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1
* Experimental Cell Research Program, Methodist Research Institute, Clarian Health Partners, Inc., Indianapolis, Indiana 46202, USA;
Department of Pulmonary and Critical Care Medicine, John Hopkins University School of Medicine, Baltimore, Maryland 21224, USA; and
Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana 46202, USA
1Correspondence: Experimental Cell Research Program, The Methodist Research Institute, 1701 N. Senate Ave., Indianapolis, IN 46219, USA. E-mail: denglish{at}msn.com
The definition of signaling pathways in endothelial cells has been
hampered by the difficulty of transiently transfecting these cells with
high efficiency. This investigation was undertaken to develop an
efficient technique for the transfection of endothelial cells for
functional analyses. Cells cotransfected with plasmid expressing green
fluorescent protein (GFP) and the plasmid of interest were isolated by
fluorescence-activated cell sorting (FACS) based on GFP expression. In
the sorted cell population, a 2.5-fold enhancement in the number of
cells expressing the gene of interest was observed, as confirmed by
FACS analysis and Western blotting. Sorted cells retained functional
properties, as demonstrated by chemotaxis to the agonist sphingosine
1-phosphate (SPP). To demonstrate the usefulness of this method for
defining cellular signaling pathways, cells were cotransfected with
plasmids encoding GFP and the carboxyl-terminal domain of the
ß-adrenergic receptor kinase (ßARKct), which inhibits signaling
through the ß
dimer of heterotrimeric G-proteins. SPP-induced
chemotaxis in sorted cells coexpressing ßARKct was inhibited by 80%,
demonstrating that chemotaxis was driven by a ß-dependent pathway.
However, no significant inhibition was observed in cells transfected
with ßARKct but not enriched by sorting. Thus, we have developed a
method for enriching transfected cells that allows the elucidation of
crucial mechanisms of endothelial cell activation and function. This
method should find wide applicability in studies designed to define
pathways responsible for regulation of motility and other functions in
these dynamic cells.—Kovala, A. T., Harvey, K. A., McGlynn,
P., Boguslawski, G., Garcia, J. G. N., English, D.
High-efficiency transient transfection of endothelial cells for
functional analysis.
Key Words: FACS sorting • chemotaxis • heterotrimeric G-protein ß subunits
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