title: Human artificial chromosome (HAC) vector with a conditional centromere for correction of genetic deficiencies in human cells.
abstract: Human artificial chromosome (HAC)-based vectors offer a promising system for delivery and expression of full-length human genes of any size. HACs avoid the limited cloning capacity, lack of copy number control, and insertional mutagenesis caused by integration into host chromosomes that plague viral vectors. We previously described a synthetic HAC that can be easily eliminated from cell populations by inactivation of its conditional kinetochore. Here, we demonstrate the utility of this HAC, which has a unique gene acceptor site, for delivery of full-length genes and correction of genetic deficiencies in human cells. A battery of functional tests was performed to demonstrate expression of NBS1 and VHL genes from the HAC at physiological levels. We also show that phenotypes arising from stable gene expression can be reversed when cells are "cured" of the HAC by inactivating its kinetochore in proliferating cell populations, a feature that provides a control for phenotypic changes attributed to expression of HAC-encoded genes. This generation of human artificial chromosomes should be suitable for studies of gene function and therapeutic applications.
authors: Laboratories of Molecular Pharmacology, National Cancer Institute, Bethesda, MD 20892, USA.
authors: Jung-Hyun
authors: JH
authors: Kim
authors: Artem
authors: A
authors: Kononenko
authors: Indri
authors: I
authors: Erliandri
authors: Tae-Aug
authors: TA
authors: Kim
authors: Megumi
authors: M
authors: Nakano
authors: Yuichi
authors: Y
authors: Iida
authors: J Carl
authors: JC
authors: Barrett
authors: Mitsuo
authors: M
authors: Oshimura
authors: Hiroshi
authors: H
authors: Masumoto
authors: William C
authors: WC
authors: Earnshaw
authors: Vladimir
authors: V
authors: Larionov
authors: Natalay
authors: N
authors: Kouprina
mesh_terms: D000818:Animals; D001324:Autoantigens / Q000378:metabolism; D016466:CHO Cells; D018797:Cell Cycle Proteins / Q000235:genetics; D002503:Centromere / Q000235:genetics*; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D022222:Chromosomes, Artificial, Human / Q000235:genetics*; D018244:Chromosomes, Artificial, Yeast / Q000235:genetics; D003001:Cloning, Molecular; D006224:Cricetinae; D003412:Cricetulus; D015870:Gene Expression; D005816:Genetic Complementation Test; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D015894:Genome, Human / Q000235:genetics; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D019426:Integrases / Q000378:metabolism; D016254:Mutagenesis, Insertional / Q000235:genetics; D009687:Nuclear Proteins / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D051794:Von Hippel-Lindau Tumor Suppressor Protein / Q000235:genetics
chemical_list: D001324:Autoantigens; D018797:Cell Cycle Proteins; D000076247:Centromere Protein A; D002868:Chromosomal Proteins, Non-Histone; C112156:NBN protein, human; D009687:Nuclear Proteins; D051794:Von Hippel-Lindau Tumor Suppressor Protein; C045073:Cre recombinase; D019426:Integrases; C497446:VHL protein, human