Cre/loxP-mediated in vivo excision of large segments from yeast genome andtheir amplification based on the 2 mu m plasmid-derived system

In vivo excision and amplification of pre-determined, large genomic segment s, directly from the genome of a natural host, provides an alternative to c onventional cloning in foreign vectors. Using this approach, we have devise d an in vivo procedure for excising large segments of Saccharomyces cerevis iae genome using Cre/loxP system of bacteriophage P1, followed by amplifica tion of excised circles, as based on the yeast 2 mu m plasmid-derived ori a nd Flp/FRT machinery. To provide the excision and replication enzymes, tran s-acting genes cre and FLP, which were under a very tight control of GAL1 a nd GAL10 promoters; respectively, were inserted by homologous recombination into the URA3 gene on chromosome V. Two parallel loxP sequences, which ser ve as the recognition sites for the Cre recombinase, were also integrated i nto the genome at pre-determined sites that are 50-100 kb apart. Moreover, 2 mu m ori, REP3 and two inverted FRTs, which serve as a conditional replic ation system, were also integrated between the loxP sites. The strain carry ing all these inserted elements was perfectly stable. Only after the induct ion by galactose of the Cre excision function, the genomic segment flanked by two loxP sites was excised and circularized. Applying this procedure, th e 50-kb LEU2-YCR011c and 100-kb LEU2-YCR035c regions of chromosome III were successfully excised from the S. cerevisiae genome, whereas the 2 mu m ori , as aided by FRT/Flp, provided the amplification function. Such excised an d amplified genomic segments can be used for the sequencing and functional analysis of any yeast genes. (C) 1998 Elsevier Science B.V. All rights rese rved.