In lab mice all over the world, genes are being turned on and off like light bulbs to find out what they do. 快猫短视频s have rewound Huntington鈥檚 disease, probed the roots of memory and staged the onset of prion disease. And that鈥檚 just in the brain. The man who made it all possible is Hermann Bujard, chairman of the Centre for Molecular Biology at the University of Heidelberg, Germany. With his colleagues, Bujard developed the Tet system which allows genes to be controlled remotely-from outside a living organism. What started as a hobby has spawned two thousand research papers and contributed to work that led to a Nobel prize last month-for somebody else. Laura Spinney spoke to him.
How do you turn genes on and off?
In the Tet system, we use an antibiotic, tetracycline, to trigger an artificial switch that turns genes on and off in cells. We can do so repeatedly, and we can also subtly control the level of a gene鈥檚 expression by increasing or reducing the dose of the antibiotic. We published the research in 1992.
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Can you give an example of how your system works?
Suppose you want to study the effect of a tumour-causing gene in an adult mouse. You would place the gene under the control of this switch. You would then add tetracycline, or even better, its derivative doxycycline (Dox), to the food or drinking water of the mother of your transgenic animal so that the gene remains inactive and the embryo develops normally. Then, at a given point in the mature animal鈥檚 life, you would withdraw Dox and observe the development of the tumour.
What does your system enable scientists to do that they couldn鈥檛 do before?
There have been attempts to control individual genes in mammalian cells before. But the components used to trigger the switch in those systems-for example, heavy metals, heat or hormones-would affect other physiological pathways as well, and thus mask the function of the transgene.
The real advantage of the Tet system is that it acts exclusively on the gene you are interested in. Moreover, at the concentrations required, Dox has no measurable effect on cells. But most importantly, tetracyclines belong to a well-studied group of compounds, some of which are widely used in medicine. We know a lot about their properties-their excellent capacity for penetrating tissues, the time they reside in a body, etc. Taken together, all these features allow you to precisely control a transgene.
Why is it important to be able to control the timing of the genetic switch?
There are a number of reasons. For example, certain genes are crucial for development. We know that if you knock out or inactivate them, this is a lethal event. The animal will not survive for you to investigate the gene鈥檚 role in later life. Or you will disturb development in some non-lethal way but the mature animal will show abnormal features against which it is impossible to distinguish the effects of the gene you are interested in. But it is also important for studying a gene鈥檚 function at different stages of a process, as exemplified by Eric Kandel鈥檚 work.
Eric Kandel of Columbia University, New York, has just won a Nobel prize for work on memory. How did he make use of the Tet system?
The Kandel lab recognised the Tet system鈥檚 potential very early on. They were investigating genes which they suspected of being involved in spatial learning. One was the gene for an enzyme thought to be required for long-term potentiation, which is believed to provide the biochemical basis for information storage in the brain. In one really impressive experiment, they allowed Tet mice to learn a certain spatial arrangement, and then flipped the switch so that the enzyme was no longer active. The mice could not remember what they had learned, which was a very exciting finding. But it raised the question: had that information been erased, or was it there but inaccessible? So they flipped the switch back again, and the mice performed as well as they had after their initial training. The memory had been there all along.
Previous experiments had indicated that the enzyme-which is called alpha calcium/calmodulin-dependent kinase II-is needed for establishing memory. But this showed that it is also essential for retrieving it. An experiment like that wouldn鈥檛 have been possible without a reversible switch mechanism. The importance of reversibility is that you can work on one individual, using it as its own control, and you can go through many cycles.
How has the Tet system been used to increase understanding of Huntington鈥檚 disease?
This is very exciting and important, because it concerns a disease which until now we considered totally irreversible. In Huntington鈥檚 disease, a mutation in what is called the huntingtin gene causes abnormal protein deposits to build up in the brain. These cause neurodegeneration, and patients experience a horrible death. Rene Hen鈥檚 group at Columbia University studied mice in which a mutant huntingtin transgene was placed under Tet control. When the gene was switched on in fully developed mice, protein deposits formed and the mice developed symptoms similar to the human disease. But when, after 18 weeks, the gene was switched off again, the aggregates went away and the symptoms actually cleared up. That means that the protein aggregates are degradable even after the disease sets in, and that neurodegeneration is at least partially reversible. This, of course, changes our view on how one could fight such a disease.
Does that work have implications for other neurodegenerative diseases, such as Parkinson鈥檚 or Alzheimer鈥檚?
With the Tet system we now have a tool for tackling these questions, because you can control the onset of a disease, its progression and potentially even its regression. I am not a neurologist, but several of these aggregation diseases look similar. At the moment we are working on a mouse model for Alzheimer鈥檚. Another type of protein aggregation leads to prion diseases. Two years ago, we contributed to a paper published by Stanley Prusiner鈥檚 group at the University of California, San Francisco showing that the rate at which a mouse develops prion disease depends on whether you over-express the prion gene. The Tet system is more sophisticated now, and more experiments are in progress. They will show whether, once the prion gene is turned off, enzymes are able to dissolve the protein aggregates and of course, most excitingly, whether the animals can recover.
Isn鈥檛 there a second version of the Tet system?
Yes. We also developed a second type of switch which acts in an opposite manner to the first. This one activates the gene only in the presence-rather than the absence-of Dox. Its advantage is that you can get more precise timing. We have recently shown that a gene in mouse liver may be activated within one hour and be switched off again within 24 hours.
Your system has also been applied to diseases outside the brain. Can you give me some examples of what has been found?
There are some interesting papers out on tumour models, where for the first time people have been able to repeatedly grow and shrink tumours and show how oncogenes control the growth and maintenance of tumours. Mike Bishop, the Nobel laureate at the University of California at San Francisco, is studying T-cell lymphoma and Ronald DePinho at Harvard Medical School is doing similar work with skin tumours. Then there is BRCA1, the gene that has been correlated with a type of breast cancer. Nobody really knows what it does. Now people have placed it under Tet control and identified genes downstream of BRCA1 that are activated or influenced by it. It鈥檚 early days, but the Tet system could have other uses-in gene therapy, for instance.
Does it bother you that so many scientists are winning laurels from your hard work?
That鈥檚 a funny question. First, I am delighted that the Tet system flies and that it has an impact on biological research. Second, it was a nice spin-off of our long-standing interest in gene regulation in bacteria. It started as a sort of hobby, and a rather good piece of molecular biology. We are also making use of the system ourselves. Don鈥檛 forget that the main focus in my lab is malaria research.
Do you think you deserve more recognition for developing the Tet system?
I am pleased with the success of the system and the recognition my co-workers and I have received. It has put me in touch with many excellent colleagues in numerous areas of biological research and I have learned a lot outside of my field-a great reward for any researcher.
Why did you sell the Tet patent to BASF Pharma, the German pharmaceutical giant?
I returned from industry to academia some years ago, and was never eager to set up a company after that, particularly with my responsibilities as chairman of the Centre for Molecular Biology. That is why we sold the rights to BASF. These days BASF is considering transferring the system to a smaller company that would have the flexibility required for further development. It has reached the point where the development should be driven in a commercial and not an academic set-up. Whether I will further involve myself in this undertaking remains to be seen.
Is the system free, or do scientists have to pay to use it?
What is important is that when BASF took over the rights to the Tet system, we agreed in our contract that it would be free for academic research. This is in contrast to what happened with the Cre-loxP system, a technology for deleting genes in a defined way. This system belongs to the American company DuPont, which for a long time required academics who wanted to use it to sign contracts that I considered unethical. More or less it meant that whatever product you developed with the system belonged to DuPont. BASF was cooperative in developing a liberal licensing policy-they didn鈥檛 intend to just sit on the patent and bury it. To date, many companies have taken out licenses. Numerous laboratories are using the system now.
You are still perfecting the system. What do you consider its limitations, and do you think you can overcome them?
In animals, the main limitations have to do with how quickly you can get the Dox into and out of a cell. You can take the drug out of the animal鈥檚 food but you cannot easily control how quickly it disappears from the body. It would be good to speed up the switching process, especially for the study of embryogenesis, in which there exist sharply defined time periods for developmental processes. There are several ways this could be done. For example, you could develop a tetracycline derivative which breaks down faster. Or you could find another chemical which would rapidly diffuse through the body and block the action of the tetracycline.
What next?
I feel the system is on its way to fulfilling its potential. There is a systematic European effort underway called the EUROSCARF Initiative to place every single yeast gene under Tet control; to create a conditional yeast cell for every gene. The data this will generate will have a tremendous impact on the study of gene function in general. But if you ask me what I find most exciting, it is the mouse models. I myself will probably leave the field to concentrate on my malaria work again. The Tet system is in good hands and I guess I will not be missed a lot.