Efficient gene transfer into human umbilical vein endothelial cells allows functional analysis of the human tissue factor gene promoter

Abstract

Tissue factor (TF) is a cellular receptor and cofactor for factor VII/VIIa which initiates the blood coagulation cascade. We have investigated the role of 5'-flanking DNA sequences in regulating the expression of the human TF gene in human umbilical vein endothelial cells (HUVEC). Using a chloramphenicol acetyltransferase (CAT) reporter gene, we attempted to transfect primary cultured HUVEC (passage 3-4) with calcium phosphate coprecipitation, DEAE Dextran, lipopolyamine-coated DNA or electroporation. Electroporation in HEPES-buffered saline of 1 x 10(7) cells at 200V and 250 microF was found to be optimal. Using these conditions, varying lengths of TF 5'-flanking sequences coupled to the CAT reporter gene were tested in transient expression studies. CAT expression corrected for variation in transfection efficiency and cell viability revealed that the sequences between -111 and +14 base pairs are essential for minimal transcriptional activity. This region contains consensus sequences for a TATA box and three Sp1 binding sites. A domain from -382 to -111bp, which contains two AP-1 consensus elements, promoted high levels of gene expression. This transcriptional activity was repressed by 50% with constructs containing sequences between -550 and -382 bp. A further 2-fold drop in transcription activity was attributed to the region between -948 and -550 bp. These results suggest that the basal transcription of the human TF gene in HUVEC is mediated through at least two negative regulatory elements upstream of the proximal promoter domain. The proximal promoter region which contains two AP-1 sites is essential for efficient transcription.

Fluid Shear Stress Induction of the Tissue Factor Promoter In Vitro and In Vivo Is Mediated by Egr-1

“…26,27 Results are expressed as shear stress activation above the basal activity of the promoter construct. Plasmids expressing Egr-1 as wild type (p930) or mutant (1.5 zinc fingers deleted, p858) driven by the CMV promoter were a gift from Dr Vikas Sukhatme, Harvard Medical School, Boston, Mass.…”

Endothelial cell DNA transfer and expression using Petri dish electroporation and the nonreplicating vaccinia virus/T7 RNA polymerase hybrid system

“…These conditions are similar to those previously published as optimal for HUVECs using cuvette electroporation. 17 As typified by results shown in Figure 2a, both Petri dish and cuvette electroporation methods resulted in less viable cells compared with an untreated control. However, the Petri dish electrode showed 23% more viable cells relative to the cuvette electrode using the applied transfection and infection conditions (Figure 2a).…”

“…16 However, electroporation methods have yielded low transfection efficiencies and limited expression levels in HUVECs. 1,3,17 Furthermore, cuvette electroporation requires manipulation of cells into a cuvette. This may introduce artifactual effects in addition to the receiving of an electric shock that may inhibit or delay subsequent gene expression and perturb cellular function.…”

Endothelial cell transfection with cationic liposomes and herpes simplex-thymidine kinase mediated killing

“…Lipofectamine transfected porcine endothelial cells with 2-15% efficiency, whereas the adenovirus transduction rate was 100%. 16 Primary cultured HUVEC cells could not be transfected with Transfectam (Promega, Madison, WI, USA), 17 but better results were reported with Lipofectin (Gibco Life Technologies, Paisley, UK) with 10-20% of cells staining positive for the reporter gene CAT (chloramphenicol acetyltransferase). 18 Our results are comparable with the best liposomal transfection rates reported so far, 18 and have the advantage of using a cationic liposome system noted for low toxicity in vivo.…”