title: Human artificial chromosome with a conditional centromere for gene delivery and gene expression.
abstract: Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoid(tet-O) (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy.
authors.affiliation: Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Sciences, Tottori University, Nishi-cho, Yonago, Tottori, Japan.
authors.forename: Yuichi
authors.initials: Y
authors.lastname: Iida
authors.forename: Jung-Hyun
authors.initials: JH
authors.lastname: Kim
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Kazuki
authors.forename: Hidetoshi
authors.initials: H
authors.lastname: Hoshiya
authors.forename: Masato
authors.initials: M
authors.lastname: Takiguchi
authors.forename: Masahiro
authors.initials: M
authors.lastname: Hayashi
authors.forename: Indri
authors.initials: I
authors.lastname: Erliandri
authors.forename: Hee-Sheung
authors.initials: HS
authors.lastname: Lee
authors.forename: Alex
authors.initials: A
authors.lastname: Samoshkin
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Masumoto
authors.forename: William C
authors.initials: WC
authors.lastname: Earnshaw
authors.forename: Natalay
authors.initials: N
authors.lastname: Kouprina
authors.forename: Vladimir
authors.initials: V
authors.lastname: Larionov
authors.forename: Mitsuo
authors.initials: M
authors.lastname: Oshimura
mesh_terms: D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002503:Centromere / Q000235:genetics*; D002645:Chickens; D022222:Chromosomes, Artificial, Human / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D009875:Operator Regions, Genetic; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D013754:Tetracyclines; D019076:Transgenes
chemical_list: D013754:Tetracyclines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
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