Cre/loxP-mediated in vivo excision of large segments from yeast genome and their amplification based on the 2 μm plasmid-derived system

Young Geol Yoon, György Pósfai, Waclaw Szybalski, Sun Chang Kim

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

In vivo excision and amplification of pre-determined, large genomic segments, directly from the genome of a natural host, provides an alternative to conventional cloning in foreign vectors. Using this approach, we have devised an in vivo procedure for excising large segments of Saccharomyces cerevisiae genome using Cre/loxP system of bacteriophage P1, followed by amplification of excised circles, as based on the yeast 2 μm plasmid- derived ori and Flp/FRT machinery. To provide the excision and replication enzymes, trans-acting genes cre and FLP, which were under a very tight control of GAL1 and GAL10 promoters, respectively, were inserted by homologous recombination into the URA3 gene on chromosome V. Two parallel loxP sequences, which serve as the recognition sites for the Cre recombinase, were also integrated into the genome at pre-determined sites that are 50-100 kb apart. Moreover, 2 gm ori, REP3 and two inverted FRTs, which serve as a conditional replication system, were also integrated between the loxP sites. The strain carrying all these inserted elements was perfectly stable. Only after the induction by galactose of the Cre excision function, the genomic segment flanked by two loxP sites was excised and circularized. Applying this procedure, the 50-kb LEU2-YCR011c and 100-kb LEU2-YCR035c regions of chromosome III were successfully excised from the S. cerevisiae genome, whereas the 2 μm ori, as aided by FRT/Flp, provided the amplification function. Such excised and amplified genomic segments can be used for the sequencing and functional analysis of any yeast genes.

Original languageEnglish
Pages (from-to)67-76
Number of pages10
JournalGene
Volume223
Issue number1-2
DOIs
Publication statusPublished - Nov 26 1998

Keywords

  • Homologous recombination
  • Large DNA fragments generateddirectly from a natural host
  • Saccharomyces cerevisiae
  • Site-specific recombination
  • Yeast chromosome III

ASJC Scopus subject areas

  • Genetics

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