Construction of a novel human artificial chromosome vector for gene delivery.
Potential problems of conventional transgenes include insertional disruption of the host genome and unpredictable, irreproducible expression of the transgene by random integration. Alternatively, human artificial chromosomes (HACs) can circumvent some of the problems. Although several HACs were generated and their mitotic stability was assessed, a practical way for introducing exogenous genes by the HACs has yet to be explored. In this study, we developed a novel HAC from sequence-ready human chromosome 21 by telomere-directed chromosome truncation and added a loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. This 21HAC vector, delivered to a human cell line HT1080 by microcell fusion, bound centromere proteins A, B, and C and was mitotically stable during long-term culture without selection. The EGFP gene inserted in the HAC vector expressed persistently. These results suggest that the HAC vector provides useful system for functional studies of genes in isogenic cell lines.
Katoh
Motonobu
M
Department of Human Genome Sciences (Kirin Brewery), Graduate School of Medical Science, Tottori University, 86 Nishimachi, Yonago, Tottori 683-8503, Japan.
Ayabe
Fumiaki
F
Norikane
Satoko
S
Okada
Teruaki
T
Masumoto
Hiroshi
H
Horike
Shin-ichi
S
Shirayoshi
Yasuaki
Y
Oshimura
Mitsuo
M
D000818:Animals; D001287:Attachment Sites, Microbiological / Q000235:genetics; D016466:CHO Cells; D002455:Cell Division; D002460:Cell Line; D002503:Centromere / Q000378:metabolism; D002645:Chickens; D022222:Chromosomes, Artificial, Human / Q000235:genetics*; D002891:Chromosomes, Human, Pair 21 / Q000235:genetics; D006224:Cricetinae; D005786:Gene Expression Regulation; D018390:Gene Targeting / Q000379:methods*; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D006822:Hybrid Cells / Q000166:cytology / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence; D018386:Kinetochores / Q000378:metabolism; D011995:Recombination, Genetic / Q000235:genetics; D016615:Telomere / Q000235:genetics