Quantitative analysis of cotransfection efficiencies in studies of ionotropic glutamate receptor complexes

Zhan Lu Ma, Markus Werner, Christoph Körber, Illah Joshi, Mohammad Hamad, Petra Wahle, Michael Hollmann

Research output: Contribution to journalArticlepeer-review

10 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)99-115
Number of pages17
JournalJournal of Neuroscience Research
Volume85
Issue number1
DOIs
Publication statusPublished - Jan 2007
Externally publishedYes

Keywords

  • Cotransfection efficiency
  • Electrophysiological analysis
  • Heteromeric assembly
  • Ionotropic glutamate receptors
  • Simultaneous transfection

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience

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