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Articles in PresS, published online ahead of print November 6, 2002
Am J Physiol Cell Physiol, 10.1152/ajpcell.00435.2002
Submitted on September 20, 2002
Accepted on October 28, 2002
1 Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL, USA; CF Research Center, University of Alabama at Birmingham, Birmingham, AL, USA
2 Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA; CF Research Center, University of Alabama at Birmingham, Birmingham, AL, USA
3 Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA; Medicine, University of Alabama at Birmingham, Birmingham, AL, USA; CF Research Center, University of Alabama at Birmingham, Birmingham, AL, USA
4 Biology, Georgia Institute of Technology, Atlanta, GA, USA
5 Physiology and Biophysics, University of Alabama at Birmingham, Birmingham, AL, USA; Cell Biology, University of Alabama at Birmingham, Birmingham, AL, USA; CF Research Center, University of Alabama at Birmingham, Birmingham, AL, USA
* To whom correspondence should be addressed. E-mail: lschwieb{at}uab.edu.
Transient transfection of epithelial cells with lipid reagents has been limited because of toxicity and lack of efficacy. In this study, we show that more recently developed lipids transfect non-polarized human airway epithelial cells with high efficacy and efficiency and little or no toxicity. Because of this success, we hypothesized that these lipids may also allow transient transfection of polarized epithelial monolayers. A panel of reagents was tested for transfer of the reporter gene, luciferase (LUC), into polarized monolayers of non-cystic fibrosis (non-CF) and CF human bronchial epithelial cells, MDCK epithelial cell monolayers, and, ultimately, primary non-CF and CF airway epithelial cells. Lipid reagents, that were most successful in initial LUC assays, were also tested for transfer of vectors bearing the reporter gene, green fluorescent protein (GFP), and for successful transfection and expression of an epithelial-specific protein, the cystic fibrosis transmembrane conductance regulator (CFTR). Electrophysiological, biochemical, and immunological assays were performed to show successful complementation of an epithelial monolayer with transiently expressed CFTR. We also present findings that help facilitate monolayer formation by these airway epithelial cell lines. Taken together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically LipofectAMINE PLUS and LipofectAMINE 2000. Tranisent transfection of epithelial monolayers provides a powerful system in which to express the cDNA of any epithelial-specific protein transiently in a native polarized epithelium to study protein function.
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