Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection

Nat Methods. 2011 Feb;8(2):159-64. doi: 10.1038/nmeth.1550. Epub 2011 Jan 9.

Abstract

Phenotypes that might otherwise reveal a gene's function can be obscured by genes with overlapping function. This phenomenon is best known within gene families, in which an important shared function may only be revealed by mutating all family members. Here we describe the 'green monster' technology that enables precise deletion of many genes. In this method, a population of deletion strains with each deletion marked by an inducible green fluorescent protein reporter gene, is subjected to repeated rounds of mating, meiosis and flow-cytometric enrichment. This results in the aggregation of multiple deletion loci in single cells. The green monster strategy is potentially applicable to assembling other engineered alterations in any species with sex or alternative means of allelic assortment. To test the technology, we generated a single broadly drug-sensitive strain of Saccharomyces cerevisiae bearing precise deletions of all 16 ATP-binding cassette transporters within clades associated with multidrug resistance.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • ATP-Binding Cassette Transporters / genetics
  • ATP-Binding Cassette Transporters / metabolism
  • Gene Deletion*
  • Gene Knockout Techniques / methods*
  • Green Fluorescent Proteins / analysis*
  • Green Fluorescent Proteins / genetics
  • Multigene Family*
  • Saccharomyces cerevisiae / chemistry
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / growth & development
  • Saccharomyces cerevisiae / metabolism

Substances

  • ATP-Binding Cassette Transporters
  • Green Fluorescent Proteins