Author: Gurjot Singh
Institution: Bangalore Medical College
Date: November 2007
Brand Nobel is a very eclectic one, with threads of all hues of life forming its rich tapestry. This is especially true about Medicine, with its very colorful list of recipients-from microbe-hunters to gene-seekers, from animal behaviorists to worm people. In essence, the prize is sometimes retro, saluting the significant past, and sometimes techno, recognizing the most cutting-edge in the arena, but always unique and significant. The new flavor this season is one that permeates all of these, enriching and transforming areas far and wide.
The Nobel Prize in Medicine or Physiology for 2007 has been awarded jointly to Mario R. Capecchi, Martin J. Evans and Oliver Smithies for their contribution in developing the field of gene targeting in mice using embryonic stem cells. This technology, commonly known as gene knockout technology, allows us to tinker with specific genes in the embryonic cells of mice, with the resulting offspring carrying the modified or silent gene. This enables us to study the role of those genes in normal and disease processes. This extremely powerful and precise technique is being applied to virtually all areas of biomedicine-from basic research to the development of new therapies.
From left, Oliver Smithies, Martin J. Evans and Mario R. Capecchi.
Dr Alexandra Joyner at the Memorial Sloan-Kettering Cancer Center said that this work "nicely marries the basic and translational research; technology is used for translational research for creating these beautiful mouse models of human diseases as well as understanding the underlying mechanisms of these diseases."
In this technique, the researchers first isolate specific cells from the mouse embryo called embryonic stem cells which are capable of giving rise to a wide variety of other tissues later on. Into these cells are added the target vectors which are short pieces of DNA similar (homologous) in sequence to the gene in consideration, but with the required modifications, plus certain markers to highlight them. These added genes undergo a kind of swapping process with those already present, in what is known as homologous recombination, so that they are inserted into the DNA now. The cells in which this has successfully happened are selected by using those markers present in the incorporated genes. These cells are now injected back into the embryo and the embryo planted into a foster mother. The offspring developed will have cells of both modified type and normal type, so it is a chimera (i.e., made up of tissues with diverse genetic constitution), with only some tissues expressing the modified gene (those that developed from the modified stem cells). These chimeras are bred with pure mice and fully expressional mice are created, which can be used to study the effects of that modified gene, and hence the normal gene.
Meeting of the Prepared Minds
The story began with the work of Dr Martin on the totipotent cells derived from the famous testicular teratomas of Dr Leroy Stevens' mice at The Jackson Laboratory. He then went on to characterize Embryonic Stem Cells and showed that they could be manipulated to transmit outside genetic material to future generations of mice, thus making possible the creation of transgenic animals. Going a step further, he and his co-workers showed that the ES cells could be engineered to transmit mutant forms of their own genes (added by using viruses) to their offspring. At the same time, across the Atlantic was going on another story, and these two were to meet soon.
The process of creating transgenic animals had been going on in many labs, with the creation of a transgenic mouse in the late 1970s by infecting mice embryos with a leukemia virus. However, in all these experiments, the outside gene got itself inserted into the genome of the animal randomly and in variable number of copies. This prevented the use of this approach for precisely tinkering with the endogenous genes.
During this period, Dr Capecchi moved to the University of Utah from Harvard. He experimented with the use of extremely small glass needles to inject DNA directly into the nuclei of living cells. He found this method to be very efficient. A crucial observation that he made during these experiments was that when multiple copies of DNA were injected into the nucleus, they always integrated as head-to-tail concatemers (i.e., were arranged in a chain or series) at a few loci. With careful experiments, it was proved that this peculiar arrangement was due to homologous recombination taking in the mammalian nucleus. Said Dr Capecchi while accepting the Kyoto Prize in 1996, "I realized immediately that, if I could harness this machinery to carry out homologous recombination between a newly introduced DNA molecule of our choice and the same DNA sequence in the cell's chromosome, I would have the ability to mutate at will any specific gene of the living cell."
In parallel with Capecchi's work, Oliver Smithies had developed the concept that homologous recombination might be used to repair mutated genes, and had worked with human globin genes. However, all this work had been done in cell cultures until then. Could they possibly use homologous recombination to target genes in the embryo and create genetically modified animals? They had heard of Evans work and decided to give it a try.
Both Capecchi's and Smithies' team started with using this technique to correct the mutant form of a gene in mice which causes a human disease called Lesch-Nyhan syndrome. They were successful in transmitting the change. Later, Capecchi's team developed methods to apply this technique to any other gene. Thus was born this technology of gene manipulation in stem cells to create progeny exhibiting and transmitting that modification. Late 1980s saw the development of a number of knockout gene models.
Impact on the Study of Health and Disease
The development of gene targeting technique marks a kind of watershed in the research into gene function. Prior to its advent, our understanding of gene function in higher organisms relied on chance mutations, linkage studies, administration of gene products and cell culture studies. This technique has revolutionized the field to the extent that we have begun to take it for granted. To date, more than ten thousand mouse genes (almost half of the total) have been knocked out, and international efforts are on going to make knockout mice available for all genes in near future. This technology has already produced mouse models of more than five hundred human diseases, including cardiovascular and neurodegenerative diseases, diabetes and cancer.
Sir Martin Evans, 66, who is presently at the Cardiff University in Wales, UK has developed mouse models of cystic fibrosis and was the first to demonstrate the use of gene therapy to cure it and, recently, has shed light on the working of breast cancer gene BRCA2 through a mutant mouse model. Evans, reached while visiting his daughter in Cambridge, England, said ''I haven't come to terms with it yet. In many ways it is the boyhood aspiration of science, isn't it? And here I am unexpectedly with it. It's amazing.''
Mario R. Capecchi, 70, at the University of Utah, Salt Lake City has been working on the genes involved in mammalian organ development and establishment of body plan. For him, the journey has been a long one, from the streets of Italy after his mother was taken to the concentration camp at Dachau during WWII, to coming to US after the war, to working under the inspiring guidance of James Watson at Harvard, to Utah, and now the Nobel Prize. In a telephone interview from Salt Lake City, Capecchi called the award ''a fantastic surprise.'' He said he was deep asleep when he got the phone call from the Nobel committee at 3 a.m. local time. ''He sounded very serious,'' Capecchi said, ''so the first reaction was, `this must be real.'''
Oliver Smithies, 82, a native of Britain now at the University of North Carolina at Chapel Hill, has used gene targeting for developing mouse models for cystic fibrosis and thalassemia. He has also developed numerous mouse models for common disease like hypertension and atherosclerosis, and added significantly to the understanding of their pathogenesis. "After working on the research for more than 20 years," Smithies said, "it's rather enjoyable being recognized at this level.'' Smithies said he hopes winning the prize will make it easier to secure funding for other work.
In conclusion, this work has fundamentally altered the way research is done in all fields of biomedicine. The development of new therapies to treat diseases will depend on the insights we get by reaping the benefits of this technology for all times to come.
References
Press Release by the Nobel Committee http://nobelprize.org/nobel_prizes/medicine/laureates/2007/press.html
Advances information on the 2007 Nobel Prize in Medicine or Physiology http://nobelprize.org/nobel_prizes/medicine/laureates/2007/adv.html
Howard Hughes Medical Institute Research News-The Making of a Scientist http://www.hhmi.org/news/nobel20071008a.html
Author: Gurjot Singh
Reviewed by: Falishia Sloan
Published by: Konrad Sawicki