Knockout mice are "still the best way to figure out the function of an unknown gene," Alexander Gragerov told BioWorld Today.
But not a particularly fast way. The existing methods for creating knockouts are all time-consuming, albeit for different reasons. Despite recent advances, gene targeting - which seeks to knock out one specific gene at a time - is useful for targeting small numbers of genes but very time consuming for larger families.
Random mutagenesis, where cells are subjected to either chemical or viral mutagens, is a higher-throughput method, but leads to another problem: finding cells that have the mutation of interest.
"To look for a phenotype is a daunting task if you have no idea what it might be for a novel gene," said Gragerov, who is director of research at Seattle, Wash., biotech company Omeros Corp. And, he added, the phenotypes can be surprisingly mild. From their own work, Gragerov joked, he has concluded that "apparently the brain is a nonessential organ - we have very few lethals."
In the Sept 4, 2007 issue of the Proceedings of the National Academy of Sciences, researchers from Omeros and the National Cancer Institute describe a library of knockout embryonic stem cells that combines mutations in more than 90 percent of genes with a fast method to identify individual gene mutations.
The researchers used insertional mutagenesis with a proprietary retroviral vector to mutate embryonic stem cells. The real trick, Gragerov said, lies in the way Omeros scientists "devised a scheme to retrieve... a particular [mutated] gene."
For their identification, the researchers essentially stacked 96-well plates into three-dimensional arrangements. Pooling embryonic stem cells into groups of roughly 500 cells per well, arranging the wells into a three-dimensional matrix, and pooling the wells again by either plate, column or row made it possible to identify wells containing a mutation of interest by quick PCR-based screening. That determined where three pools with a desired mutation intersected. Wells containing the mutant ES cells could be identified in less than a week, at which point the specific cells were isolated using a two-step process of sorting and sequencing.
The scientists first tested their approach by isolating knockouts that had mutations in G-protein coupled receptors or GPCRs. This receptor family is a drug targeting mainstay, and with several hundred members it seems likely that there are plenty of targets left to be discovered. But it is difficult to make knockouts of GPCRs, many of which are encoded by small genes without any introns, via insertional mutagenesis.
The researchers found that the combination of the specific retroviral vector they used and their retrieval method allowed them to find embryonic stem cells with an insertion in about 90 percent of the GPCR genes. Insertions were found at almost equal rates in GPCRs that are expressed in embryonic stem cells and those that are not, and in an additional set of experiments, the scientists had a similar success rate for targeting another gene family, nuclear receptors. Based on these results, Gragerov said he believes that as many as 90 percent of mouse genes are mutated in the Omeros mutant ES cell library, which contains about 10 million individual clones.
However, "not every clone will produce an animal," Gragerov clarified. In the paper, they isolated ES cell clones for 85 percent of the target genes, and his team estimates the probability of being able to make a knockout animal of a given gene at another 85 percent, bringing an overall likelihood for making a knockout of a given target gene to 70 percent.
Gragerov and his colleagues ultimately bred 60 knockout strains. Gragerov said the 60 were more than enough for Omeros' purposes: Though the company is "open to different suggestions" for licensing the technology, its primary focus is on using the method for its own target validation. And that, Gragerov said, is well-served by the strains they did make: "We have more targets than we can handle." Some of the targets identified by the method are in Omeros's drug discovery pipeline, with compounds acting on those targets in preclinical development.
Gragerov said the company is "expanding on the technology to make inducible knockouts," and that the paper describing the expansion has been submitted for peer review. He noted that the inducible knockout technology Omeros is working on is not limited to being combined with retroviral mutagenesis, but "could be used with other techniques."