Being a mouse geneticist in the 1980s just wasn’t fair. If you worked in bacteria, yeast, or fruit flies, genetic discoveries were proceeding at a heady pace. Find a mutant fruit fly with a leg popping out of its head and soon enough you could winnow your way through its genes to find the one that had caused this aberration.
It was possible to link mutations to genes in the small critters because of their rapid breeding times and vast numbers of offspring. Fruit flies also had the advantage of giant salivary gland chromosomes where gene alterations could actually be seen under the microscope. However if you happened to be interested in mammals like mice, things moved along at a snail’s pace.
But in 1989 envious mouse geneticists had a glimpse of a dream come true. A few pioneering researchers found a way to do in the mouse what was being been done in flies. Except that they did it in reverse. Instead of starting with a mutant and then zeroing in on the altered gene, they started with an altered gene and observed the effect on the mouse.
Typically the information carried by the altered gene was ‘knocked out’ so mice bearing these genes were dubbed ‘knockout mice’. The technique won the 2007 Nobel Prize for Physiology or Medicine for its inventors; Martin Evans, Mario Capecchi and Oliver Smithies (see, Knockout mice earn U.S.-British trio Nobel Medicine Prize, Cosmos Online).
It is no exaggeration to say that mouse knockouts revolutionised genetics. We share 99 per cent of our genes with mice. Figuring out what genes are doing in mice genes gives a fair notion as to what to what they do for us. Research in diseases like cancer, heart disease, respiratory diseases and Alzheimer’s has launched into new realms because of the ability to study the function of genes in knock out mice.
Today knockout technology is a routine and robust technology. In September 2006, the U.S. National Institutes of Health (NIH) launched the knockout mouse project or KOMP, a mission to knockout every one of the mouse’s 22,400 genes. But it is worth reflecting that just twenty-five years ago, the notion of knocking out mouse genes seemed so pie in the sky, that granting agencies flatly refused to fund the projects proposed by two of the Nobel Prize winners.
The prize-winning technique relied on putting together three legs of a stool.
The first leg was ‘transgenics’ – a technique that allowed geneticists to finally break down the barrier that prevented them from tinkering with the genes of mammals. By the mid 1970s mouse geneticists had had enough of watching the fly people tinkering with genes at will. They realised nature provided a purpose-built gene tinkering device: viruses.
Viruses are expert at inserting their genes into mouse chromosomes. Researchers packaged new genes into viruses and infected the cells of developing mouse embryos. The introduced genes or transgenes ended up in various tissues of the mice and sometimes they hit the genetic jackpot: they ended up in the sperm or eggs, which meant they became permanent genetic fixtures of future generations.
Soon researchers started generating ‘transgenic’ livestock, like fast-growing pigs that had extra copies of the growth hormone gene or cows that produced a pharmaceutical hormone in their milk. Transgenics broke down the barrier to tinkering with the genes of mammalian cells, but it was a limited tool.
A chromosome is like a street where every gene lives at a precise address. When new genes went in like squatters willy-nilly, they could create havoc. To truly begin to decipher the instructions carried by mouse genes, researchers needed a way to alter their function with surgical precision. Once again, nature had just the mechanism: ‘homologous recombination’, which was to provide the second leg of the stool.
Homologous recombination was known to occur when sex cells were being formed. Just prior to producing eggs or sperm, the chromosomes inherited from mum and dad get together and swap genes; blue eyes for brown and so on. In 1982, Mario Capecchi – one of those indomitable mouse geneticists – realised that even mouse skin cells grown in Petri dishes could carry out the gene swap: it was just very uncommon.
For Capecchi, now at the University of Utah, the odds were good enough to give it a try, though it wasn’t an idea he could sell to the U.S. government funding body, the NIH. Despite this obstacle, in 1986 he achieved it! He was able to repair a gene in mouse skin cells by swapping a faulty piece of a gene with a functional piece. Those rare cells that corrected the gene through gene swapping were able to resist the antibiotic neomycin and survived.
In responding to his next grant proposal, the NIH wrote, “We are glad you didn’t follow our advice”.
Oliver Smithies, of Duke University in North Carolina, independently showed homologous recombination at work in mouse cells. For which he was also recognised as a co-recipient of the Nobel Prize.
Capecchi and Smithies had proven homologous recombination could take place in mouse cells – albeit very infrequently, one in a thousand cells would do it. Because that rare cell could now grow in the presence of the antibiotic neomycin, it could be detected. It was a useful trick for swapping genes in cells growing in a dish, but it wouldn’t be much good for swapping genes in a mouse embryo. If only one in a thousand cells did the swap, that was just too much of a long shot. The researchers need a way of upping the odds. Enter embryonic stem cells.
Embryonic stem cells
Mouse embryonic stem cells provided the third leg of the stool. The secret of making embryonic stem cells was mastered by Martin Evans of Cardiff University in Wales, the third of the trio of winners of the 2007 Nobel Prize for Physiology o Medicine.
Like bacteria, embryonic stem cells can be grown in their millions in Petri dishes and by growing the cells in the presence of antibiotics, it is possible to select the rare cell that has swapped an old gene for a new one. But there is also something unique about embryonic stem cells. Like primordial putty, they can be dabbed onto the side of a developing mouse embryo and help form every tissue. When embryonic stem cells from a brown mouse are dabbed onto a developing black embryo, the result is a spectacular mottled brown and black mouse. Some of the stem cells also end up hitting the genetic jackpot; they end up being the sperm or egg cells. When that happens, the genes of the stem cells will be transmitted to future offspring.
With these embryonic stem cells in hand, Capecchi, Evans and Smithies all proceeded to the end game: to create knockout mice. In these mice, the normal gene would be swapped for a defective copy, so allowing researchers to understand the function of the gene. The technique was clear-cut. First it was a matter of knocking out the gene in embryonic stem cells. Capecchi developed an ingenious method of upping the odds so as to pick the stem cells that carried out precise gene swapping as opposed to randomly inserting new genes into their chromosomes.
The replacement gene was equipped with an antibiotic resistance gene tucked in the middle (neomycin resistance) and a poison gene on its flank (thymidine kinase). Cells that failed to take up the offered new gene into their chromosome died because they had no antibiotic resistance. Cells that inserted the replacement gene willy-nilly, died because they also took the poison flanking gene. Only those rare cells that carried out a precise gene swap survived because they took up the matching gene (and its internal antibiotic resistance gene) but not the poison flanking gene.
Models of disease
Once the knockout stem cells had been made, it was a matter of injecting them into developing mouse embryos, and then waiting for the litter of mottled mice. The mottled mice are mated to each other, and some of their offspring will be pure ‘brown’ mice – born from egg and sperm derived from the stem cells and carrying the gene knockout.
The first knockout mice appeared in 1989. One of these knock-out mice provided a model for the disease Lesch Nyhan syndrome since it lacked the gene mutated in these people; another took a hit in a gene associated with cancer. Soon there were knockout mice for genes implicated in cystic fibrosis, various forms of cancer, atherosclerosis, heart disease and a multitude of other illnesses.
Today, the technique has become virtually routine. Not bad for an idea that just twenty-five years ago was the stuff of dreams. John Mattick, a professor at the University of Queensland observed, “It’s a paradox. When you’re in the middle of research it seems to progress so slowly; then you look back and see how fast things change. I marvel at the perseverance of those who developed this technology; it has opened up the other half of genetics.”
Interactive graphic explaining knockout mice – Wellcome Trust