PMID- 11099466
OWN - NLM
STAT- MEDLINE
DCOM- 20010208
LR  - 20231213
IS  - 0892-6638 (Print)
IS  - 0892-6638 (Linking)
VI  - 14
IP  - 15
DP  - 2000 Dec
TI  - High-efficiency transient transfection of endothelial cells for functional 
      analysis.
PG  - 2486-94
AB  - 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.
FAU - Kovala, A T
AU  - Kovala AT
AD  - Experimental Cell Research Program, Methodist Research Institute, Clarian Health 
      Partners, Inc., Indianapolis, Indiana 46202, USA.
FAU - Harvey, K A
AU  - Harvey KA
FAU - McGlynn, P
AU  - McGlynn P
FAU - Boguslawski, G
AU  - Boguslawski G
FAU - Garcia, J G
AU  - Garcia JG
FAU - English, D
AU  - English D
LA  - eng
GR  - P01 HL 50864/HL/NHLBI NIH HHS/United States
GR  - R01 61751/PHS HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - FASEB J
JT  - FASEB journal : official publication of the Federation of American Societies for 
      Experimental Biology
JID - 8804484
RN  - 0 (Luminescent Proteins)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - EC 2.7.11.11 (Cyclic AMP-Dependent Protein Kinases)
RN  - EC 2.7.11.15 (beta-Adrenergic Receptor Kinases)
RN  - EC 3.6.5.1 (Heterotrimeric GTP-Binding Proteins)
SB  - IM
MH  - Animals
MH  - Aorta
MH  - Cattle
MH  - Cell Separation
MH  - Chemotaxis/*genetics
MH  - Cyclic AMP-Dependent Protein Kinases/*genetics/metabolism
MH  - *Endothelium, Vascular
MH  - Green Fluorescent Proteins
MH  - Heterotrimeric GTP-Binding Proteins/metabolism
MH  - Luminescent Proteins/genetics
MH  - Recombinant Fusion Proteins/metabolism
MH  - Signal Transduction
MH  - Transfection/*methods
MH  - beta-Adrenergic Receptor Kinases
MH  - Red Fluorescent Protein
EDAT- 2000/12/02 11:00
MHDA- 2001/03/03 10:01
CRDT- 2000/12/02 11:00
PHST- 2000/12/02 11:00 [pubmed]
PHST- 2001/03/03 10:01 [medline]
PHST- 2000/12/02 11:00 [entrez]
AID - 14/15/2486 [pii]
AID - 10.1096/fj.00-0147com [doi]
PST - ppublish
SO  - FASEB J. 2000 Dec;14(15):2486-94. doi: 10.1096/fj.00-0147com.