title: Accumulative gene integration into a pre-determined site using Cre/loxP.
abstract: Site-specific gene recombination systems, such as Cre/loxP, have been used for genetic modification of cells and organisms in both basic and applied research. We previously developed an accumulative gene integration system (AGIS), in which target gene cassettes could be repeatedly integrated into a pre-determined site on a plasmid or cellular genome by recombinase-mediated cassette exchange (RMCE), using Cre and mutated loxPs. In the present study, we designed a simplified AGIS. For gene integration into a target site, the previous system used two loxP sites in the acceptor DNA, whereas the new system uses a single loxP site. The gene integration reactions were repeated four times in vitro using Cre protein and specific plasmids. The expected integration reactions mediated by Cre occurred at the loxP sites, resulting in integration of four target genes. The system was also used for genomic integration of reporter genes using Chinese hamster ovary (CHO) cells. The reporter genes were efficiently introduced into the CHO genome in a Cre-dependent manner, and transgene expression was detected after the integration reaction. The expression levels of the reporter genes were enhanced, corresponding to the increase of transgene copy number. Recombinase-mediated AGIS provides a useful tool for the modification of cellular genomes.
authors.affiliation: Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Obayashi
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Kawabe
authors.forename: Hirokatsu
authors.initials: H
authors.lastname: Makitsubo
authors.forename: Ryoko
authors.initials: R
authors.lastname: Watanabe
authors.forename: Yujiro
authors.initials: Y
authors.lastname: Kameyama
authors.forename: Shuohao
authors.initials: S
authors.lastname: Huang
authors.forename: Yuta
authors.initials: Y
authors.lastname: Takenouchi
authors.forename: Akira
authors.initials: A
authors.lastname: Ito
authors.forename: Masamichi
authors.initials: M
authors.lastname: Kamihira
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D016678:Genome; D010957:Plasmids; D045522:Recombinases / Q000235:genetics* / Q000378:metabolism*; D019076:Transgenes / Q000235:genetics*
chemical_list: D045522:Recombinases