2007 Nobel Prize, Medicine: Gene Modifications in Mice
On October 2, 2007, the Nobel Prize in Physiology or Medicine was awarded jointly to Mario R. Capecchi, Martin K. Evans, and Oliver Smithies. The Nobel laureates were lauded for their findings of “principles for introducing specific gene modifications in mice by the use of embryonic stem cells” (1). Their combination of two significant scientific tools – embryonic stem cells and DNA recombination in mammals – led to the discovery of a new technology: gene targeting. Gene targeting is used to inactivate single genes through processes known as “knockout” experiments. By “knocking out” single genes, researchers can carefully study the roles of individual genes. DNA can be modified in the mouse genome by gene targeting to produce hundreds of different mouse models of human disorders. Consequently, gene targeting in mice can be useful in many areas of biomedicine, including research and the development of new therapies for diseases (1).
Capecchi and Smithies initially worked by themselves on a study that focused on the modification of genes by the process of homologous recombination. This process increases genetic variation in a population through the exchange of DNA in chromosome pairs, each containing a maternal and paternal chromosome component. Homologous recombination occurs naturally in organisms during the process of meiosis, but it can also be used artificially as a molecular biology technique for introducing change into an organism’s genome. Two strands of DNA are physically rearranged by first lining up the two similar DNA sequences, crossing over material between the sequences, then breaking and repairing the DNA to produce an exchange of genetic material between the strands. Capecchi, a professor at the University of Utah, and Smithies, a professor at the University of North Carolina at Chapel Hill, hoped to use homologous recombination to modify genes in mammalian cells (1). The pair demonstrated that homologous recombination could occur between newly-introduced DNA and chromosomes in mammalian cells (2, 3). To create gene-targeted mammals, however, researchers must use cells in the germline so that modified DNA can be inherited. Working with germline cells, as opposed to somatic cells, ensures that the altered DNA will pass to the progeny because gametes are derived from germ cells (1).
Evans’s previous work with undifferentiated cells that could give rise to germ cells was a critical link to Capecchi and Smithies’s study. Evans, of Cardiff University in the United Kingdom, discovered that cells derived from early mouse embryos could be used to deliver genetic material into the mouse germ line. These cells are commonly known as embryonic stem cells. To prove this, Evans created “mosaic” embryos by injecting embryos from one mouse strain with embryonic stem cells from another mouse strain. Surrogate mothers gave birth to the mosaic offspring, which were then mated with each other to produce offspring that contained embryonic stem cell-derived genes. Evans then modified embryonic stem cells using retroviruses that embedded their genetic material into the chromosomes. A retrovirus contains viral RNA and reverse transcriptase (DNA polymerase). It functions by using the reverse transcriptase to transcribe its genome from RNA to DNA, which is then integrated into the host’s genome. The virus then replicates as part of the cell’s DNA. Mosaic mice were once again bred, but this time they contained retroviral DNA from the embryonic stem cells. Evans demonstrated that the DNA was successfully transferred into the germ line, confirming that the embryonic stem cells generated mice that carried new genes (1).
The combination of Evans’s work with that of Capecchi and Smithies enabled the production of the first gene-targeted embryonic stem cells. Capecchi and Smithies demonstrated that genes could be targeted by homologous recombination in cultured cells, and Evans contributed the necessary vehicle for delivering genetic material to the mouse germ line – embryonic stem cells (4). Since the first gene-targeted mice were created in 1989, gene targeting has developed into a versatile and powerful technology. Mutations in DNA can now be introduced at specific times and into cells and organs at all points in mice development.
Gene targeting is currently very promising for use in medical studies. Countless facets of mammalian physiology can be examined through this process. Nearly two decades after their initial discovery, Capecchi, Smithies, and Evans continue to use gene targeting to better understand the roles of genes involved in mammalian development. Capecchi has been researching the genes involved in organ development and the establishment of the body plan in mammals (5). To date, his work in genetics has helped explain several medical phenomena, including the causes of human inborn malformations. Evans and Smithies have used gene targeting to develop mouse models for human diseases, including cystic fibrosis, and to test the implications of gene therapy. In particular, Smithies and his co-workers have identified the defective gene responsible for cystic fibrosis, one of the most common monogenetic diseases. They named the gene Cystic Fibrosis Transmembrane conductance Regulator (CFTR) (6). When CFTR was knocked out in mice, many features of the human manifestation of cystic fibrosis were generated, including defective chloride transport in airways and intestines, failure to thrive, and pathological alterations of gastrointestinal glands. This model of cystic fibrosis can be used to research the mechanisms of the disease and the effectiveness of potential therapies. This study, among the first to create a model of human disease by gene targeting in mice, has inspired an increase in the number of knockout models for other diseases, such as cancer and inherited heart disease. With this recent work, it is evident that gene targeting in mice has opened up a variety of possibilities in biomedicine. The future is sure to hold a greater understanding of gene function, which will translate into progress in the fields of human health and disease treatment.
References:
1. The Nobel Prize in Physiology or Medicine 2007. Available at http://nobelprize.org/nobel_prizes/medicine/laureates/2007/ (15 November 2007).
2. O. Smithies, R.G. Gregg, S.S. Boggs, M.A. Koralewski, R.S. Kucherlapati, Nature 317, 230 (1985).
3. T.R. Thomas, K.R. Folger, M.R. Capecchi, Cell 44, 419 (1986).
4. M.J. Evans, M.H. Kaufman, Nature 292, 154 (1981).
5. A. M. Boulet, A. M. Moon, B. R. Arenkiel, M. R. Capecchi, Dev. Biol. 273, 361 (2004).
6. B.H. Koller, et al., PNAS 88, 10730 (1991).