title: Knocking out multigene redundancies via cycles of sexual assortment and fluorescence selection.
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.
authors.affiliation: Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Yo
authors.initials: Y
authors.lastname: Suzuki
authors.forename: Robert P
authors.initials: RP
authors.lastname: St Onge
authors.forename: Ramamurthy
authors.initials: R
authors.lastname: Mani
authors.forename: Oliver D
authors.initials: OD
authors.lastname: King
authors.forename: Adrian
authors.initials: A
authors.lastname: Heilbut
authors.forename: Vyacheslav M
authors.initials: VM
authors.lastname: Labunskyy
authors.forename: Weidong
authors.initials: W
authors.lastname: Chen
authors.forename: Linda
authors.initials: L
authors.lastname: Pham
authors.forename: Lan V
authors.initials: LV
authors.lastname: Zhang
authors.forename: Amy H Y
authors.initials: AH
authors.lastname: Tong
authors.forename: Corey
authors.initials: C
authors.lastname: Nislow
authors.forename: Guri
authors.initials: G
authors.lastname: Giaever
authors.forename: Vadim N
authors.initials: VN
authors.lastname: Gladyshev
authors.forename: Marc
authors.initials: M
authors.lastname: Vidal
authors.forename: Peter
authors.initials: P
authors.lastname: Schow
authors.forename: Joseph
authors.initials: J
authors.lastname: Lehár
authors.forename: Frederick P
authors.initials: FP
authors.lastname: Roth
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D055786:Gene Knockout Techniques / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000032:analysis* / Q000235:genetics; D005810:Multigene Family; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics / Q000254:growth & development / Q000378:metabolism
chemical_list: D018528:ATP-Binding Cassette Transporters; D049452:Green Fluorescent Proteins