title: Refined human artificial chromosome vectors for gene therapy and animal transgenesis.
abstract: Human artificial chromosomes (HACs) have several advantages as gene therapy vectors, including stable episomal maintenance, and the ability to carry large gene inserts. We previously developed HAC vectors from the normal human chromosomes using a chromosome engineering technique. However, endogenous genes were remained in these HACs, limiting their therapeutic applications. In this study, we refined a HAC vector without endogenous genes from human chromosome 21 in homologous recombination-proficient chicken DT40 cells. The HAC was physically characterized using a transformation-associated recombination (TAR) cloning strategy followed by sequencing of TAR-bacterial artificial chromosome clones. No endogenous genes were remained in the HAC. We demonstrated that any desired gene can be cloned into the HAC using the Cre-loxP system in Chinese hamster ovary cells, or a homologous recombination system in DT40 cells. The HAC can be efficiently transferred to other type of cells including mouse ES cells via microcell-mediated chromosome transfer. The transferred HAC was stably maintained in vitro and in vivo. Furthermore, tumor cells containing a HAC carrying the suicide gene, herpes simplex virus thymidine kinase (HSV-TK), were selectively killed by ganciclovir in vitro and in vivo. Thus, this novel HAC vector may be useful not only for gene and cell therapy, but also for animal transgenesis.
authors.affiliation: Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University, Tottori, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kazuki
authors.forename: H
authors.initials: H
authors.lastname: Hoshiya
authors.forename: M
authors.initials: M
authors.lastname: Takiguchi
authors.forename: S
authors.initials: S
authors.lastname: Abe
authors.forename: Y
authors.initials: Y
authors.lastname: Iida
authors.forename: M
authors.initials: M
authors.lastname: Osaki
authors.forename: M
authors.initials: M
authors.lastname: Katoh
authors.forename: M
authors.initials: M
authors.lastname: Hiratsuka
authors.forename: Y
authors.initials: Y
authors.lastname: Shirayoshi
authors.forename: K
authors.initials: K
authors.lastname: Hiramatsu
authors.forename: E
authors.initials: E
authors.lastname: Ueno
authors.forename: N
authors.initials: N
authors.lastname: Kajitani
authors.forename: T
authors.initials: T
authors.lastname: Yoshino
authors.forename: K
authors.initials: K
authors.lastname: Kazuki
authors.forename: C
authors.initials: C
authors.lastname: Ishihara
authors.forename: S
authors.initials: S
authors.lastname: Takehara
authors.forename: S
authors.initials: S
authors.lastname: Tsuji
authors.forename: F
authors.initials: F
authors.lastname: Ejima
authors.forename: A
authors.initials: A
authors.lastname: Toyoda
authors.forename: Y
authors.initials: Y
authors.lastname: Sakaki
authors.forename: V
authors.initials: V
authors.lastname: Larionov
authors.forename: N
authors.initials: N
authors.lastname: Kouprina
authors.forename: M
authors.initials: M
authors.lastname: Oshimura
mesh_terms: D000818:Animals; D002460:Cell Line; D022222:Chromosomes, Artificial, Human; D002891:Chromosomes, Human, Pair 21; D003001:Cloning, Molecular; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D051379:Mice; D011995:Recombination, Genetic