title: Stable maintenance of de novo assembled human artificial chromosomes in embryonic stem cells and their differentiated progeny in mice.
abstract: De novo assembled alphoid(tetO)-type human artificial chromosomes (HACs) represent a novel promising generation of high capacity episomal vectors. Their function and persistence, and any adverse effects, in various cell types in live animals, have not, however, been explored. In this study we transferred the alphoid(tetO)-HAC into mouse ES cells and assessed whether the presence of this extra chromosome affects their pluripotent properties. Alphoid(tetO)-HAC-bearing ES cells were indistinguishable from their wild-type counterparts: they retained self-renewal potential and full capacity for multilineage differentiation during mouse development, whereas the HAC itself was mitotically and transcriptionally stable during this process. Our data provide the first example of fully synthetic DNA behaving like a normal chromosome in cells of living animals. It also opens a new perspective into functional genetic studies in laboratory animals as well as stem cell-based regenerative medicine.
authors.affiliation: a Institute of Cytology Russian Academy of Sciences ; St-Petersburg , Russia.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Liskovykh
authors.forename: Sergey
authors.initials: S
authors.lastname: Ponomartsev
authors.forename: Elena
authors.initials: E
authors.lastname: Popova
authors.forename: Michael
authors.initials: M
authors.lastname: Bader
authors.forename: Natalay
authors.initials: N
authors.lastname: Kouprina
authors.forename: Vladimir
authors.initials: V
authors.lastname: Larionov
authors.forename: Natalia
authors.initials: N
authors.lastname: Alenina
authors.forename: Alexey
authors.initials: A
authors.lastname: Tomilin
mesh_terms: D000818:Animals; D001755:Blastocyst / Q000166:cytology / Q000378:metabolism; D016466:CHO Cells; D002454:Cell Differentiation; D022222:Chromosomes, Artificial, Human / Q000235:genetics / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D005260:Female; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D051379:Mice; D008810:Mice, Inbred C57BL; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D013724:Teratoma / Q000378:metabolism / Q000473:pathology
keywords: Bsd, blasticidin; DAPI, 4′, 6-Diamidino-2-Phenylindole; EGFP, enhanced green fluorescent protein; ES cells, embryonic stem cells; FISH, fluorescent in situ hybridization; HAC, human artificial chromosome; HAT, hypoxanthine-aminopterin-thymidine; MMCT, microcell mediated chromosome transfer; PBS, phosphate buffered saline; PFA, paraformaldehyde; dpc, days post coitum; embryonic stem cells; gene therapy; human artificial chromosomes; iPS cells, induced pluripotent stem cells