Nucleic Acids Research Advance Access published online on April 10, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm163
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Methods Online |
A recombineering based approach for high-throughput conditional knockout targeting vector construction
1Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK and 2National Cancer Institute-Frederick, Frederick, MD 21702, USA
*To whom correspondence should be addressed. Pentao Liu. Tel: +44 (0) 1223-4968050; Fax: +(44) (0) 1223-496802; Email: pl2{at}sanger.ac.uk or Don Court. Tel: +(301) 846-5940; Fax: +(301) 846-6988; Email: court{at}ncifcrf.gov
Received January 28, 2007. Revised March 5, 2007. Accepted March 5, 2007.
Functional analysis of mammalian genes in vivo is primarily achieved through analysing knockout mice. Now that the sequencing of several mammalian genomes has been completed, understanding functions of all the genes represents the next major challenge in the post-genome era. Generation of knockout mutant mice has currently been achieved by many research groups but only by making individual knockouts, one by one. New technological advances and the refinements of existing technologies are critical for genome-wide targeted mutagenesis in the mouse. We describe here new recombineering reagents and protocols that enable recombineering to be carried out in a 96-well format. Consequently, we are able to construct 96 conditional knockout targeting vectors simultaneously. Our new recombineering system makes it a reality to generate large numbers of precisely engineered DNA constructs for functional genomics studies.