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The loxP-kanMX-loxP marker

A new efficient gene disruption cassette for repeated use in budding yeast.

Ulrich Güldener, Susanne Heck, Thomas Fiedler, Jens Beinhauer and Johannes H. Hegemann

Heinrich-Heine-Universität, Institut fur Mikrobiologie, Geb. 26.12.01 Raum 64, Universitätsstr. 1, 40225 Düsseldorf, Germany, Tel.: +49-211-81-13733, Fax.: +49-211-81-15370, e-mail: hegemann@uni-duesseldorf.de

Abstract

The dominant kanr marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the convential yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous kanr marker with those from the Cre-loxP recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanr marker is excised by an efficient recombination between the loxP sites leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the kanr marker gene and will be of great advantage for the functional analysis of gene families.

Map of plasmid pUG6 carrying the loxP-kanMX-loxP disruption module

The 2 loxP sites are flanking as direct repeats the kanrmarker gene (kanr) plus TEF2 promoter (T.-P.) and TEF2 terminator (T.-T.) in plasmid pFAG-kanMX4 (A. Wach et al.,1994, YEAST 10:1793-1808). Unique restriction enzymes are indicated in bold letters.


Gene disruption using the loxP-kanMX-loxP disruption cassette

For a gene disruption experiment two oligonucleotides were used that carry at their 3' end a segment (arrow) homologous to sequences left and right of the loxP-kanMX-loxP module on plasmid pUG6 and at their 5' end a segment (shaded box) homologous to the Open Reading Frame (ORF) to be disrupted. Plasmid pUG6 was used as PCR template to generate the disruption cassette.


kanr Marker rescue by expression of the Cre recombinase

The diploid kan+ yeast strain with the relevant genotype ORF/ORF:: loxP-ka nMX-loxP was transformed with plasmid pSH47 (selection marker: URA3). Transformants were grown on glucose plates and then shifted to galactose media to induce expression of the Cre recombinase. The Cre-induced recombination proce ss between the 2 loxP sites removes the marker gene resulting in the genotype ORF/ORF::loxP. Additionally 2 other Cre-plasmids are availabl e: pSH62 (HIS3) and pSH63 (TRP1).



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