Havy N. NguyenKei ichi IshidohHiroshi KinoshitaTakuya NihiraOsaka UniversityMahidol University2019-08-232019-08-232018-07-01Journal of Microbiological Methods. Vol.150, (2018), 47-5418728359016770122-s2.0-85047523770https://repository.li.mahidol.ac.th/handle/20.500.14594/45110© 2018 Functionally related genes often form a large gene cluster on fungal genomes. To analyze, by heterologous expression system, overall pathway in which a series of related genes are involved, the whole gene cluster should be introduced intact into the host strain. However, the construction of a genomic library based on cosmid or bacterial artificial chromosome, and screening of a clone harboring the target region are time consuming and usually require additional cloning of missing regions. The available PCR-based methods are convenient, but are likely to cause unexpected errors during long-range PCR. Therefore, in this study we developed a method for targeted cloning of a large gene cluster based on Cre/loxP-mediated recombination. loxP sequences were integrated at both edges of the targeted region, and the region was excised and cloned as a circular fosmid by in vitro Cre recombination. To facilitate the Cre/loxP-based method, a competent host-vector system was developed, including a double auxotrophic Lecanicillium PTk3 (ΔpyrG trp1 − ku80 − ) strain and two vectors for introducing the loxP sequences, pUTlox and pCCPlox. A targeted region longer than 45 kb in length was successfully cloned by the Cre/loxP-based method.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyImmunology and MicrobiologyMedicineArticleSCOPUS10.1016/j.mimet.2018.05.017