It seems as if each major discovery in genetics gives rise to even more questions than it answers. The discovery that DNA contains a genetic blueprint led to the race to crack the code in which it is written. The cracking of that code and the sequencing of the genome led to another big question: What do all those genes do?
It's a question scientists around the world are still answering—using a technique developed by Mario Capecchi, an HHMI researcher at the University of Utah. With Oliver Smithies at the University of North Carolina, Chapel Hill and Sir Martin Evans at Cardiff University in the United Kingdom, Capecchi won the 2007 Nobel Prize in Physiology or Medicine for developing the technology used to create knockout mice, experimental animals in which one or more genes have been selectively turned off, or "knocked out." By observing mice without a functioning gene, scientists are able to determine gene function. Some knockout mice develop diseases analogous to human conditions, enabling experimental study of disease progression and treatment.
In the 1970s, when Mario Capecchi was setting up his laboratory at the University of Utah—where he'd relocated from Harvard—the idea that researchers could successfully target a specific gene was considered off the wall. Even getting a cell to take up extra DNA was considered a dicey proposition. In 1977, Capecchi read a paper by two Columbia researchers who had succeeded in getting cultured cells to take up experimentally applied DNA fragments. They'd mixed the DNA with calcium phosphate and applied it to the cells. Most of the time, the DNA was destroyed by enzymes in the cell cytoplasm, but in about one in a million cells, the applied DNA made it to the cell's nucleus and was expressed.
Capecchi thought that the limiting step was probably getting the DNA through the cytoplasm and into the nucleus, so he developed a needle that let him inject the DNA directly into the nucleus. This increased DNA take-up about 106-fold. And in some of those cells, the DNA was taken up by a process called homologous recombination, where the introduced DNA swapped out the related segment of DNA from the host cell. That is, it replaced the cell's own gene.
As Capecchi was developing techniques to introduce mutated genes into cultured cells, Sir Martin Evans was at work on a separate problem: how to culture embryonic stem (ES) cells from mice, and then use these cells to grow mouse tissues or a whole mouse. When Evans presented his work, Capecchi saw the opportunity to target genes in a mouse ES cell and then grow mice without the functioning gene. Oliver Smithies, who was working on gene targeting with a different method, had the same thought, and both men obtained mouse ES cells from Evans. Both scientists also succeeded in introducing targeted mutations into cultured mammalian cell lines.
In 1987, after setbacks that included losing an entire mouse colony to disease, Capecchi published a description of his first knockout mouse; other labs quickly followed suit. Today, when scientists want to study a new gene, one of the first things they do is create a knockout. Repositories such as the Jackson laboratory contain hundreds of knockout mice that scientists can simply order, and the United States, the European Union, and Canada have embarked on an ambitious project to create mouse embryonic stem cell lines with each of 20,000 proteins knocked out. Francis S. Collins, director of the National Human Genome Research Institute at the National Institutes of Health, calls the Knockout Mouse Project the best acknowledgement of the significance of the work pioneered by Capecchi, Evans, and Smithies.
Photo: Ramin Rahamian