Science Editor

In the July 24, 2009, issue of Science, academic and industrial scientists reported the creation of knockout rats through the use of zinc-finger nuclease technology.

In a field that loves to attach the term revolutionary breakthrough to advances that are sometimes anything but, there can be no question that knockout technology has been truly transformative. When the Nobel committee recognized the inventors of knockout technology in 2007, it wrote in an explanation that the technology "has now been used by so many research groups and in so many contexts that it is impossible to make a brief summary of the results."

But current knockout methods do not work equally well in all species. The methodology that originally was developed in mice, which entails the use of embryonic stem cells, is basically limited to them. Through the use of fetal fibroblast and somatic cell nuclear transfer technologies, it has been possible to knock out genes in several larger mammals including cows, pigs and sheep.

But to the chagrin of researchers, neither method has worked in rats, and finding a method that does has been "somewhat of a holy grail in the world of transgenic animals," noted Roland Buelow, CEO of Palo Alto, Calif.-based start-up Open Monoclonal Technologies Inc. and the senior author of the new Science paper.

Rats are more closely related to humans than mice are. And although they are much closer in size to a mouse than a human, rats are larger than mice by just enough to make many physiological processes a much better model of what happens in humans.

In their paper, the authors, from the Medical College of Wisconsin, INSERM, Sangamo Biosciences and Sigma Aldrich as well as Open Monoclonal Technologies, used zinc-finger nuclease technology to knock out three different genes - an engineered green fluorescent protein gene as well as endogenous rat genes immunoglobulin M and the cell trafficking gene Rab38 - in rat embryos.

Zinc fingers are naturally occurring transcription factors. They have been used to knock out genes in adult cells by combining zinc-finger proteins that target a specific sequence with an enzyme that induces double-stranded DNA breaks. When such double-stranded breaks are induced, the cell's own machinery repairs them - in this case, with the template provided by the zinc-finger proteins.

While the method is established in adult cells of mice and humans, as well as other species, it was far from clear that it would work with embryonic cells - which is probably why no one else had tried before. Buelow described the approach as essentially taking to the embryo "with a pair of scissors," for DNA, which, by all rights, should kill the embryo.

But work it did. In fact, the simplest method of directly injecting an mRNA or plasmid with the gene of interest, inducing a double-stranded break, and implanting the repaired embryos into foster mothers "worked beautifully," Buelow said, with anywhere between 5 percent and 75 percent of progeny harboring the mutation in the experiments.

On a basic scientific level, the work opens up the possibility that knockout rats may soon be as easy to come by as knockout mice - maybe easier. "This new method is independent of embryonic stem cells and simpler than nuclear transfer," Buelow said. "And that, of course, opens the door to many animal models."

He added that since the submission of the Science manuscript the authors have been able to knock out other genes besides those described in the paper, "and in all instances, it works very well."

Open Monoclonal Technologies already has commercial plans with the knockouts described in the paper. Monoclonal antibodies are big business, and to produce fully human antibodies by animals it is critical that the animals' own antibody production be silenced. Such production is currently governed by a thicket of restraints that are partly scientific and partly legal, stemming from the licensing agreements of existing technologies. Calling the new method a "new monoclonal antibody platform with unrestricted development options," Buelow said that the company expects to have the technology ready for partners looking for licensing agreements by year-end.

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