A set of loxP marker cassettes for Cre-mediated multiple gene disruption in Schizosaccharomyces pombe

Biosci Biotechnol Biochem. 2004 Mar;68(3):545-50. doi: 10.1271/bbb.68.545.

Abstract

For functional analysis, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene, while the number of marker genes is limited in Schizosaccharomyces pombe. Here we describe a loxP-flanked ura4(+) cassette and Cre recombinase vector for a Cre-loxP-mediated marker removal procedure in S. pombe. This loxP-ura4-loxP cassette can be used for disruption of hmt1(+) as a model target gene. We have constructed two vectors which express Cre recombinase under the control of the nmt1 or nmt41 promoter. Excisive recombination at loxP sites in the chromosome was promoted efficiently and accurately when the Cre recombinase was expressed under the control of the nmt41 promoter. In addition, ura4(+) could be excised from the genome by Cre recombinase, when a single loxP site was adjacent to ura4. The use of the Cre-loxP system proved to be a practical strategy to excise a marker gene for repeated use in S. pombe.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • ATP-Binding Cassette Transporters / genetics
  • Base Sequence
  • Cadmium Chloride / toxicity
  • Gene Expression Regulation, Fungal
  • Gene Silencing
  • Genetic Markers
  • Genetic Vectors / genetics*
  • Integrases / genetics*
  • Integrases / metabolism
  • Isoenzymes / genetics
  • Molecular Sequence Data
  • Mutagenesis, Insertional / methods*
  • Plasmids / genetics
  • Promoter Regions, Genetic
  • Recombination, Genetic*
  • Schizosaccharomyces / genetics*
  • Schizosaccharomyces pombe Proteins / genetics
  • Thiamine / metabolism
  • Viral Proteins / genetics*
  • Viral Proteins / metabolism

Substances

  • ATP-Binding Cassette Transporters
  • Genetic Markers
  • Isoenzymes
  • NMT1 protein, S pombe
  • Schizosaccharomyces pombe Proteins
  • Viral Proteins
  • hmt1 protein, S pombe
  • ura4 protein, S pombe
  • Cre recombinase
  • Integrases
  • Cadmium Chloride
  • Thiamine