title: Modified Cre-loxP recombination in Aspergillus oryzae by direct introduction of Cre recombinase for marker gene rescue.
abstract: Marker rescue is an important molecular technique that enables sequential gene deletions. The Cre-loxP recombination system has been used for marker gene rescue in various organisms, including aspergilli. However, this system requires many time-consuming steps, including construction of a Cre expression plasmid, introduction of the plasmid, and Cre expression in the transformant. To circumvent this laborious process, we investigated a method wherein Cre could be directly introduced into Aspergillus oryzae protoplasts on carrier DNA such as a fragment or plasmid. In this study, we define the carrier DNA (Cre carrier) as a carrier for the Cre enzyme. A mixture of commercial Cre and nucleic acids (e.g., pUG6 plasmid) was introduced into A. oryzae protoplasts using a modified protoplast-polyethylene glycol method, resulting in the deletion of a selectable marker gene flanked by loxP sites. By using this method, we readily constructed a marker gene-rescued strain lacking ligD to optimize homologous recombination. Furthermore, we succeeded in integrative recombination at a loxP site in A. oryzae. Thus, we developed a simple method to use the Cre-loxP recombination system in A. oryzae by direct introduction of Cre into protoplasts using DNA as a carrier for the enzyme.
authors.affiliation: National Research Institute of Brewing, Hiroshima, Japan. mizutani@nrib.go.jp
authors.forename: Osamu
authors.initials: O
authors.lastname: Mizutani
authors.forename: Kazuo
authors.initials: K
authors.lastname: Masaki
authors.forename: Katsuya
authors.initials: K
authors.lastname: Gomi
authors.forename: Haruyuki
authors.initials: H
authors.lastname: Iefuji
mesh_terms: D001236:Aspergillus oryzae / Q000235:genetics*; D017353:Gene Deletion; D015870:Gene Expression; D005827:Genetics, Microbial / Q000379:methods*; D019426:Integrases / Q000235:genetics* / Q000378:metabolism*; D008967:Molecular Biology / Q000379:methods*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic
chemical_list: D011994:Recombinant Proteins; C045073:Cre recombinase; D019426:Integrases