The loxP-kanMX-loxP marker
A new efficient gene disruption cassette for repeated use in budding
|Yeast - useful tools
Ulrich Güldener, Susanne Heck, Thomas Fiedler, Jens Beinhauer and Johannes H.
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: email@example.com
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
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
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).
- Güldener, U., Heck, S., Fiedler, T., Beinhauer, J. and Hegemann, J.H. (1996).
A new efficient gene disruption cassette for repeated use in in
budding yeast. Nucleic Acids Res. 24:2519-2524 (1996)