High-efficiency transient transfection of endothelial cells for functional analysis.
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 beta-adrenergic receptor kinase (beta ARKct), which inhibits signaling through the beta gamma dimer of heterotrimeric G-proteins. SPP-induced chemotaxis in sorted cells coexpressing beta ARKct was inhibited by 80%, demonstrating that chemotaxis was driven by a beta gamma-dependent pathway. However, no significant inhibition was observed in cells transfected with betaARKct 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
AT
Experimental Cell Research Program, Methodist Research Institute, Clarian Health Partners, Inc., Indianapolis, Indiana 46202, USA.
Harvey
K A
KA
McGlynn
P
P
Boguslawski
G
G
Garcia
J G
JG
English
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D
D000818:Animals; D001011:Aorta; D002417:Cattle; D002469:Cell Separation; D002633:Chemotaxis / Q000235:genetics*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics* / Q000378:metabolism; D004730:Endothelium, Vascular; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction; D014162:Transfection / Q000379:methods*; D051552:beta-Adrenergic Receptor Kinases; D000097573:Red Fluorescent Protein
D017868:Cyclic AMP-Dependent Protein Kinases; D049452:Green Fluorescent Proteins; D020962:Heterotrimeric GTP-Binding Proteins; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D051552:beta-Adrenergic Receptor Kinases; D000097573:Red Fluorescent Protein