Linked Life Data

17075894

Source:http://linkedlifedata.com/resource/pubmed/id/17075894

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2006-12-26
pubmed:abstractText
Transient transfection of cultured mammalian cells is widely employed in the study of ionotropic glutamate receptors. Heteromeric expression is usually achieved by simultaneous transfection of various combinations of glutamate receptor subunit-encoding cDNAs. This approach is based on an "all-or-none" assumption, rarely verified experimentally, that any given cell expresses all subunits present during transfection. A similar assumption implicitly is made when cotransfection of a cDNA encoding a fluorescent marker protein is applied to distinguish transfected from untransfected cells. A further frequent assumption alleges that the ratio between cDNAs used in cotransfection experiments directs the assembly of receptor complexes in heterologous expression systems. To check the validity of these assumptions for ionotropic glutamate receptors as model transmembrane receptors, we generated fluorescently labeled receptor subunits and introduced them into HEK-293 cells by the calcium phosphate method. Analyzing the expression of multiple fusion proteins by confocal microscopy, we evaluated the coexpression efficiencies for various glutamate receptor cDNA combinations, cDNA amounts, and cDNA ratios. Several factors were found to influence the individual, cumulative, and cotransfection efficiencies, including the cDNA ratio, the nature of the expressed protein, and the specific combination of cotransfected cDNAs. After simultaneous transfection with equal amounts of several cDNAs, we demonstrate the consistent generation of several distinct populations of cells that express different receptor subunit combinations. The evidence we present suggests that cotransfected cells should always be independently tested for the expression of all target subunits before picking cells for the analysis of specific heteromeric receptor assemblies.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0360-4012
pubmed:author
pubmed:issnType
Print
pubmed:volume
85
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
99-115
pubmed:dateRevised
2010-1-13
pubmed:meshHeading
pubmed:year
2007
pubmed:articleTitle
Quantitative analysis of cotransfection efficiencies in studies of ionotropic glutamate receptor complexes.
pubmed:affiliation
Department of Biochemistry I-Receptor Biochemistry, Ruhr University Bochum, Bochum, Germany.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't