title: A novel system for simultaneous or sequential integration of multiple gene-loading vectors into a defined site of a human artificial chromosome.
abstract: Human artificial chromosomes (HACs) are gene-delivery vectors suitable for introducing large DNA fragments into mammalian cells. Although a HAC theoretically incorporates multiple gene expression cassettes of unlimited DNA size, its application has been limited because the conventional gene-loading system accepts only one gene-loading vector (GLV) into a HAC. We report a novel method for the simultaneous or sequential integration of multiple GLVs into a HAC vector (designated as the SIM system) via combined usage of Cre, FLP, Bxb1, and φC31 recombinase/integrase. As a proof of principle, we first attempted simultaneous integration of three GLVs encoding EGFP, Venus, and TdTomato into a gene-loading site of a HAC in CHO cells. These cells successfully expressed all three fluorescent proteins. Furthermore, microcell-mediated transfer of HACs enabled the expression of those fluorescent proteins in recipient cells. We next demonstrated that GLVs could be introduced into a HAC one-by-one via reciprocal usage of recombinase/integrase. Lastly, we introduced a fourth GLV into a HAC after simultaneous integration of three GLVs by FLP-mediated DNA recombination. The SIM system expands the applicability of HAC vectors and is useful for various biomedical studies, including cell reprogramming.
authors.affiliation: Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo, Japan.
authors.forename: Teruhiko
authors.initials: T
authors.lastname: Suzuki
authors.affiliation: Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Yonago, Tottori, Japan; Chromosome Engineering Research Center, Tottori University, Yonago, Tottori, Japan.
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kazuki
authors.affiliation: Chromosome Engineering Research Center, Tottori University, Yonago, Tottori, Japan.
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
authors.affiliation: Stem Cell Project, Tokyo Metropolitan Institute of Medical Science, Kamikitazawa, Setagaya-ku, Tokyo, Japan; Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo, Japan.
authors.forename: Takahiko
authors.initials: T
authors.lastname: Hara
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D022222:Chromosomes, Artificial, Human; D015870:Gene Expression; D023061:Gene Order; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D005822:Genetic Vectors; D059765:Homologous Recombination; D006801:Humans; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D019076:Transgenes