Albert Lasker
Basic Medical Research Award

The release of the human genome sequence in draft form makes this a landmark year in the history of biology. Now we know that we have 30,000 or so genes (or is it 50,000?). We are now faced with several important questions, which include: First, what are the functions of these genes and the proteins that they code for? And, second, how can we use this information to improve human health?

Until the ability to knock out genes in the mouse was developed, determining the function of human genes seemed largely out of reach, tantalizingly so. For example, we might know of a human protein that is found only in certain cells of the brain and suspect what it might do, but how can we find out? Or, we might know of a gene in the fruit fly that is necessary for its development and see that humans have a very similar gene. Does it perform a similar function in humans? A powerful way to link a gene to function is to study the behavior of a mutant that lacks that gene and then see what the mutant can and cannot do. It's somewhat like disabling an automobile by removing one part and then inferring the function of the part that was removed. But we can't knock out genes in a human, so how can such mutants be produced?

The mighty mouse has come to the rescue. Its genes are typically 95 percent identical in sequence to ours, and we share the vast majority of our genes with the mouse. Despite the obvious differences between human and mouse in morphology and in some physiological processes, these differences are greatly outweighed by our similarities: they have kidneys and brains like ours; they have an immune system and develop a lot like humans; and they get diseases such as cancer and others that affect their cardiovascular and nervous systems like us. In some respects, mice are "pocket-sized humans." The bottom line is that the mouse provides the opportunity, dreamed about for decades, to make the link between a mammalian gene and its function. How is this done?

Building on more than one hundred years of genetic and embryological studies of the mouse, Mario Capecchi, Martin Evans, and Oliver Smithies have created a magic wand by which it is possible to modify any mouse gene with exquisite precision—to completely delete it or to produce a specifically altered form of the gene.

The same technology also makes it possible to go the other direction—instead of knocking out a mouse gene, it's possible to restore function to a gene that is defective.

Let's now look at the process by which a mouse knockout is constructed.

A key piece of starting material is a mouse gene that's already been cloned: it might be a mouse gene corresponding to a human gene or a mouse gene corresponding to a fruit fly or nematode gene. The goal is to construct a mouse that lacks this gene. The second key piece of starting material is a special mouse cell line where the gene is going to be knocked out.

There are three steps for constructing a mouse knockout. In the first, a cloned gene is manipulated in a test tube to delete all or part of the gene. This is routine molecular biology. In step two, the mutated DNA is introduced into special mouse cells, where the mutated DNA replaces a normal gene copy in the chromosome. The crucial aspect of this process is that the mutant gene has to find the related sequences in the chromosome, so-called homologous DNA sequences, and then undergo recombination to switch places with the good gene. The ability of the introduced DNA to find the homologous DNA sequences is called "gene targeting." There was no evidence for gene targeting in animal cells growing in culture and great doubt about whether this could be done. This is where Capecchi and Smithies made their most important contributions. In the third step, the cells with the targeted, inactivated gene are grown into a mouse that has this inactivated gene. It was Martin Evans who isolated the cell lines that made this possible and showed that genetic changes introduced into these cells in culture could be transmitted through the germ line and into mutant, progeny mice.

Let's now look at our awardees.

Verona, Italy has given us not only Romeo and Juliet, but Mario Capecchi. His early days as a child included living in orphanages and on the street in war-torn Italy from 4–9 years of age, then growing up in a nurturing Quaker environment in Pennsylvania. I refer interested people to articles that are available on the Internet. Capecchi did his graduate work at Harvard with Jim Watson and was enormously productive, making textbook discoveries on molecular mechanisms underlying protein synthesis. This was a golden age of molecular biology. Mario learned his lessons well, and when he established his own laboratory at the University of Utah in 1973, he sought to bring molecular genetics to animal cells growing in culture and to learn how to manipulate the genes of these cells. This led him to undertake a series of studies beginning in 1977 that demonstrated gene targeting in animal cells and culminated in the construction of one of the first knockout mice in 1989. His first indications of homologous recombination in animal cells were published in 1982 and fueled a series of logical and remarkable studies that provide the standard methods for knocking out mouse genes.

Oliver Smithies was trained as a biochemist, but throughout his scientific career, homologous recombination kept on cropping up, and he came to think about how it could be used to fix defective genes. Smithies was born in Halifax, England and raised in the United Kingdom. After studying at Oxford University, he came to the University of Wisconsin for postdoctoral studies in 1951 and was on the faculty there for 28 years, from 1960 to 1988. He is presently at the University of North Carolina at Chapel Hill, and may well have flown here in his own little plane to attend this luncheon. After important early contributions springing from his development of a method for fractionating proteins, he became intrigued by the structure and evolution of mammalian genes, which meant that he became involved in cloning these genes.

In the early 1980s, Smithies began to wonder whether homologous recombination—gene targeting—could be carried out experimentally to correct a defective gene, for example, a mutant globin gene. For this type of genetic correction to occur, exogenously introduced DNA would have to target to the homologous chromosomal DNA sequence and recombine with it. But was this possible? No one had demonstrated gene targeting in animal cells.

In 1985 Smithies and colleagues demonstrated that they could introduce a DNA segment containing part of the globin gene into cells and then find cells in which this DNA segment had targeted to the chromsomal globin gene. This was a tour-de-force of sophisticated molecular genetics. His strategy was completely different from that used by Capecchi and though laborious, the demonstration of targeting was unequivocal.

These studies from the Capecchi and Smithies laboratories provided one of the essential ingredients for constructing gene knockouts in mice, the ability to target genes in cultured animal cells. The crucial next step was to take mouse cell lines modified in this manner and produce mice from them.

The history of mammalian embryology is intellectually rich and filled with great practical applications. It was nurtured by the agricultural industry, among others, and involved important work with rabbits and mice. The UK can lay claim to many important contributions in this area, and thus Martin Evans is part of a distinguished tradition. Evans was born in the UK and graduated from Cambridge in 1963. He then went to University College London, where he studied vertebrate development using frogs. After working with a certain type of cancer cell line that could differentiate in cell culture and be used to generate whole mice, Evans set out to isolate normal cells from an early mouse embryo that would have similar properties. Work from Richard Gardner argued for the existence of such cells, but culturing them had been elusive. In 1981, Martin Evans and Matt Kaufman and, independently, Gail Martin, in the U.S. were successful in isolating such cells, which have become known as embryonic stem cells, "ES cells." Evans then carried out an important series of experiments with his students Allan Bradley and Elizabeth Robertson that demonstrated that these ES cells could contribute to the mouse germ line. They further showed that genetically manipulated ES cells could transfer their genetic changes to progeny mice. The importance of ES cells was immediately recognized by Capecchi and Smithies, who learned how to grow ES cells and demonstrated that they could carry out targeted genetic alterations with them.

The first knockout mice constructed by gene targeting were published in 1989, and the rest is history. More than 4,000 different knockout mice have been constructed in the last dozen years, and many more are in the works! To keep on top of this fast-moving field, I suggest looking at the Jackson Laboratory's website, where you can find columns called "It's a Knockout!" and "KO of the Month."

The ability to modify the genetic make-up of a mouse by design provides a wealth of information on the function of the gene that is knocked out. Every aspect of mammalian physiology is being penetratingly analyzed by this technique. Particularly notable are the discoveries made on how the immune system functions, which have enormous implications for human health. Knockout mice made it possible to demonstrate unequivocally the molecular basis for prion diseases such as mad-cow disease. Knockout technology is also used to create mice that have versions of human diseases such as cystic fibrosis, muscular dystrophy, atherosclerosis, and many others. These mice make it possible to follow the course of a disease and provide an opportunity to identify and test drugs to ameliorate or cure these diseases.

The ability to precisely tailor mouse genes has completely revolutionized the practice of biomedical science for the last decade and is likely to become even more important in the decades to come. We are certain to reap an enormous bounty of information from knockout mice and reap great benefits for the improvement of human health.