Three novel antibiotic marker cassettes for gene disruption and marker switching in Schizosaccharomyces pombe.
The ease of construction of multiple mutant strains in Schizosaccharomyces pombe is limited by the number of available genetic markers. We describe here three new cassettes for PCR-mediated gene disruption that can be used in combination with commonly used fission yeast markers to make multiple gene deletions. The natMX6, hphMX6 and bleMX6 markers give rise to resistance towards the antibiotics nourseothricin (NAT), hygromycin B and phleomycin, respectively. The cassettes are composed of exogenous sequences to increase the frequency of integration at targeted loci, and have a structure similar to the commonly used pFA6a-kanMX6 modular plasmid system. This allows a simple exchange of the kanMX6 marker in existing strains with any of the three new cassettes. Alternatively, oligonucleotide primers designed for the modular kanMX6 cassettes can be used to make the transforming PCR fragments for gene disruption. We illustrate the construction of a mutant strain with six independent gene disruptions, using the novel antibiotic cassettes in combination with existing genetic markers.
Hentges
Pierre
P
Genome Damage and Stability Centre, University of Sussex, Brighton BN1 9RQ, UK.
Van Driessche
Benoit
B
Tafforeau
Lionel
L
Vandenhaute
Jean
J
Carr
Antony M
AM
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D000935:Antifungal Agents / Q000494:pharmacology; D001426:Bacterial Proteins / Q000235:genetics*; D025141:Drug Resistance, Fungal / Q000235:genetics*; D017353:Gene Deletion; D005819:Genetic Markers; D006921:Hygromycin B / Q000494:pharmacology; D010692:Phleomycins / Q000494:pharmacology; D012568:Schizosaccharomyces / Q000187:drug effects* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics*; D013309:Streptothricins / Q000494:pharmacology; D014170:Transformation, Genetic
D000900:Anti-Bacterial Agents; D000935:Antifungal Agents; D001426:Bacterial Proteins; D005819:Genetic Markers; D010692:Phleomycins; D029702:Schizosaccharomyces pombe Proteins; D006921:Hygromycin B; D013309:Streptothricins