Puromycin- and methotrexate-resistance cassettes and optimized Cre-recombinase expression plasmids for use in yeast

TY - JOUR

T1 - Puromycin- and methotrexate-resistance cassettes and optimized Cre-recombinase expression plasmids for use in yeast

AU - MacDonald, Chris

AU - Piper, Robert C.

PY - 2015/5/1

Y1 - 2015/5/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84928582251&partnerID=8YFLogxK

U2 - 10.1002/yea.3069

DO - 10.1002/yea.3069

M3 - Article

C2 - 25688547

AN - SCOPUS:84928582251

SN - 0749-503X

VL - 32

SP - 423

EP - 438

JO - Yeast (Chichester, England)

JF - Yeast (Chichester, England)

IS - 5

ER -