Mouse models feature: It's a knockout

Making mutant mice was once a challenge, but in the Speedy Gonzales business of biotech and medical research, it's now routine, and a time-consuming distraction from the real game of investigating gene function.

The do-it-yourself mouse is being overtaken by an international 'we-do-it-for you' mouse-making business worth an estimated $US100 million. It produces mouse models to specification, for researchers in academic, government and corporate research laboratories around the world. As a pioneer in stem-cell research, Australia is right on the commercial pace: embryonic stem cells are the delivery systems for the genes that make knockout and knock-in mice.

Two Australian companies are already up and running in the 'designer' mouse business. Perth's Ozgene began producing mouse models of human disorders for clients in Australia, Japan, Singapore, Canada, Germany, Italy and the UK three years ago. Melbourne's IngenKO, a spin-off from the Monash Institute for Reproduction and Development, started up 15 months ago, and it supplies knockout and mutant knock-in mice to academic researchers, and to biotech and pharmaceutical companies.

Today it's mice, but tomorrow it will be rats and small primates. The evolutionary gulf between animal models and real-world human disorders is about to narrow dramatically. IngenKO's sibling spin-off from MRI, CopyRat, is likely to launch a world-first service making supplying designer rats early in 2003. Dr Rob Daniels, CopyRat's chief operating officer, believes his company is a front-runner in the rat race. Meanwhile, a third Monash spin-off, Maccine, plans to bring animal models within a hairy arm's length of humans, by producing transgenic primate models of human disorders -- the 'mac' in the corporate name refers to the rhesus macaque monkey.

Maccine's CSO, Prof Charanjit Bambra, says the technology is now available to produce transgenic macaques commercially for biotech and pharmaceutical companies that want to complete a final safety and efficacy check before testing new drug compounds and bio-therapeutics in human volunteers.

'Quite simple' Mice are inexpensive to breed and maintain, and provide quick answers. But the 80 million-year evolutionary gulf between mouse and man means that what is true for mice is not always be true for humans. IngenKO's CSO Dr Danny Kitsberg says making knockout or knock-in (KO/KI) mice has become "quite a simple thing to do" with the availability of detailed gene sequences from genomics databases. "We're offering a one-stop-shop for clients," he says.

Kitsberg says it usually takes 10 days after a client delivers a specified mutation to produce a genetic construct containing the mutation, and insert it into mouse ES cells. It then takes three mouse generations -- nine months -- to produce the first KO/KI mice. "We can do two things in parallel -- we produce one mouse line in which the gene is knocked out in every tissue, and then make a line in which we knock it out only in specific tissues," he says.

The second type of knockout model relies on the use of promoters -- gene switches -- that quarantine expression of the mutant gene to specific tissues or organs. The strategy ensures that if the mutation has lethal consequences for embryonic development, the targeted-tissue mutant is close behind in development, as a back-up. The contract guarantees clients will be delivered at least three mating pairs of mice expressing the mutation.

Kitsberg says IngenKO is also beginning to offer phenotype models, in which the same mutation is introduced into different mouse strains, so researchers can explore how different genetic backgrounds may influence expression of the mutant gene. The techniques for producing KO/KI mice have been developed over more than a decade; in addition to their short generation interval, mice have the advantages of being the model for animal genetics research for nearly a century.

Gene tidal wave The Human Genome Project has spawned an tidal wave of new genes of unknown function -- "The more genes discovered, the greater the rush to create knockout mice," Kitsberg says. "With whole new families of genes being discovered, it's creating a huge volume of new IP. There are likely to be big legal problems for companies that are developing mouse models for the same genes."

But with their bigger bodies, rats are anatomically and metabolically closer to humans. Their larger internal organs makes them easier subjects for surgery, and they are better subjects, in particular, for exploring immune-system disorders. CopyRat's Rob Daniels says knockout and knock-in rats will add value to the drug-discovery chain that begins with genomics and ends with new clinical treatment for human disorders.

He says the quickest and most favoured way of discovering what a gene does is to express it in a cell line in vitro, and use new high-throughput gene-expression tools like DNA microarrays to see whether it is over- or under-expressed in a particular disorder. "When you've been through all that, and have an idea which genes are involved in certain disorders, you then need to understand which one is causing the disease," says Daniels. Confirming the problem gene usually involves reproducing the specific mutation seen in the human disorder in a knockout or knock-in rodent.

Rats favoured "Historically, the rat has been the favoured animal for most biomedical research into human disease. It's larger, so you can do surgical interventions and take metabolic parameters that can't be done in mice," Daniels says. "There is a whole range of historic data relevant to human disorders that is based on rat studies, so we'll now be able to reproduce specific [human] alterations in the rat genome and know that they will more closely reflect the human condition.

"Rats live longer than mice, so they're a better model if you need to monitor the progress of a disease, or physiological changes associated with ageing. Rats have a number of metabolic pathways that more closely resemble those in humans, and just about every new drug is screened initially in rats."

Daniels sees rat models augmenting, rather than competing with mouse models. He says the two big US players in the designer mouse field, Lexicon Genetics and Deltagen, have changed focus and become established as fully integrated companies, that develop their own mouse models.

"When dealing with academic researchers, they provide a service at lower cost than their rivals, in return for a share of any IP that emerges from their research," Daniels says.

"In this way, they have been able to access a very large range of potential drug targets.

"It's a model we could follow in Australia with IngenKO and CopyRat, but the fact that these guys are going down the integration route leaves open the market for fee-for-service rodents.

"Providing a quick and efficient service has a lot to recommend itself."

Big market

Ozgene director and CEO Dr Frank Koentgen has been making knockout mice since 1987, and saw a business opportunity after doing some contract work for several major pharmaceutical companies in the 1990s. "We're competing with Lexicon and Deltagene, which are both capitalised at around $4 billion," he says. "We see a multi-million dollar market developing here in Australia.

"If our business continues to grow as it has recently, it will increase several-fold. We've just won a deal to supply knockout mice to a very large research institution in the US.

"We're not the cheapest in the business, but people come to us because we have a very good publication record in prestige journals like Nature, Science and Proceedings of the National Academy of Science. They know we can do the work, do it well, and in the fastest possible time.

"People also know I was the first person to generate knockout mice for the C57 Black 6 strain, and that achievement is still recognised by the scientific community." Ozgene has a strategic alliance with Adelaide biotech company Bionomics, and produced the world's first mouse model of epilepsy for the company.

Maccine's Bambra says his company is likely to produce primate models for major human health disorders like obesity, cardiovascular disease, type 1 and type 2 diabetes, central nervous system disorders, and HIV-AIDS. He says a primate model is likely to be particularly useful for evaluating stem-cell therapies before they are tested in humans. "We'll need to be able to knock out genes or organs in primates and prove that stem cells will do what they are supposed to do," he says.

"Primates are going to be very useful for experiments that involve manipulating the immune response, because the macaque and human immune systems are very similar.

"The capabilities for producing the monkeys are already in existence. It's now a question of harnessing the technology, getting a high-class set-up, and the proper commitment to do this sort of work."