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Nucleic Acids Research 2005 33(18):e163; doi:10.1093/nar/gni164
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Published online 27 October 2005

© The Author 2005. Published by Oxford University Press. All rights reserved
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions{at}oxfordjournals.org

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Gene CATCHR—Gene Cloning And Tagging for Caenorhabditis elegans using yeast Homologous Recombination: a novel approach for the analysis of gene expression

Holly E. Sassi1,2, Stephanie Renihan1, Andrew M. Spence1,2 and Ramona L. Cooperstock1,*

1Department of Medical Genetics and Microbiology, University of Toronto 1 King's College Circle, Toronto, Canada, M5S 1A8 2Collaborative Program in Developmental Biology, University of Toronto 1 King's College Circle, Toronto, Canada, M5S 1A8

*To whom correspondence should be addressed. Tel: +1 416 946 7917; Fax: +1 416 978 6885; Email: r.cooperstock{at}utoronto.ca

Received August 31, 2005. Revised September 29, 2005. Accepted September 29, 2005.

Expression patterns of gene products provide important insights into gene function. Reporter constructs are frequently used to analyze gene expression in Caenorhabditis elegans, but the sequence context of a given gene is inevitably altered in such constructs. As a result, these transgenes may lack regulatory elements required for proper gene expression. We developed Gene CATCHR, a novel method of generating reporter constructs that exploits yeast homologous recombination (YHR) to subclone and tag worm genes while preserving their local sequence context. YHR facilitates the cloning of large genomic regions, allowing the isolation of regulatory sequences in promoters, introns, untranslated regions and flanking DNA. The endogenous regulatory context of a given gene is thus preserved, producing expression patterns that are as accurate as possible. Gene CATCHR is flexible: any tag can be inserted at any position without introducing extra sequence. Each step is simple and can be adapted to process multiple genes in parallel. We show that expression patterns derived from Gene CATCHR transgenes are consistent with previous reports and also describe novel expression data. Mutant rescue assays demonstrate that Gene CATCHR-generated transgenes are functional. Our results validate the use of Gene CATCHR as a valuable tool to study spatiotemporal gene expression.


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