Transient transfection of polarized epithelial monolayers with CFTR and reporter genes using efficacious lipids.
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 nonpolarized 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, which 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. Together, these data show that polarized monolayers are transfected transiently with more recently developed lipids, specifically LipofectAMINE PLUS and LipofectAMINE 2000. Transient transfection of epithelial monolayers provides a powerful system in which to express the cDNA of any epithelium-specific protein transiently in a native polarized epithelium to study protein function.
Tucker
Torry A
TA
Department of Physiology and Biophysics, University of Alabama at Birmingham, 35294, USA.
Varga
Karoly
K
Bebok
Zsuzsa
Z
Zsembery
Akos
A
McCarty
Nael A
NA
Collawn
James F
JF
Schwiebert
Erik M
EM
Schwiebert
Lisa M
LM
D000818:Animals; D002411:Cation Exchange Resins; D018929:Cell Culture Techniques / Q000379:methods*; D016764:Cell Polarity / Q000187:drug effects / Q000235:genetics; D018946:Chemokine CCL5 / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D049454:Firefly Luciferin / Q000235:genetics; D017930:Genes, Reporter / Q000235:genetics*; D005822:Genetic Vectors / Q000187:drug effects / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D008055:Lipids / Q000235:genetics*; D008164:Luminescent Proteins; D011233:Precipitin Tests; D020545:Respiratory Mucosa / Q000187:drug effects / Q000378:metabolism; D014162:Transfection / Q000379:methods*
C505032:CFTR protein, human; D002411:Cation Exchange Resins; D018946:Chemokine CCL5; C411955:FuGene; D008055:Lipids; C086724:Lipofectamine; D008164:Luminescent Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D049452:Green Fluorescent Proteins; D049454:Firefly Luciferin