The mouse is the leading vertebrate model because its genome can be altered by both random transgenesis and homologous recombination with targeting constructs. Both approaches have been hindered by the size and site limitations implicit in conventional Escherichia coli DNA-engineering methods. Homologous recombination in E. coli, or 'recombineering', has overcome these limitations for bacterial artificial chromosome (BAC) transgenesis1,2,3. Here we applied Red/ET recombineering (using the lambda Redα/Redβ recombinase pair)4,5,6 to generate a 64 kilobase targeting construct that carried two selectable cassettes permitting the simultaneous mutation of the target gene, Mll, at sites 43 kb apart in one round of mouse embryonic stem (ES) cell targeting. The targeting frequency after dual selection was 6%. Because the two selectable cassettes were flanked by FRT or loxP sites, three more alleles can be generated by site-specific recombination. Our approach represents a simple way to introduce changes at two or more sites in a genetic locus, and thereafter generate allele combinations. The size of BAC templates offers new freedom for the design of targeting constructs. Combined with the use of two selectable cassettes placed far apart, BAC-based targeting constructs may be applicable to tasks such as regional exchanges, deletions, and insertions.
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We wish to thank Konstantinos Anastassiadis, William Brown, Frank van der Hoeven, Robin Lovell-Badge, and Daniela Nebenius-Oosthuizen for discussions and help. This work was partly funded by a grant from the Volkswagen Foundation, Program on Conditional Mutagenesis. The work was initiated at the European Molecular Biology Laboratory (EMBL), Heidelberg.
The Red/ET DNA engineering technology is the subject of two issued patents authored by Drs. Zhang and Stewart. These patents are owned by EMBL which issued exclusive rights to GeneBridges GmbH, a company cofounded by Drs. Zhang and Stewart to develop the commercial implications of the patents. Dr. Zhang is CSO and Dr. Stewart chairperson of GeneBridges GmbH. Both are shareholders.
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Testa, G., Zhang, Y., Vintersten, K. et al. Engineering the mouse genome with bacterial artificial chromosomes to create multipurpose alleles. Nat Biotechnol 21, 443–447 (2003). https://doi.org/10.1038/nbt804
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