Inducible expression of green fluorescent protein in porcine tracheal epithelial cells by the bovine tracheal antimicrobial peptide promoter.
Tracheal antimicrobial peptides (TAP) are expressed primarily in respiratory epithelial cells of cattle. The TAP expression is inducible upon challenge with bacteria and bacterial lipopolysaccharide (LPS). In pigs, a promoter that can be activated by bacterial infection has yet to be identified. The objective of this study was to use green fluorescent protein (GFP) as a reporter gene to determine the function and inducibility of the bovine TAP promoter in porcine primary tracheal epithelial cells. Thus, evaluating the feasibility of using this promoter to direct transgene expression in porcine cells. The percentage of GFP expressing cells increased in response to LPS challenge in both a dose-dependent and time-dependent manner (p < 0.05). Moreover, when the intensity of the GFP fluorescence was measured, it was observed that the percentage of cells that have a high intensity of GFP fluorescence, also increased gradually as LPS dose increased, the difference between the unchallenged (control) and challenged group become statistically significant at the concentration of 100 ng/mL after 36 h LPS challenge (p < 0.05). The level of induced-expression driven by the TAP promoter was 67.8 +/-12.2% that of the cytomegalovirus (CMV) promoter. The intensity of GFP fluorescence by the TAP promoter was 39.8 +/- 7.6% when compared to the expression driven by the CMV promoter. These data suggest the TAP promoter functions at a lower, but comparable, level to the strong CMV promoter. Our data demonstrated that the bovine TAP promoter was functional in porcine primary tracheal epithelial cells. The ability of the TAP promoter to control gene expression in an inducible manner in the porcine respiratory tract presents an important application potential in transgenic animal studies.
Dyce
Paul W
PW
Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1.
DeVries
Robert J
RJ
Walton
John
J
Hacker
Roger R
RR
Li
Julang
J
D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism; D023181:Antimicrobial Cationic Peptides / Q000096:biosynthesis* / Q000235:genetics*; D002417:Cattle / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D018274:Electroporation / Q000379:methods; D005786:Gene Expression Regulation / Q000502:physiology*; D017930:Genes, Reporter / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D020545:Respiratory Mucosa / Q000378:metabolism*; D013552:Swine / Q000235:genetics / Q000378:metabolism; D014018:Tissue Distribution; D014132:Trachea / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
D023181:Antimicrobial Cationic Peptides; D008164:Luminescent Proteins; D011994:Recombinant Proteins; C068629:tracheal antimicrobial peptide, bovine; D049452:Green Fluorescent Proteins