Human artificial chromosome with a conditional centromere for gene delivery and gene expression.
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.
Iida
Yuichi
Y
Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Sciences, Tottori University, Nishi-cho, Yonago, Tottori, Japan.
Kim
Jung-Hyun
JH
Kazuki
Yasuhiro
Y
Hoshiya
Hidetoshi
H
Takiguchi
Masato
M
Hayashi
Masahiro
M
Erliandri
Indri
I
Lee
Hee-Sheung
HS
Samoshkin
Alex
A
Masumoto
Hiroshi
H
Earnshaw
William C
WC
Kouprina
Natalay
N
Larionov
Vladimir
V
Oshimura
Mitsuo
M
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
D013754:Tetracyclines; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins
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