Efficient gene deletion systems in the Schizosaccharomyces pombe.
Kyotaro Hirashima (1), Tomoko Iwaki (2), Hideki Tohda (1), Hiromichi Kumagai (1), Yuko Giga-Hama (1), Kaoru Takegawa (2)
(1) ASPEX Division, Asahi Glass Co., Ltd., 1150 Hazawa Kanagawa, Yokohama, 221-8755, Japan; (2) Kagawa Univ. Mikicho Kagawa Japan, 761-0795
Efficient gene deletion systems are important tools required for the genome-wide functional gene analysis in many organisms, including fission yeast Schizosaccharomyces pombe ( S. pombe ). For repeated marker removal procedure, we recently constructed a lox P-flanked ura4 + auxotrophic marker cassette to replace the target gene locus using Cre -recombinase. After the introduction of marker cassette to the locus, excision at lox P sites in the chromosome was promoted efficiently and accurately when the Cre -recombinase was expressed under the control of nmt41 promoter. Although this method is applicable to any selection markers, there still remains one lox P sequence in chromosome. Here we also describe the development of another method for recycling of selection markers using cis -homologous recombination. Ura4 + marker cassette flanked by one of the adjacent sequence of target gene was also introduced into the target gene locus. Deletion mutant was selected by 5-fluoroorotic acid containing media. Sequence analysis of isolated clones revealed that foreign nucleotides including ura4 + marker was completely deleted without any insertion. This deletion probably caused by cis -homologous recombination of overlapping sequence. The set of Cre/lox P-mediated and cis -homologous recombination marker removal procedures described here proved to be a practical strategy to create large-scale chromosomal deletion strains in S. pombe. This study was founded by NEDO, Japan.