A Win by a Knockout

Three Scientists Win the Nobel Prize in Medicine for Work on Eliminating Genes in Mice
The Nobel Prize in Medicine for 2007 has been awarded to Mario Capecchi and Martin Evans in the U.S. and Oliver Smithies in Great Britain. And they definitely deserved it. Their combined research led to scientists being able to get rid of individual genes reliably in mice. The American scientists showed us how to eliminate the gene. And the British scientist showed us which cells to use. It is hard to believe that this sort of thing has only been around since 1989. This is because knocking out a gene (as it is called) is important in so much of biology. For example, if you type "knockout mouse" into PubMed (a search engine for scientific journals), you get back 46,666 different journal articles that use the technique. Wow. Knockout technology is involved in almost every aspect of modern biology. When scientists propose a gene is involved in a certain disease, they need to try to prove it by knocking that gene out in mice. When they propose one is involved in development, they need to again knock it out in mice to show how. And the list of experiments goes on and on. Knocking out genes has been absolutely critical for understanding how many of our genes work and what happens when they go wrong. The technology also lets scientists make lots of mice that can serve as models for disease. There is one for cystic fibrosis. And another for Parkinson's. And hemochromatosis. And various cancers. And… As you can probably tell, using knockout technology has fundamentally contributed to a better understanding of who we are. Here are a few random examples of important and cool knockout experiments. Please note that these are just a few of the tens of thousands of reports that are out there.
Knocking Out a Gene
So how do scientists knock out a gene? Basically they take advantage of something called homologous recombination to replace a working gene with a broken one in a single cell. They do this in an embryonic stem cell so that they can eventually turn the single cell into a whole mouse. Sounds simple enough. But it is really complicated in practice.

Knockout technology
uses recombination to
swap a broken gene
for a working one.
Mixing and matching DNA To replace a good gene with a broken one, scientists need to take advantage of something called homologous recombination. Homologous recombination is basically the way a cell mixes and matches two similar DNA sequences. It is an important process in making each one of us unique (click here to learn more). To take advantage of this process, scientists first need to get a copy of the gene they want to knock out. Once they have this, they then stick a big piece of DNA into the middle of the gene to inactivate it. Sort of like throwing a monkey wrench into a machine to stop the machine from working. Next scientists put this inactivated gene into a cell and hope that homologous recombination will happen. It undoubtedly will, but it won't happen frequently. So scientists need some way to pick out the few cells where recombination has happened. The way they do this is by taking advantage of something very similar to the antibiotic resistance of bacteria. Basically what scientists do is to make sure they put a gene onto the monkey wrench that will make a cell resistant to some chemical. Neomycin is a common chemical they use. After adding the inactivated gene to some cells, the scientists then grow those cells in the presence of neomycin. Only the cells where the inactivated gene has been inserted into the cell's DNA survive. Unfortunately, the inactivated gene will sometimes stick itself anywhere into the cell's DNA. This isn't useful because the working gene is still there too. We only want the cells where the inactivated gene swaps out the working gene in the cell. The final step, then, is for the scientists to look at the DNA of a bunch of cells to find the ones where the cell's gene has been replaced by the inactivated gene. With a little luck, they'll find a few cells where homologous recombination has been successful. Of course, we still have just one cell. We need to grow this thing into a whole mouse to really figure out what a gene does.

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Using the Right Cell
So here we are with a cell with a copy of a certain gene knocked out. The other key to a knock out experiment is to use the right kind of cell. An embryonic stem (ES) cell. We all start out as a single fertilized egg that develops into many different kinds of cells. What this means is that a single fertilized egg cell can become any other kind of cell. Scientists say that this cell is pluripotent. Cells that come from this single cell retain this ability until the embryo reaches around 50 or 100 cells. These are ES cells. The idea is that if scientists knock a gene out in an ES cell, then they can grow a knockout mouse from that cell. If only it were that simple! What scientists actually do is add the ES cell with the knocked out gene to an early mouse embryo and grow that embryo into a mouse. Since all of the cells in this embryo can become any cell type, these cells from different sources can grow up into a single mouse just fine (click here to learn more). So the embryo has some cells with the gene knocked out and some with the gene still there. The mouse that comes from that embryo will too. Scientists call this mixed-cell mouse a mosaic. The next step is to take advantage of nature. What scientists do is look for mice that happened to have the gene knocked out in their sperm or egg cells. The scientists then breed this mouse with a garden variety mouse. The pups born will now have the gene knocked out, right? Well, one copy will be. Remember, we have two copies of most of our genes—one from mom and one from dad. The same thing is true for a mouse. So what we now have are mice with one of their gene copies knocked out. Again scientists take advantage of nature to knock out the other copy. What they do is breed the brothers and sisters from this litter. Because of the way genetics works, around 1 in 4 mice in these litters will have both genes knocked out. We're finally done—we knocked a gene out in about a bazillion easy steps. Now the experiments can begin. What did knocking out the gene do to the mouse? How well does the mouse mimic a disease? Can we use the mouse to test medicines for a specific disease? What happens if we knockout another gene in the same mouse? The technology has now become so sophisticated that scientists can control when and where the gene is knocked out. They can even just tweak the gene instead of blasting it apart to look at what subtle changes to a gene do to a mouse. All because of the three scientists who won the Nobel Prize in Medicine this year. Definitely well deserved.
Scientists use embryonic
stem cells to make
a knockout mouse.