key role in the development of medical therapy."

Biologists were quick to praise the research. "I cannot imagine doing the study of medicine or disease without gene targeting," says Waclaw Szybalski, a geneticist at the University of Wisconsin-Madison and a former colleague of Smithies. Says Szybalski, who cut his teeth in genetics before the structure of DNA was even known and was the first to alter the DNA sequence in a human chromosome, "With mice -- you cannot do this with a human -- you can take genes out one after another, and find out what is wrong. And you can study in that way the architecture of genetic material, find out how things work."

Two mice, shown alert with open eyes before a puff of air.  During puff, top mouse still has open eyes, bottom mouse's eyes are closeGene targeting can restore broken genes. The mouse on top has a broken Hoxb1 gene, which controls development of facial nerves, so it cannot wiggle its whiskers or retract its ears in response to a puff of air. Using gene targeting, a gene fragment was inserted In a similar mouse (bottom) to recreate an ancient gene that once directed the growth of facial nerves. This mouse reacted normally to a puff of air by blinking, wiggling its whiskers and retracting its ears. The experiment indicates that gene insertion can repair broken genes. Courtesy Petr Tvrdik, University of Utah

Better late than never?
"I think [the prize] was long overdue," says Rick Woychik, director of Jackson Laboratory in Maine. "I can't imagine an award that was more appropriate, based on the impact of the technology of Smithies, Evans and Capecchi, on the biomedical community." Woychik stresses that gene targeting can do more than just destroy a gene -- it can also create genes that operate only under specific conditions, or make an abnormal amount of protein.

Jackson Laboratory is both a biomedical research institute and a storehouse that sells mice with interesting genetics to researchers, which puts Woychik in a good position to grasp the significance of gene targeting. "The ability to do very specific engineering -- to not just knock out ... but also introduce subtle changes in the gene -- helps you get a more complete picture of the function of the gene. The ability to conditionally modify, to knock down or knock out, to do this in specific cell types, or at specific times of development ... the things you can do in a mouse with gene targeting are truly phenomenal."

By the early 1980s, scientists could randomly inactivate or insert genes into chromosomes, but they could not controllably change a particular gene. (Random insertion is still used in agricultural genetics, to make crops that resist herbicides, for example, but it's a blunt instrument that cannot be used to fix or inactivate specific genes.) Then, between 1982 and 1989, Capecchi, Smithies and Evans invented and combined elements of a precise targeting system that can make mice with essentially any genetic defect.

Splicing genes
Unfortunately, the story of the Nobel Prize for Medicine or Physiology, Round 2007, starts with a jargon-saturated process called "homologous recombination." This sequence of matching, cutting and rejoining occurs when chromosomes divide during the formation of sperm or eggs, and it is one of several ways that parental genes get mixed 'n matched.

circle drops down and joins with horizontal barsCells have enzymes that self-repair chromosomes, using the process of homologous combination. The Nobel winners took advantage of this process to precisely target genes. Notice how the "engineered construct" contains a section that matches parts of the target sequence. An enzyme cuts the target and the construct exchanges DNA with the target. A similar process could be used to delete all or part of a gene.

In 1982, Mario Capecchi, a Howard Hughes Medical Institute researcher at the University of Utah, showed that mammalian cells could, through homologous recombination, incorporate new DNA into a chromosome.

Capecchi wondered if homologous recombination could be used to change specific genes, and he was not the only one. At the University of Wisconsin-Madison, geneticist Oliver Smithies began trying to use homologous recombination to alter specific gene sequences at specific locations on the chromosomes.

Colleagues were dubious, and grant reviewers described their quest as too difficult to receive funding. But Smithies and Capecchi kept at it, and in 1985, Smithies published proof that he could target and change a specific gene. Smithies, who moved to the University of North Carolina in 1989, found that most of the cells that did adopt the new DNA took it up in the wrong place, so he had to devise a laborious method for extracting only the cells where the insertion was on target.

2 machines with cords connecting to small, clear, plastic boxes
During his years at University of Wisconsin-Madison, new Nobelist Oliver Smithies not only did the work that won the Nobel Prize, but also created other genetic-research devices, including this "gene zapper," which electrically jolts a cell to allow the insertion of genetic material. Smithies has referred to himself as an "inventor." Courtesy Bruce Winkler

Sweet Salt Lake
Out in Salt Lake City, Capecchi persisted, showing much the same drive to succeed that helped him on the streets of Italy during World War II. After the Nazis sent his mother to a concentration camp, he survived for years by begging and stealing.

To get DNA into the nucleus, Capecchi simply injected it, and then used antibiotics to isolate the successfully transformed cells. At the start of the experiment, the cells had a partial gene for antibiotic resistance. Successfully inserting the rest of the gene would give cells a working antibiotic-resistance gene, and they -- but no other cells -- could survive a dose of antibiotic.

The system worked, and like Smithies, Capecchi was able to target a genetic change. Capecchi discovered a two-step, "positive-negative" sorting mechanism to identify the transformed cells. The "positive" step increases the percentage of correct cells, and the "negative" step removes any remaining failures.

Once the deliberate engineering of mammal cells became possible, a new challenge arose: Could one make an animal containing only the new genetics?

Embryonic imagination
The solution came from Martin Evans, at Cardiff University (United Kingdom), who was working with embryonic stem (ES) cells. ES cells appear at the center of a very young embryo and produce daughter cells that eventually specialize into any cell of the body. In 1981, Evans reported the discovery of ES cells in mice, and by 1986 he proved that the alterations he'd made to the ES cells could be transmitted to reproductive cells, which could create a stable line of transgenic animals.

From there, it was a short step to use gene targeting to make animals carrying the targeted genes. First, alter ES cells. Second, place those embryonic stem cells in a mouse. Third, grow the mice and use conventional breeding to create mice containing only the altered genes. These are the knockout mice that have become so central to biology.

Nobel notes
In recognizing Evans's work with mouse embryonic stem cells as part of the award for gene targeting, the Nobel committee recognized almost in passing a critical scientific advance: the discovery of ES cells. In 1998, a report on human embryonic stem cells by James Thomson of the University of Wisconsin launched intensive research efforts to understand how ES cells develop into specialized cells, such as heart muscle cells or neurons. Custom-made cells derived from stem cells may some day substitute for diseased cells as part of "regenerative" medicine. Thomson's discovery aroused a stubborn politico-scientific wrangle in the United States, and federal funding restrictions have slowed research.

Curiously, last year's Nobel for Medicine went to a different method for analyzing the function of genes: RNA interference is a technique for blocking specific sequences of RNA. Because RNA "reads" DNA and becomes the template for proteins, blocking ("silencing") the RNA from a certain gene prevents that protein from being made, and thus can reveal a gene's function.

By 1989, the techniques of Capecchi, Smithies and Evans had already resulted in four strains of mice with different genetic alterations. The ability to do gene targeting in mice made the animal critical to genetics research, and for good reason, says Woychik. "In the vast majority of cases, if you study a human gene, there is a functional counterpart for that gene in the mouse. ... A lot of sequence variations in those genes contribute to the same type of diseases in humans as in mice." (Although some progress has been made in targeting rat genes, the process is far simpler in mice.)

Beyond the knockout
By now, thousands of knockout mice have been produced with gene targeting, and the scientific establishment has decided to complete the job using mass-production techniques to cut costs and make a library of mice (or mouse embryonic stem cells) that each lack one gene. Here's how the Knockout Mouse Project explains the focus on altering mouse genes:

"Mouse models have added to our understanding of human obesity, cancer, cardiovascular disease, diabetes, Parkinson's and Alzheimer's, to name just a few. The value of the mouse as a model organism is derived from the fact that the mouse has similar developmental, physiological, biochemical, and behavioral patterns to humans. It is worth noting that the similarities between human and mouse are supported at the genotypic level -- 99 percent of mouse genes have homologs [genes with similar structure] in humans."

While knockouts are used to find the function of an unknown gene, gene targeting can also:

correct a defective gene

introduce a completely new gene (a "knockin")

reduce, but not eliminate, the gene's activity (a "knockdown")

over-express the gene, so that several copies one particular same protein

introduce a bit of regulatory DNA to change when, where or how the gene functions

make a mutated protein instead of the proper one

introduce a defective human gene to make an "animal model" of human disease

Using this last approach, in 1992, Smithies and colleagues used gene targeting to transfer the gene for cystic fibrosis into mice, creating one of the first genetically engineered mouse models of a human disease (see #1 in the bibliography). "Cystic fibrosis mice have arrived!" was how the journal Human Molecular Genetics greeted the announcement in 1992.

There are plenty of other animal disease models. What have gene-targeted mice done for us lately?

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