Genetic amplification of the transcriptional response to hypoxia as a novel means of identifying regulators of angiogenesis
Section snippets
Results and discussion
Adenoviral vectors were designed for the expression of the related transcription factors HIF1A and EPAS1 in mammalian cells. To this end, cDNA sequences for the genes HIF1A and EPAS1 were configured into the adenoviral transfer plasmid AdApt-gfp for expression from the powerful cytomegalovirus promoter. The plasmids, named AdApt-HIF1A-gfp and AdApt-EPAS1-gfp, were tested for their ability to drive expression of functional transcriptional factors in a luciferase reporter transfection assay [13].
Cells and culture
Peripheral blood mononuclear cells were isolated from buffy coats of normal individuals over a Ficoll–Paque gradient, and CD14+ monocytes were sorted using MACS beads (Miltenyi Biotech). Monocytes were cultured at 5 × 105 cells/ml in Iscove's medium supplemented with 2% human AB serum in Teflon bags for a period of 7 days, during which time they spontaneously differentiate, assuming the characteristic macrophage morphology. Macrophages were harvested from Teflon bags, plated onto 10-cm Primeria
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