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Nucleic Acids Research Advance Access originally published online on April 10, 2007
Nucleic Acids Research 2007 35(8):e64; doi:10.1093/nar/gkm163
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Nucleic Acids Research, 2007, Vol. 35, No. 8 e64
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A recombineering based approach for high-throughput conditional knockout targeting vector construction

Waiin Chan1, Nina Costantino2, Ruixue Li1, Song Choon Lee1, Qin Su1, David Melvin1, Donald L. Court2,* and Pentao Liu1,*

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.


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S.-C. Lee and P. Liu
Construction of Gene-Targeting Vectors by Recombineering
CSH Protocols, September 1, 2009; 2009(9): pdb.prot5291 - pdb.prot5291.
[Abstract] [Full Text]


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