Friday, May 28, 2010

Mouse project to find each gene's role

Published online 25 May 2010 | Nature 465, 410 (2010) | doi:10.1038/465410a

Mouse project to find each gene's role

International Mouse Phenotyping Consortium launches with a massive funding commitment.

Alison Abbott
Making thousands of strains of knockout mice will build a powerful database.Image courtesy of MRC Harwell

An ambitious effort to identify the function of every gene in the mouse genome looks set to provide scientists with the ultimate mouse model of human disease.

The International Mouse Phenotyping Consortium (IMPC) has yet to find all of the US$900 million it needs to complete its task over the next decade. But at a meeting on mouse models of human diseases in London, where the project was unveiled last week, scientists announced a commitment of $110 million from the US National Institutes of Health (NIH) in Bethesda, Maryland over the next five years.

"The project will explain the genes — it is going to be transformative for biology," says James Battey, director of the National Institute on Deafness and Other Communication Disorders. This is one of the nine NIH institutes that have agreed to set aside $11 million from their budgets to match the $11 million put up by Francis Collins from his own budget as NIH director.

The IMPC aims to take mice of identical genetic background and to create viable strains in which one of the 20,000 or so genes in the mouse genome is knocked out, or deactivated. The knockout strains will then be put through rigorous, systematic phenotypic screens, which will check for physical and behavioural differences. The information will be stored in a purpose-built, open-access database.

Scientists would, for example, be able to turn to the database to learn more about an unfamiliar gene signalled in a genome-wide association study in humans as being possibly relevant to a particular disease. Making the mutant animal and phenotyping it in a lab could take three years.

According to Battey, the NIH thinks that its investment in the large-scale programme will actually save money in the long run, because it already spends "hundreds of millions of dollars per year" supporting small-scale grants for individual scientists wanting to create and phenotype their own particular gene of interest.

“No question, the mouse is where the action is.”

But the launch of the visionary programme comes at a time of global financial crisis and may have trouble finding additional funders. Some scientists had looked to the European Commission, which sponsored the meeting and has spent close to €250 million (US$305 million) over the past ten years to pioneer systematic phenotyping of mice, and to generate mutant mice. But Leszek Borysiewicz, chief executive of the UK Medical Research Council (MRC) in London, warned at the meeting that the commission's politicians would need a lot of con­vincing that mouse genomics was more deserving of funds than other scientific projects.

Scientists at the meeting, however, argue that the IMPC's goals will eventually be achieved, even if the timetable has to be stretched, simply because the work is so vital. The mouse genome was the first to be sequenced after the human because of its importance as a laboratory model. "But it soon became clear that it is impossible to predict function directly from sequence," says Paul Schofield, a geneticist at the Uni­versity of Cambridge, UK, who helped to organize the meeting. "Also, there are black holes in the genomes where we simply don't know what the genes do — the mouse phenotype database would give us traction."

Mouse 'clinics' have sprung up around the world to screen mutant mice for crude phenotypes — such as heart defects — and to perform secondary screens to investigate the changes in more detail. But it is already clear that even this is not enough. Gene expression, and the resultant phenotype, are profoundly influenced by environment, and many of these mice are raised in a stressed environment for the purposes of experiments — for example, being fed high-fat diets or subjected to infection. Much more will be learnt by comparing phenotype screens carried out on mutant mice raised in a normal environment, say project scientists.

"It makes sense to coordinate the systematic phenotyping of the complete genome internationally, like we did the human and mouse genome," says geneticist Mark Moore, a consultant for the NIH, the MRC and the Wellcome Trust UK medical funding charity, who heads the IMPC. The consortium, which will involve clinical as well as basic research scientists, will do primary phenotypic screens on 4,000 knockout mice in its first five years. Any researcher may request a particular gene to be prioritized for knocking out in this first phase, although a proportion will also be selected at random.

The IMPC is already discussing with the scientific community exactly what types of phenotyping should be carried out, and what environmental challenges the mice should face in later stages of the project. It is also considering including a colony of ageing mice: "This would increase costs because the mice would have to be housed for so long, but age-related diseases are at the top of everyone's agendas," says Moore.
Source of drugs

"The IMPC sounds expensive but it is not compared with other genomic resources," adds Moore. "The database only needs to help industry to develop a handful of multibillion-dollar blockbuster drugs and it will have paid for itself."

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"The initiative will save us time and money, which will help us provide drugs in return," agrees David Frendewey, an associate director of Regeneron Pharmaceuticals, headquartered in Tarry­town, New York. "No question, the mouse is where the action is." Yet turning drug companies' interest into hard currency won't be easy. "Industry will participate in kind, but maybe not with cash — we are no longer a bottom­less pit of money," says Malcolm Skingle, director of academic liaison at GlaxoSmith­Kline in Stevenage, UK.

And genome engineer Francis Stewart of the Technical University of Dresden, Germany, says that focusing on the commercial benefits of the project misses the point. "Through the IMPC, we have a historic opportunity to systematically learn everything about a mammal, for the first time," he says. "Let's not lose the vision by side-tracking into applications issues just to please politicians.

Tuesday, May 11, 2010

Genomics goes beyond DNA sequence

Genomics goes beyond DNA sequence

A technology that simultaneously reads a DNA sequence and its crucial modifications makes its debut.

Alla Katsnelson

What makes two individuals different? Biologists now know that the genome sequence holds only a small part of the answer, and that key elements of development and disease are controlled by the epigenome — a set of chemical modifications, not encoded in DNA, that orchestrate how and when genes are expressed. But whereas faster, cheaper and more accurate sequencing technologies have developed rapidly, techniques to map the epigenome have lagged behind.
DNA polymerase (shown flanking the double helix) can reveal genomic and epigenomic detail.LAGUNA DESIGN/SPL

Sequencing company Pacific Biosciences, based in Menlo Park, California, has now developed an integrated system that simultaneously reads a genome sequence and detects an important epigenetic marker called DNA methylation. "I think it's an important step forward, although I think it is a baby step," says Joseph Ecker, a plant geneticist at the Salk Institute for Biological Studies in La Jolla, California, who was not involved in the work.

DNA methylation — the addition of methyl groups to individual bases — is just one of many epigenetic markers of DNA and its associated proteins. Others include modification of the histone proteins that DNA winds around to form chromatin — the tightly packed cluster that makes up chromosomes — and the activation of small non-coding RNA molecules.

DNA methylation, which reduces gene expression, is linked to key developmental events, as well as many types of cancer. It is the best-studied epigenetic modification, mainly because tools have existed to study it, says Susan Clark, an epigeneticist at the Garvan Institute of Medical Research in Sydney, Australia.

The gold-standard method for detecting DNA methylation, which Clark's group developed more than 15 years ago, is bisulphite sequencing, in which unmethylated versions of the base cytosine are chemically converted into another base, uracil. Sequencing the converted DNA allows scientists to reconstruct a genome-wide methylation map. But the technique has several drawbacks. Not only is it expensive and time consuming, it also damages DNA, reducing the map's accuracy. And it doesn't detect methylation at adenine bases, which are very prevalent in organisms such as bacteria.

Pacific Biosciences' approach for detecting DNA methylation, published this month in Nature Methods1, builds on the company's sequencing technology. The system uses an enzyme called DNA polymerase to read a strand of DNA and build a complementary strand out of nucleo­tides labelled with fluorescent mol­ecules. As each component is added to the growing strand, it produces a flash of light — the colour of the light corresponds to the identity of the base, and thus reveals the sequence of the template DNA.

Analysing the pulses of light, and the time between them, can also show whether methylation is affecting polymerase activity. This has now been exploited to detect methyl­adenine, methylcytosine and a poorly understood modification called 5-hydroxymethylcytosine. "We foresee with this technology that in the future there will be a unification of the fields of epigenomics and genomics," says Stephen Turner, the company's founder and chief technology officer.
Game changer?

Although the data are promising, obstacles remain. "There are distinct advantages, but we're not rushing out tomorrow to apply this because it's not prime time for human methyl­ome mapping," says Ecker.

One problem is that although the technique is great at distinguishing adenine from methyl­adenine, it doesn't quite reach single-base resolution for cytosine and methylcytosine. It also lacks one of the key promised benefits of Pacific Biosciences' sequencing technology: its ability to read long sequences of DNA, up to 8,000–10,000 base pairs, which makes it easier to assemble the data into complete genomes. Instead, the reported methylation read-length is only about 1,000 base pairs.

“This is exactly the technology you could use to look for epigenomic changes in specific cell types.”

Turner says that the company is working to solve these problems. It will ship the first sequencers that use fluorescent labelling this year, and plans to add the methylation mapping capability next year.

"What needs to be done now is to make it robust and accurate," says Clark, a steering-committee member of the Inter­national Human Epigenome Consortium, a bid launched in January to map the epigenome in multiple cell types2. "There's a lot of trouble­shooting that needs to be done to get it to be accurate enough to be able to compete with bisulphite sequencing."

Several companies are working on similar technologies. UK-based Oxford Nanopore Technologies published a report last year showing that it could detect methylated DNA at a single-molecule level3. But that system and others are still at an earlier stage of development.

Some say that the promise for such a technique is huge. Bisulphite sequencing for a single human genome can cost up to US$100,000, says Robert Martienssen, a geneticist at Cold Spring Harbor Laboratory in New York. With the latest technique, the cost of a full-genome methylation map would drop to $100–1,000, he says. "That will change everything."

There is no shortage of epigenetic questions ripe for probing. One example is in tumour biology, where different cancer cells are likely to have different methylation patterns. Another is how cells in a single organism take on different functions despite having identical genomes. "This is exactly the technology you could use to look for epigenomic changes in specific cell types," says Martienssen, who is also on the International Human Epigenome Consortium's steering committee.

Ecker says researchers still haven't pinned down the significance of, say, having a methylation mark in one position and not another, and what's really needed is more studies that unify genomic and epigenomic information. "As you get more genomes to compare, then of course the differences take on some meaning," he says. "We're just lacking numbers at this point."