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Nucleic Acids Research, 2000, Vol. 28, No. 9 E41-e41
© 2000 Oxford University Press

Retro-recombination screening of a mouse embryonic stem cell genomic library

Knut Woltjen, Gerard Bain and Derrick E. Rancourt*

Southern Alberta Cancer Research Center, Departments of Oncology and Biochemistry and Molecular Biology, University of Calgary, Calgary, Alberta T2N 4N1, Canada

Targeted gene disruption is an important tool in molecular medicine, allowing for the generation of animal models of human disease. Conventional methods of targeting vector (TV) construction are difficult and represent a rate limiting step in any targeting experiment. We previously demonstrated that bacteriophage are capable of acting as TVs directly, obviating the requirement for ‘rolling out’ plasmids from primary phage clones and thus eliminating an additional, time consuming step. We have also developed methods which facilitate the construction of TVs using recombination. In this approach, modification cassettes and point mutations are shuttled to specific sites in phage TVs using phage–plasmid recombination. Here, we report a further improvement in TV generation using a recombination screening-based approach deemed ‘retro-recombination screening’ (RRS). We demonstrate that phage vectors containing specific genomic clones can be genetically isolated from a {lambda}TK embryonic stem cell genomic library using a cycle of integrative recombination and condensation. By introducing the gam gene of bacteriophage {lambda} into the probe plasmid it is possible to select for positive clones which have excised the plasmid, thus returning to their native conformation following purification from the library. Rapid clone isolation using the RRS protocol provides another method by which the time required for TV construction may be further reduced.

* To whom correspondence should be addressed. Tel: +1 403 220 2888; Fax: +1 403 283 8727; Email: rancourt@ucalgary.ca Present address: Gerard Bain, Aventis Pharmaceuticals Inc., 26 Landsdowne Street, Cambridge, MA 02139-4234, USA


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