Bergmann, M., Barnes, P.J. and Newton, R.
Notes: The interleukin (IL)-1ß stimulated release of granulocyte macrophage colony stimulating factor (GM-CSF) from lung epithelial cells was explored in this study. To test the promoter activity of GM-CSF, the promoter and enhancer regions were amplified by PCR from human peripheral blood mononuclear cells and cloned into the pGEM®-T vector. After verification by sequencing, the promoter and enhancer were cloned individually and together into the pGL3-Basic Vector. Additionally, an Xho I/Sal I fragment containing the HSV tk promoter, a gene conferring neomycin resistance, and a poly-A tail were cloned into the Sal I site of pGL3 to allow the production of stable transfectants. To perform stable transfections, 20µl of the Tfx™-50 transfection reagent was incubated with 8µg of plasmid in serum-free medium for 15 minutes at room temperature. Preconfluent human A549 type II alveolar carcinoma cells were incubated with the transfection mix for 2 hours after washing with serum-free medium. The cells were then cultured in fresh medium for 16 hours before the addition of 0.5mg/mL G-418. After 21 days, foci of cells developed and were harvested for use in luciferase assays. The cells were plated into 24-well plates, grown to confluency and incubated in serum-free medium. Stimulation by 1ng/ml IL-1ß or 1µM PMA for 12 hours was followed by lysate production and measurement of luciferase activity using the Luciferase Assay System and a Turner luminometer. Luciferase readings were standardized against total protein measurements. The PolyATract® System IV was also used prior to a Northern Blot to obtain purified mRNA. (2742)