Cite as: Cold Spring Harb. Protoc.; 2009; doi:10.1101/pdb.prot5291

This Protocol
Right arrow Full Text
Right arrow Update/discuss this protocolDiscussion icon
Right arrow Alert me when this protocol is cited
Right arrow Alert me when comments are published
Right arrow Alert me if a correction is posted
Services
Right arrow Similar protocols in this database
Right arrow Similar articles in PubMed
Right arrow Alert me to new releases of protocols
Right arrow Save to Personal Folders
Right arrow Download to citation manager
Right arrow Printer-friendly versionPrinter-friendly version
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lee, S.-C.
Right arrow Articles by Liu, P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lee, S.-C.
Right arrow Articles by Liu, P.
Related Collections
Right arrow Genetics, general
Right arrow Laboratory Organisms, general
Right arrow Mouse
Right arrow Molecular Biology, general
Right arrow High-Capacity Vectors
Right arrow BACs
Right arrow Plasmids
Right arrow Vectors
Right arrow Transgenic Technology, general
Right arrow Transgenic Mice
Right arrow Transgenic Mice, general
Right arrow DNA Delivery/Gene Transfer
Right arrow DNA Delivery/Gene Transfer, general
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

protocolProtocol

Construction of Gene-Targeting Vectors by Recombineering

Song-Choon Lee and Pentao Liu1

Mouse Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, United Kingdom

1Corresponding author (pl2{at}sanger.ac.uk).


INTRODUCTION

The critical step in generating a knockout mouse is gene-targeting vector construction. Recombineering technology has greatly streamlined the vector construction process. This protocol describes a method for making conditional knockout (cko) targeting vectors using the pSim18 plasmid. This plasmid carries the three {lambda} phage Red genes (Gam, Bet, and Exo) under the control of the pL promoter, which is in turn regulated by the temperature-sensitive CI857 repressor. Hence, the heat-inducible recombineering functions can be easily delivered to bacterial artificial chromosomes (BACs) using a simple plasmid transformation, allowing one to manipulate any cloned mouse genomic region in Escherichia coli for cko vector construction. The conditional targeting vectors described in this protocol generate a flexible reporter/null/conditional allele in the mouse.


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?