Nucleic Acids Research, 2002, Vol. 30, No. 6 e23
© 2002 Oxford University Press
A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast
Heinrich-Heine-Universität, Institut für Mikrobiologie, Universitätsstrasse 1, Geb. 26.12.01.64, 40225 Düsseldorf, Germany
Heterologous markers are important tools required for the molecular dissection of gene function in many organisms, including Saccharomyces cerevisiae. Moreover, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene. We recently introduced a new and efficient gene disruption cassette for repeated use in budding yeast, which combines the heterologous dominant kanr resistance marker with a Cre/loxP-mediated marker removal procedure. Here we describe an additional set of four completely heterologous loxP-flanked marker cassettes carrying the genes URA3 and LEU2 from Kluyveromyces lactis, his5+ from Schizosaccharomyces pombe and the dominant resistance marker bler from the bacterial transposon Tn5, which confers resistance to the antibiotic phleomycin. All five loxP–marker gene–loxP gene disruption cassettes can be generated using the same pair of oligonucleotides and all can be used for gene disruption with high efficiency. For marker rescue we have created three additional Cre expression vectors carrying HIS3, TRP1 or bler as the yeast selection marker. The set of disruption cassettes and Cre expression plasmids described here represents a significant further development of the marker rescue system, which is ideally suited to functional analysis of the yeast genome.
* To whom correspondence should be addressed. Tel: +49 211 81 13733; Fax: +49 211 81 13567; Email: hegemann{at}uni-duesseldorf.dePresent addresses:U. Gueldener, MIPS, GSF, Ingolstaedter Landstrasse 1, 85764 Neuherberg, GermanyJ. Heinisch, Universität Hohenheim, Institut für Lebensmitteltechnologie (150), Fachgebiet Gärungstechnologie, Garbenstrasse 25, 70599 Stuttgart, Germany +AF298780, AF298782, AF298785, AF298788–AF298790, AF298792–AF298794
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