Linked Life Data

11576873

Source:http://linkedlifedata.com/resource/pubmed/id/11576873

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
4
pubmed:dateCreated
2001-9-28
pubmed:abstractText
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 as well as increasing nuclear transport of plasmids, resulting in significant but 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 green fluorescent protein (GFP) transgene delivered via Lipofectamine, by testing the effects of culturing C3A human hepatoblastoma cells with 5-Azacytidine (AzaC), an irreversible inhibitor of DNA methyltransferase. To control for loss 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 (MAP) 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 level of GFP expression.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Oct
pubmed:issn
1389-0344
pubmed:author
pubmed:issnType
Print
pubmed:day
31
pubmed:volume
18
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
185-92
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
2001
pubmed:articleTitle
Methylation of episomal plasmids as a barrier to transient gene expression via a synthetic delivery vector.
pubmed:affiliation
Biotechnology Process Engineering Center and Division of Bioengineering and Environmental Health, 16-436 Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S.