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Sanger-Led Team Reports on Knockout Mouse Resource

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – An international team led by investigators at the Wellcome Trust Sanger Institute reported online today in Nature that it has come up with a strategy for systematically creating conditional gene knockouts in mice.

The high-throughput strategy relies on homologous recombination between modular, computationally designed vectors and genomic DNA from mouse embryonic stem cells. Using this approach, the researchers have made mouse embryonic stem cells containing reporter-tagged, conditional knockout versions of more than 9,000 mouse genes — roughly 40 percent of genes in the mouse genome. Moreover, those involved in the study say the same genome engineering approach should be feasible for use in other model organisms and in human cells.

"High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells," senior author Allan Bradley, director emeritus at the Sanger Institute, and co-authors wrote, "and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome."

"We have taken careful steps to ensure we deliver quality resources of maximum utility that will stand the test of time," Bradley added in a statement. "Indeed, we expect our systems will be increasingly adopted by researchers using human and other cells to seek advances in the understanding of disease."

After computationally finding key exons for each gene of interest, the researchers used oligonucleotides designed by their software to come up with modular vectors based on bacterial artificial chromosomes to target the genes.

They dropped these lacZ-inducible, reporter-tagged vectors, produced in several steps, into the genome of mouse embryonic stem cells from the inbred C57BL/6N strain in a high-throughput manner by electroporating several stem cell samples in parallel.

"Computational allele design, 96-well modular vector construction, and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale," they wrote.

For the current study, the team reported on conditional knockouts for 9,000 mouse genes, though the work represents part of a broader effort to make tagged knockouts for all 21,000 or so protein-coding genes in the mouse genome.

Information on the International Knockout Mouse Consortium, which includes the European Conditional Mouse Mutagenesis program, the National Institutes of Health Knockout Mouse program, and other efforts centered in the US, UK, and Canada, is available online.

"Biomedical research needs biological resources on a scale that match genomics resources," Colin Fletcher, program director for the NIH Knockout Mouse Program, who was not directly involved in the study, said in a statement. "Such knockout resources are the foundation for producing thousands of valuable mouse mutants for future large-scale international phenotyping programs and will serve the biological and biomedical research community worldwide."

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