Puromycin- And Methotrexate-Resistance Cassettes and Optimized Cre-recombinase Expression Plasmids for Use in Yeast

Yeast. 2015 May;32(5):423-38. doi: 10.1002/yea.3069. Epub 2015 Mar 19.

Abstract

Here we expand the set of tools for genetically manipulating Saccharomyces cerevisiae. We show that puromycin-resistance can be achieved in yeast through expression of a bacterial puromycin-resistance gene optimized to the yeast codon bias, which in turn serves as an easy-to-use dominant genetic marker suitable for gene disruption. We have constructed a similar DNA cassette expressing yeast codon-optimized mutant human dihydrofolate reductase (DHFR), which confers resistance to methotrexate and can also be used as a dominant selectable marker. Both of these drug-resistant marker cassettes are flanked by loxP sites, allowing for their excision from the genome following expression of Cre-recombinase. Finally, we have created a series of plasmids for low-level constitutive expression of Cre-recombinase in yeast that allows for efficient excision of loxP-flanked markers.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Codon
  • Drug Resistance, Fungal*
  • Genetic Markers
  • Integrases / genetics*
  • Integrases / metabolism
  • Methotrexate / pharmacology*
  • Plasmids / genetics*
  • Plasmids / metabolism
  • Protein Engineering
  • Puromycin / pharmacology*
  • Saccharomyces cerevisiae / drug effects*
  • Saccharomyces cerevisiae / genetics*
  • Saccharomyces cerevisiae / metabolism

Substances

  • Codon
  • Genetic Markers
  • Puromycin
  • Cre recombinase
  • Integrases
  • Methotrexate