Linked Life Data

9518670

Source:http://linkedlifedata.com/resource/pubmed/id/9518670

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1998-4-17
pubmed:abstractText
Cationic lipid-mediated gene transfer has been shown to be a competent albeit inefficient mechanism of promoting cellular gene transfer. One way to improve the efficacy of cationic lipid-mediated transgene expression is to optimize conditions for complex formation between the lipids and nucleic acids. In this report we describe the beneficial effects of using phosphate buffer to precondition lipofectin (a 1:1 (w/w) mixture of N-[1-(2,3-dioleyloxy)propyl]-n,n, n-trimethylammonium chloride (DOTMA), and dioleoyl phosphatidylethanolamine (DOPE)) prior to complexing with plasmid DNA or mRNA. Under such optimized conditions we studied the kinetics of DNA- and RNA-mediated transgene expression in a human osteosarcoma cell line (HOS). Preincubation of lipofectin in phosphate buffer resulted in up to 26- and 56-fold increases in luciferase expression from plasmid DNA and mRNA, respectively. Addition of chloroquine (50 microM), which enhanced plasmid-mediated gene delivery 3-fold, was synergistic with phosphate resulting in an additional 46-fold increase in luciferase expression. The preincubation with phosphate shortened both the time required for cellular uptake and the time to achieve maximal transgene expression. Optimal transfection was achieved in the presence of 30-80 mM phosphate, at pH 5.6-6.8 under which the phosphate anion is divalent. The effect of phosphate anion was specific in that monovalent Cl- and acetate anions were not stimulatory. These results demonstrate that divalent phosphate anion plays a stimulatory role during complex formation and transfection when cationic lipids come in contact with negatively charged nucleic acids and cell membranes. These findings delineate specific conditions which dramatically enhance transfection efficiency for both DNA and mRNA, and provide an effective procedure for gene transfection studies.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Mar
pubmed:issn
0006-3002
pubmed:author
pubmed:copyrightInfo
Copyright 1998 Elsevier Science B.V.
pubmed:issnType
Print
pubmed:day
2
pubmed:volume
1369
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
320-34
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1998
pubmed:articleTitle
Phosphate-enhanced transfection of cationic lipid-complexed mRNA and plasmid DNA.
pubmed:affiliation
Department of Neurosurgery, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA. kariko@mail.med.upenn.edu
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't