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Titolo:
Methylation of episomal plasmids as a barrier to transient gene expressionvia a synthetic delivery vector
Autore:
Hong, K; Sherley, J; Lauffenburger, DA;
Indirizzi:
16 436 MIT, Ctr Biotechnol Proc Engn, Cambridge, MA 02139 USA 16 436 MIT Cambridge MA USA 02139 hnol Proc Engn, Cambridge, MA 02139 USA 16 436 MIT, Div Bioengn & Environm Hlth, Cambridge, MA 02139 USA 16 436 MIT Cambridge MA USA 02139 Environm Hlth, Cambridge, MA 02139 USA
Titolo Testata:
BIOMOLECULAR ENGINEERING
fascicolo: 4, volume: 18, anno: 2001,
pagine: 185 - 192
SICI:
1389-0344(20011031)18:4<185:MOEPAA>2.0.ZU;2-H
Fonte:
ISI
Lingua:
ENG
Soggetto:
EMBRYONIC STEM-CELLS; DE-NOVO METHYLATION; DNA METHYLATION; 5-METHYLCYTOSINE CONTENT; DENOVO METHYLATION; MAMMALIAN-CELLS; IN-VIVO; METHYLTRANSFERASE; DIFFERENTIATION; MUTATION;
Keywords:
methylation; transgene expression; plasmid; GFP;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
40
Recensione:
Indirizzi per estratti:
Indirizzo: Lauffenburger, DA 16 436 MIT, Ctr Biotechnol Proc Engn, Cambridge, MA 02139 USA 16 436 MIT Cambridge MA USA 02139 mbridge, MA 02139 USA
Citazione:
K. Hong et al., "Methylation of episomal plasmids as a barrier to transient gene expressionvia a synthetic delivery vector", BIOMOL ENG, 18(4), 2001, pp. 185-192

Abstract

Efficient and sustained transgene expression are desirable features for many envisioned gene therapy applications, yet synthetic vectors tested to date are rarely successful in achieving these properties. Substantial research efforts have focused on protection of plasmid DNA from nuclease attack aswell as increasing nuclear transport of plasmids, resulting in significantbut still limited gains. We show here that a further barrier to efficient and sustained expression exists for synthetic vectors: plasmid. DNA methylation. We have investigated this barrier for transient expression of a greenfluorescent protein (GFP) transgene delivered via Lipofectamine, by testing the effects of culturing OA human hepatoblastoma cells with 5-Azacytidine(AzaC), an irreversible inhibitor of DNA methyltransferase. To control forloss of plasmids by dilution during mitosis, transfected cells were growth-arrested for 1 week and their subsequent GFP expression quantified by FACS. In the presence of AzaC, a significantly greater fraction of transfected cells remained GFP-positive and possessed higher levels of GFP production relative to AzaC-untreated cells. Additionally, we have applied a Methyl-Assisted PCR (MA-P) assay to quantify a subset of methylated CpG sites in the GFP gene. When MAP was performed on plasmids isolated from transfected cells, the extent of methylation was found to be inversely related to the levelof GFP expression. (C) 2001 Elsevier Science B.V. All rights reserved.

ASDD Area Sistemi Dipartimentali e Documentali, Università di Bologna, Catalogo delle riviste ed altri periodici
Documento generato il 08/07/20 alle ore 07:58:34