However, as these antibiotic cassettes all share the same promoter and terminator sequences, it is difficult to perform sequential gene targeting due to the shared homology. Thus, use of antibiotic resistance markers has become standard for yeast organisms, as the working strain does not require any particular auxotrophic genetic background. However, when using auxotrophic cassettes, strains must have mutations in the corresponding endogenous gene, which could affect cell metabolism, stress response and cell growth. pombe and LEU2 from Saccharomyces cerevisiae have also been extensively used in fission yeast. Auxotrophic gene cassettes, such as ura4 + from S. These cassettes are composed of a promoter and terminator from the fungus Ashbya gossypii and genes conferring selectable resistance to G418 (geneticin or neomycin), nourseothricin (ClonNat or Nat), hygromycin B and bleomycin respectively. In fission yeast, commonly used drug selection cassettes are kanMX6, natMX6, hygMX6 ( hphMX6) and bleMX6. Generated strains can be further crossed to isolate multiple genetic alternations that can be selected for using combination of appropriate markers. A strain of induced genotype can be isolated simply by plating transformants onto corresponding selection plates. In yeast, genes can be deleted and replaced with selection markers readily, simply by inducing homology directed repair with a selection cassette amplified with primers that contain short, flanking sequences homologous to a target gene. A haploid life cycle along with efficient homologous recombination system and simple reproductive processes make it an excellent system to study genetics. The plasmid constructs and techniques described here are invaluable tools for sequential gene targeting and will simplify construction of fission yeast strains required for study.įission yeast Schizosaccharomyces pombe is a popular model organism for fundamental biological research. Finally, utilizing the negative selectable markers, we showed efficient induction of point mutations in an endogenous gene by a two-step transformation method. To remove counter-selectable markers, we introduced the Cre-loxP irreversible recombination method. However, a number of DNA replication checkpoint and homologous recombination mutants, including rad3∆, cds1∆, rad54∆ and rad55∆, exhibited sensitivity to FdU even though those strains did not carry the HSV-TK gene. We noted that the efficiency of the counter selection by FdU was enhanced by treatment with hydroxyurea. By fusing an antibiotic resistance gene to HSV-TK, we were able to select on the relevant antibiotic as well as counter-select on FdU media to confirm the desired genomic modification had been made. We also utilized the anti-selection gene HSV-TK, which results in cell death in strains grown on the drug 5-Fluoro-2’-deoxyuridine (FdU, Floxuridin or FUDR). In addition, we have generated a series of plasmids to facilitate the C-terminal tagging of genes with desired epitopes. The newly created selection cassettes did not recombine with the tef-containing MX6 cassettes already present in the genome, allowing for sequential gene targeting using the PCR-based method. We achieved this by swapping the tef promoter and terminator sequences in the established antibiotic resistance MX6 cassette series for alternative promoters and/or terminators. Here we have generated new selection cassettes that do not recombine with those traditionally used. It can therefore be challenging to perform multiple genetic modifications by PCR-based targeting, as existing resistance cassettes in strains can be favored for recombination due to shared homology between the cassettes. However, the currently available cassettes all share the same tef promoter and terminator sequences. In yeasts, this is achieved by PCR amplification of the cassette flanked by short homology sequences, which can be incorporated by homology directed repair. For selection of desirable genotypes, antibiotic resistance cassettes are widely integrated into the genome near genes of interest. Fission yeast is one of the most commonly used model organisms for studying genetics.
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