Knockout win

Print edition : November 16, 2007

MARIO R. CAPECCHI. - TIM KELLY/BLOOMBERG NEWS

A trio of biologists Mario R. Capecchi, Sir Martin J. Evans and Oliver Smithies is awarded the Nobel Prize in Physiology or Medicine 2007.

IN the post-genetic engineering era of modern biology, particularly in the last two decades or so, the mouse has acquired the status of the experimental animal, or the animal model system, of choice for the entire spectrum of research: from basic science to the development of new therapies in biomedicine. This is thanks to the path-breaking discoveries of this years Nobel Prize-winning trio of biologists two British and one Italian-American.

Seventy-year-old Mario R. Capecchi of the Howard Hughes Medical Institute and the University of Utah, the United States; 66-year-old Sir Martin J. Evans of Cardiff University, the United Kingdom; and 82-year-old Oliver Smithies of the University of North Carolina at Chapel Hill, the U.S., will share the $1.5 million Nobel Prize in Physiology or Medicine for discovering and elucidating the principles of introducing gene modifications in mice and their progenies using the embryonic stem (ES) cell technique.

In 2001, the three together were awarded the prestigious Lasker Prize for biology. Their work has led to the powerful technology called gene targeting in mice, which forms the backbone of present-day biological research. In gene targeting, gene alterations are made in the germ line of a mouse, enabling the raising of offspring that carry and express the modified gene.

Depending on whether genes have been deleted or inserted into the mouse genome, such gene-altered mice are referred to as knockout (KO) and knock-in (KI) mice respectively. (Gene modifications in the latter are, however, not targeted but only inserted randomly at a specified locus of the genome.) With this technology, it is now possible to modify, in any manner to establish the role of specific genes in health and disease, the deoxyribonucleic acid (DNA) in the mouse genome, which has considerable overlap with the human genome. Aspects of this technology have been licensed to Lexicon Pharmaceuticals. Various techniques to produce KO mice are extensively patented in the U.S., and KO mice are themselves patentable in many countries, including the U.S.

Mammals have over 20,000 genes, and from 1989 when the first KO mice were reported to date, more than 10,000 specific KO mice have been created and studied. Such knockout experiments, where single genes are inactivated, are used to study which genes are involved in embryo development, adult physiology, aging and specific health disorders. KO mice for all genes should become available in the near future with the ongoing efforts in the field. The technology of gene targeting, or the KO technology, has already produced more than 500 mouse models of human disorders, including cardiovascular and neurodegenerative diseases, diabetes and cancer.

The technology exploits a natural biological phenomenon called homologous recombination, which appears to have been conserved throughout evolution. Joshua Lederberg demonstrated this in bacteria about half a century ago and won the Nobel in 1958 for his work. Genetic information about development and body functions is contained in the DNA, which is packaged in the chromosomes that occur in pairs, one inherited from paternal DNA and the other from maternal DNA. The natural process of homologous recombination allows exchange of DNA sequences within such chromosome pairs, which is what is responsible for genetic variation in the population.

In the 1980s, Capecchi and Smithies were seeking ways to specifically modify the mammalian genome the former to insert new genes into cells and the latter to correct defective disease-causing genes. Both, independent of each other, struck upon the idea that homologous recombination could be exploited to introduce short DNA sequences into the chromosomes of mouse cells cultured in the laboratory. Smithies and Capecchi both chose a gene called HPRT, which is easily identified and which is involved in a rare inherited human disease called Lesch-Nyhan Syndrome. Standard methods were available for selectively growing cells with functional HPRT enzymes and had already been used for some years.

Sir Martin J. Evans-AP

Their work collectively demonstrated that all cellular genes, irrespective of their activity, could be targeted with exogenous DNA through homologous recombination, and that too with extraordinary precision. It is interesting to note here that Capecchis application to the U.S. National Institutes of Health for funding for his proposal to test the feasibility of gene targeting (through homologous recombination) was rejected on the grounds that the proposed scheme was extremely unlikely to work because of the low probability of the introduced DNA sequence finding a matching sequence with 330 base pairs in the host genome.

But the discovery of both Smithies and Capecchi was limited, to the extent that the modifications could be brought about only at the level of mammalian cells. What was needed to effect such alterations in the germ line the set, or sequence, of cells that can grow into a complete life system of the mouse so that the changes could be inherited and gene-targeted animals could be created as progenies. The ES cell culture that Evans was developing provided the necessary vehicle for them to carry the in vitro gene modifications over to in vivo.

(A stem cell is a cell that can proliferate extensively, is capable of self-replication and can create new, differentiated cells. Somatic cells enable replication and renewal of only specific tissues in the adult organism. Early ES cells, on the other hand, are capable of multiplying into all the types of cells that make up an organism. Hence, the enormous current interest in ES cells for developing therapies at the cellular level for various kinds of health disorders and for the regeneration of dysfunctional tissues and organs.)

Working with mouse embryo carcinoma (EC) cells, Evans found that though these cells came from tumours (and hence proliferated), they could give rise to almost any type of cells. He had the idea of transferring genetic material to the mouse germ line using these EC cells. However, the idea did not succeed because EC cells carried abnormal chromosomes that produced abnormal cultures and, therefore, did not contribute to the growth of a proper germ line. With further research, he found that ES cells could be used to establish chromosomally normal cultures. Whether these cells would contribute to the germ line still remained to be established.

Evans injected ES cells of one mouse strain into the embryos of another strain. As expected from his earlier experiments, the chromosomes of the two strains combined, and these embryos, made up of the cells of both strains (or mosaic embryos), were implanted in surrogate mothers to be carried to full term. The mosaic offspring were then mated, and the resulting pups were found to carry the genes derived from the ES cells. These genes could now be inherited by future generations according to Mendelian principles.

Evans carried his experiments further, towards his original objective of transferring new genetic material into the mouse genome. He modified the ES cells genetically by inserting retroviral DNA into them and transferring them into the mouse germ line via the mosaic mice. Thus mice that carried new genetic material had been created using ES cells.

All the pieces required for the birth of gene targeting technology were now available: Capecchi and Smithies had demonstrated that homologous recombination could be used to target genes in cultured cells and Evans had shown that ES cells were the ideal vehicles to carry the modifications to the level of the germ line and thence to inheritance between generations. In their paper published in Nature in 1987, Evans and his co-workers wrote that their success opened up the possibility of deriving strains carrying specifically induced alterations in other genes and suggested, citing the works of Capecchi and Smithies, that it may also eventually be possible to produce specific alterations in endogenous genes through homologous recombination with cloned copies modified in vitro.

Oliver Smithies at his university office.-KAREN TAM/AP

Oliver Smithies at

As recounted by the laureates in their interviews to the editor-in-chief of the official Nobel website, how these techniques came together is an interesting story and a fine illustration of how free exchange of ideas and collaborative efforts in the true spirit of science are fundamental to its progress and healthy growth. The fact that the three have got an award together not once but twice only reiterates the importance of the confluence of the contributions of each one of them, which has led to the realisation of this important technology. Also, the three met through science and are very good friends. It may seem an irony that aspects of this technology are now patented, thus restricting their free use for refinements and improvements.

Both Capecchi and Smithies heard of Evans ES cells and decided to try them. Evans had gone to the U.S. to work in Richard Mulligans laboratory at the Whitehead Institute for a month.

Now, because I had a short time and was definitely going to learn technology there, I had said no seminars, no visiting, and I will not see or speak to anybody else, said Evans in his interview. But when he got a phone call from Smithies, he apparently said, Oliver, you are the only person who I will come and visit. I went over [for] the weekend with the [ES] cells in my pocket. And then, when I got back from the States, a week or so later, Mario Capecchi had got in touch with me and he came over for a week with his wife to learn how to deal with the cells, Evans added.

And this is how Capecchi got interested in meeting Evans. I had heard about the results at a Gordon Conference the previous summer, says Capecchi. So I actually called him, and my wife and I went there and actually spent a couple of weeks during Christmas to learn about ES cells. So that was extremely important for us to do that and get first hand experience in how to grow them, how to manipulate them, and also how to inject them into pre-implantation embryosSo we actually got direct instructions from the people who actually worked them out. That included Martin and Liz Roberston, says Capecchi.

Following this significant meeting and newly gained knowledge, Capecchi refined the strategies for targeting genes and developed a new method, called positive-negative selection technique (see illustration), that was generally applicable. He and his collaborator, K.R. Thomas, introduced a neomycin resistance gene (neo{+r}) into the functional part of the HPRT gene in the ES cells and showed that the clones of the transfected cells had lost HPRT but gained neo{+r} activity. The first reports on the work in which homologous recombination in ES cells was used to produce gene-targeted mice came out in 1989. And the rest is now an important part of the history of modern biology.

There have been several important developments and refinements to the technology. It is now possible to introduce mutations that can be activated at spatially and temporally segregated points that is, in specific cells and organs or during specific phases of development and in the adult animal which go by the name of conditional knockouts or conditional mutagenesis.

For instance, a system in mice developed by Klaus Rajewsky of the Harvard Medical School, called Cre/lox, allows the targeted gene to be switched off at the time of birth. This development is highly significant because up to 15 per cent of genes are known to be necessary for embryonic development, and because a KO gene would not survive until birth, this could be important to prevent the development of a particular disorder that shows up only later in life.

Almost every aspect of mammalian physiology can now be studied by gene targeting. In particular, Capecchis later work has revealed the roles of genes involved in mammalian organ development, in the body plans blueprint, as it were. His work has also been concerned with causes of several birth-defects and malformations. Likewise, Evans has developed mouse models for the inherited disease cystic fibrosis and used them to study disease mechanisms and gene therapy. Smithies, too, has developed mouse models, for thalassaemia, hypertension and atherosclerosis.

What promise does this revolutionary technology hold for the future?

There are now better and better techniques for manipulating the genome in more and more sophisticated ways. And my guess is, it will also become mutiplex so that you will not only be working with a few genes but with many genes at the same time, said Capecchi in his interview. You will be able to manipulate their expression, and place of expression as well as the time of expression, and also probably modulate how much gene product is being made, he added.

My own feeling, he said, is that even though, to me it was always a gamble, you know, how complicated a mouse is and is it actually penetrable by this kind of technology and at the same time can we make the technology sophisticated enough to be able to handle essentially very complex questions. Eventually what we would like to do is to be able to extend it to studying other mammalian organs so [that] we are not simply restricted with respect to how does a mouse work, and its analogy to humans, but also to be able to utilise it to study more processes in evolution and how different traits have come up during evolution. These are the kind of questions that we are looking forward to in the future.

I think, said Evans, it is very interesting that ES cells, which can be grown in culture and which can differentiate in a mouse, can also differentiate in vitro. And that has given two platform technologies. One is the ability to manipulate the mouse genome. But the other one is the one thats coming through in recent years, which [is] the opportunity, may be, to use the human ES cells as a platform for regenerative medicine. And I think all of the ethical angst and anxiety is going to melt away once we have got the programming going properly.

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