title: Protecting a transgene expression from the HAC-based vector by different chromatin insulators.
abstract: Human artificial chromosomes (HACs) are vectors that offer advantages of capacity and stability for gene delivery and expression. Several studies have even demonstrated their use for gene complementation in gene-deficient recipient cell lines and animal transgenesis. Recently, we constructed an advance HAC-based vector, alphoid(tetO)-HAC, with a conditional centromere. In this HAC, a gene-loading site was inserted into a centrochromatin domain critical for kinetochore assembly and maintenance. While by definition this domain is permissive for transcription, there have been no long-term studies on transgene expression within centrochromatin. In this study, we compared the effects of three chromatin insulators, cHS4, gamma-satellite DNA, and tDNA, on the expression of an EGFP transgene inserted into the alphoid(tetO)-HAC vector. Insulator function was essential for stable expression of the transgene in centrochromatin. In two analyzed host cell lines, a tDNA insulator composed of two functional copies of tRNA genes showed the highest barrier activity. We infer that proximity to centrochromatin does not protect genes lacking chromatin insulators from epigenetic silencing. Barrier elements that prevent gene silencing in centrochromatin would thus help to optimize transgenesis using HAC vectors.
authors.affiliation: Laboratories of Molecular Pharmacology, National Cancer Institute, Bethesda, MD, 20892, USA.
authors.forename: Nicholas C O
authors.initials: NC
authors.lastname: Lee
authors.forename: Artem V
authors.initials: AV
authors.lastname: Kononenko
authors.forename: Hee-Sheung
authors.initials: HS
authors.lastname: Lee
authors.forename: Elena N
authors.initials: EN
authors.lastname: Tolkunova
authors.forename: Mikhail A
authors.initials: MA
authors.lastname: Liskovykh
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Masumoto
authors.forename: William C
authors.initials: WC
authors.lastname: Earnshaw
authors.forename: Alexey N
authors.initials: AN
authors.lastname: Tomilin
authors.forename: Vladimir
authors.initials: V
authors.lastname: Larionov
authors.forename: Natalay
authors.initials: N
authors.lastname: Kouprina
mesh_terms: D000818:Animals; D016466:CHO Cells; D045744:Cell Line, Tumor; D002843:Chromatin / Q000235:genetics*; D022222:Chromosomes, Artificial, Human; D006224:Cricetinae; D003412:Cricetulus; D004276:DNA, Satellite / Q000235:genetics; D015870:Gene Expression; D020868:Gene Silencing; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics; D006367:HeLa Cells; D006801:Humans; D012343:RNA, Transfer / Q000235:genetics; D019076:Transgenes
chemical_list: D002843:Chromatin; D004276:DNA, Satellite; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D012343:RNA, Transfer