Phosphate-buffered saline-based nucleofection of primary endothelial cells

Abstract

Although various non-viral transfection methods are available, cell-toxicity, low transfection efficiency and high-cost remain hurdles for in vitro gene delivery in cultured primary endothelial cells. Recently, unprecedented transfection efficiency for primary endothelial cells has been achieved due to the newly developed nucleofection technology that utilizes a combination of novel electroporation conditions and specific buffer components that stabilize the cells in the electrical field. Despite its superior transfection efficiency and cell viability, high cost of the technology has discouraged the cardiovascular researchers to liberally adopt this new technology. Here, we report that a phosphate-buffered saline (PBS)-based nucleofection method can be used for efficient gene delivery into primary endothelial cells and other types of cells. Comparative analyses of transfection efficiency and cell viability for primary arterial, venous, microvascular and lymphatic endothelial cells were performed by using PBS. Compared to the commercial buffers, PBS can support equally remarkable nucleofection efficiency to both primary and non-primary cells. Moreover, PBSmediated nucleofection of siRNA showed more than 90% knockdown of the expression of target genes in primary endothelial cells. Together, we demonstrate that PBS can be an unprecedented economical alternative for the high-cost buffers for nucleofection of various primary and non-primary cells.

Peroxisome Proliferator-activated Receptor-α-mediated Transcription of miR-199a2 Attenuates Endothelin-1 Expression via Hypoxia-inducible Factor-1α

“…The transfection was highly effective in the absence of nucleofection agent (31). Transfected cells were transferred to complete medium and incubated overnight.…”

Placenta Growth Factor-induced Early Growth Response 1 (Egr-1) Regulates Hypoxia-inducible Factor-1α (HIF-1α) in Endothelial Cells

“…Transient Transfection-HPMVEC (1 ϫ 10 6 ) were resuspended in 100 l of unsupplemented plain RPMI 1640 medium containing appropriate siRNA constructs (50 nM) or luciferase reporter plasmids or several expression plasmids and transfected by nucleofection using the S-05 program in a nucleofector device (Lonza, Basel, Switzerland) (32). The ␤-galactosidase plasmid (0.5 g) was cotransfected with reporter constructs (0.5 g) to monitor transfection efficiency.…”

Protein function analysis: rapid, cell-based siRNA-mediated ablation of endogenous expression with simultaneous ectopic replacement

“…Optimization of transfection efficiency must be balanced with cell viability following electroporation, a factor that is also cell type-specific. Electroporation of MCF-7 cells under our conditions results in roughly 50% cell viability, which is not uncommon for electroporation (Kang et al 2009). Near complete efficiency usually leads to low viability, which is easily overcome with the use of an appropriate number of cells.…”