A 2-mu m DNA-based marker recycling system for multiple gene disruption inthe yeast Saccharomyces cerevisiae

A molecular FRT (Flp recombinase recognition target)-based cassette system for multiple gene disruption in the yeast Saccharomyces cerevisiae was deve loped. FRT DNA sequences were designed with different core mutations and su bsequently cloned in direct orientation upstream and downstream of a marker gene to serve as template for the amplification of a set of different gene disruption cassettes. After each disruption, the marker can be easily elim inated from its integration site by ill vivo sits-specific recombination be tween the two identical, mutated FRT sequences flanking the marker, leaving behind one FRT sequence with a particular point mutation. Since recombinat ion between two FRTs with a different core mutation is extremely rare, the possibility of chromosome rearrangements, due to site-specific recombinatio n between residual FRTs, is very low. In strains containing 2-mu m ([cir(+) ]) the site-specific reaction is catalysed by the endogenous Flp gene produ ct, whereas in strains without 2-mu m ([cir(0)]), the FLP gene is carried o n the cassette, together with the marker gene. This system can be applied f or haploid and diploid [cir(+)] and [cir(0)] strains. Copyright (C) 1999 Jo hn Wiley & Sons, Ltd.