title: Improved transgene integration into the Chinese hamster ovary cell genome using the Cre-loxP system.
abstract: Genetic engineering of cellular genomes has provided useful tools for biomedical and pharmaceutical studies such as the generation of transgenic animals and producer cells of biopharmaceutical proteins. Gene integration using site-specific recombinases enables precise transgene insertion into predetermined genomic sites if the target site sequence is introduced into a specific chromosomal locus. We previously developed an accumulative site-specific gene integration system (AGIS) using Cre and mutated loxPs. The system enabled the repeated integration of multiple transgenes into a predetermined locus of a genome. In this study, we explored applicable mutated loxP pairs for AGIS to improve the integration efficiency. The integration efficiencies of 52 mutated loxP sequences, including novel sequences, were measured using an in vitro evaluation system. Among mutated loxP pairs that exhibited a high integration efficiency, the applicability of the selected pairs to AGIS was confirmed for transgene integration into the Chinese hamster ovary cell genome. The newly found mutated loxP pairs should be useful for Cre-mediated integration of transgenes and AGIS.
authors.affiliation: Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Inao
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kawabe
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Takuro
authors.initials: T
authors.lastname: Yamashiro
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Yujiro
authors.initials: Y
authors.lastname: Kameyama
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Xue
authors.initials: X
authors.lastname: Wang
authors.affiliation: Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Ito
authors.affiliation: Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan; Department of Chemical Engineering, Faculty of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan. Electronic address: kamihira@chem-eng.kyushu-u.ac.jp.
authors.forename: Masamichi
authors.initials: M
authors.lastname: Kamihira
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001287:Attachment Sites, Microbiological / Q000235:genetics*; D016466:CHO Cells; D003412:Cricetulus; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D016678:Genome / Q000235:genetics*; D019426:Integrases / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D011995:Recombination, Genetic; D019076:Transgenes / Q000235:genetics*
chemical_list: C045073:Cre recombinase; D019426:Integrases
keywords: Accumulative site-specific gene integration; Chinese hamster ovary cells; Cre/loxP; Mutated loxP; Recombinase-mediated cassette exchange