title: Integration-free iPS cells engineered using human artificial chromosome vectors.
abstract: Human artificial chromosomes (HACs) have unique characteristics as gene-delivery vectors, including episomal transmission and transfer of multiple, large transgenes. Here, we demonstrate the advantages of HAC vectors for reprogramming mouse embryonic fibroblasts (MEFs) into induced pluripotent stem (iPS) cells. Two HAC vectors (iHAC1 and iHAC2) were constructed. Both carried four reprogramming factors, and iHAC2 also encoded a p53-knockdown cassette. iHAC1 partially reprogrammed MEFs, and iHAC2 efficiently reprogrammed MEFs. Global gene expression patterns showed that the iHACs, unlike other vectors, generated relatively uniform iPS cells. Under non-selecting conditions, we established iHAC-free iPS cells by isolating cells that spontaneously lost iHAC2. Analyses of pluripotent markers, teratomas and chimeras confirmed that these iHAC-free iPS cells were pluripotent. Moreover, iHAC-free iPS cells with a re-introduced HAC encoding Herpes Simplex virus thymidine kinase were eliminated by ganciclovir treatment, indicating that the HAC safeguard system functioned in iPS cells. Thus, the HAC vector could generate uniform, integration-free iPS cells with a built-in safeguard system.
authors: Division of Molecular and Cell Genetics, Department of Molecular and Cellular Biology, School of Life Sciences, Faculty of Medicine, Tottori University, Yonago, Japan.
authors: Masaharu
authors: M
authors: Hiratsuka
authors: Narumi
authors: N
authors: Uno
authors: Kana
authors: K
authors: Ueda
authors: Hajime
authors: H
authors: Kurosaki
authors: Natsuko
authors: N
authors: Imaoka
authors: Kanako
authors: K
authors: Kazuki
authors: Etsuya
authors: E
authors: Ueno
authors: Yutaro
authors: Y
authors: Akakura
authors: Motonobu
authors: M
authors: Katoh
authors: Mitsuhiko
authors: M
authors: Osaki
authors: Yasuhiro
authors: Y
authors: Kazuki
authors: Masato
authors: M
authors: Nakagawa
authors: Shinya
authors: S
authors: Yamanaka
authors: Mitsuo
authors: M
authors: Oshimura
mesh_terms: D000818:Animals; D016466:CHO Cells; D060846:Cell Engineering / Q000379:methods*; D022222:Chromosomes, Artificial, Human / Q000235:genetics*; D006224:Cricetinae; D003412:Cricetulus; D005347:Fibroblasts / Q000166:cytology; D041722:Genes, Transgenic, Suicide / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D051379:Mice; D018139:Simplexvirus / Q000235:genetics; D013937:Thymidine Kinase / Q000235:genetics
chemical_list: D013937:Thymidine Kinase