title: Mouse embryonic stem cells with a multi-integrase mouse artificial chromosome for transchromosomic mouse generation.
abstract: The mouse artificial chromosome (MAC) has several advantages as a gene delivery vector, including stable episomal maintenance of the exogenous genetic material and the ability to carry large and/or multiple gene inserts including their regulatory elements. Previously, a MAC containing multi-integration site (MI-MAC) was generated to facilitate transfer of multiple genes into desired cells. To generate transchromosomic (Tc) mice containing a MI-MAC with genes of interest, the desired genes were inserted into MI-MAC in CHO cells, and then the MI-MAC was transferred to mouse embryonic stem (mES) cells via microcell-mediated chromosome transfer (MMCT). However, the efficiency of MMCT from CHO to mES cells is very low (<10(-6)). In this study, we constructed mES cell lines containing a MI-MAC vector to directly insert a gene of interest into the MI-MAC in mES cells via a simple transfection method for Tc mouse generation. The recombination rate of the GFP gene at each attachment site (FRT, PhiC31attP, R4attP, TP901-1attP and Bxb1attP) on MI-MAC was greater than 50% in MI-MAC mES cells. Chimeric mice with high coat colour chimerism were generated from the MI-MAC mES cell lines and germline transmission from the chimera was observed. As an example for the generation of Tc mice with a desired gene by the MI-MAC mES approach, a Tc mouse strain ubiquitously expressing Emerald luciferase was efficiently established. Thus, the findings suggest that this new Tc strategy employing mES cells and a MI-MAC vector is efficient and useful for animal transgenesis.
authors.affiliation: Division of Laboratory Animal Science, Research Center for Bioscience and Technology, Tottori University, 86 Nishi-cho, Yonago, Tottori, 683-8503, Japan.
authors.forename: Yuki
authors.initials: Y
authors.lastname: Yoshimura
authors.forename: Kazuomi
authors.initials: K
authors.lastname: Nakamura
authors.forename: Takeshi
authors.initials: T
authors.lastname: Endo
authors.forename: Naoyo
authors.initials: N
authors.lastname: Kajitani
authors.forename: Kanako
authors.initials: K
authors.lastname: Kazuki
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kazuki
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Kugoh
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ohbayashi
mesh_terms: D000818:Animals; D016466:CHO Cells; D002678:Chimera; D022221:Chromosomes, Artificial, Mammalian / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D005854:Germ Cells; D006801:Humans; D019426:Integrases / Q000235:genetics*; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D008822:Mice, Transgenic; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D011995:Recombination, Genetic; D019076:Transgenes / Q000235:genetics
chemical_list: D019426:Integrases