Researchers create a knockout resource for mouse genetics
16 June 2011
The results are founded on a novel, efficient production line that is able to target each specific gene in turn. The consortium has already knocked out 9000 genes as part of an international effort to knock out all 21 000 in the mouse genome.
The cells generated by this approach will enable researchers to ask and answer questions about the roles of genes at the scale of the whole mouse and human genome. The gold standard method to uncover these roles is to mutate a gene in mouse embryonic stem cells and study the biochemical and developmental behaviour of the mutated cells in test tubes or in mice.
Until the new production system was developed, conducting gold standard research on this scale was impossible. There was a crucial problem to be overcome: how do you scale this approach to tackle the whole mouse genome?
"We have pioneered novel methods that enable us to deliver the most complex and accurate high-throughput functional genomics platform yet attempted," says Dr Bill Skarnes, Wellcome Trust Sanger Institute researcher and lead author of the study. "We believe that our work raises the standards of achievement and expectation for genome-scale programmes.
"It is an investment for the future: the genome-engineering technologies developed here for the mouse will drive future model systems, including work on human stem cells."
Genomics was transformed in the 1990s from individual-based research to large-scale commodity resources. An equivalent success was needed for mouse mutagenesis - to provide resources efficiently and consistently and to release them freely. Previously attempted strategies to develop mouse models on a large scale suffered the twin disadvantages of not producing precise genetic changes and favouring only the genes that were active during the experiment, leaving the remainder unaltered.
The present work solves these problems. The team exploited a system called homologous recombination within mouse embryonic stem cells, which can deliver very precise alteration of any gene in the genome. It is founded on choosing recombinant DNA molecules, which are used as vectors to target genes efficiently.
Some genes are essential to life of the cell or organism, however. Disruption of such genes might cause the cell to die and the mutation to be 'lost' from the project. Crucially, to ensure that all genes can be disrupted, the team developed DNA vectors that create a mutation only when required. In this way, the gene targeted by the mutation can be identified, but the mutation is activated only when it is to be studied.
In the essential step to realize its ambitions of a comprehensive, freely available resource, the team designed and delivered a pipeline that systematically designs and constructs the vectors, and efficiently introduces the engineered DNA molecules into the mouse embryonic stem cell line developed specifically for these projects.
Finally, by employing a modular approach to the vector design, several other valuable resources are created en route to the generation of targeted embryonic stem cells. The paper reports that the consortium had produced vectors for more than half of the genes in the mouse genome. All of these outputs are being made available to the mouse research community through the International Knockout Mouse Consortium’s website.
"We are producing mutations in embryonic stem cells with greater efficiency and speed than we predicted and at well above the historical average," says Allan Bradley, senior author of the study and Director Emeritus of the Wellcome Trust Sanger Institute. "We have taken careful steps to ensure we deliver quality resources of maximum utility that will stand the test of time. Indeed, we expect our systems will be increasingly adopted by researchers using human and other cells to seek advances in the understanding of disease."
The methods the team have developed will also accelerate studies on human stem cells - cells that have the potential to grow into many different types of adult tissue. Research into producing such induced pluripotent stem cells from adult tissues, forgoing the need for embryonic stem cells, is expected to be vital in understanding human disease and therapies. The systems developed for mouse stem cells are transferable to human cells and could drive research into mutation in the human genome and its biological and medical consequences.
"Biomedical research needs biological resources on a scale that matches genomics resources," explains Colin Fletcher, PhD, Program Director of the Knock Out Mouse Program at the National Institutes of Health, a part of the international knockout effort. "Such knockout resources are the foundation for producing thousands of valuable mouse mutants for future large-scale international phenotyping programmes and will serve the biological and biomedical research community worldwide."
The findings are reported today in the journal 'Nature'.
Image credit: Genome Research Limited.
Reference
Skarnes WC et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 2011 [epub ahead of print]
