siRNA Knockdown of Gene Expression in Endothelial Cells

Cultured endothelial cells are renowned for being difficult to transfect, whether for the purpose of exogenous over-expression of plasmid DNA or for genetic knockdown via silencing RNA. Therefore, optimal conditions are absolutely necessary for achieving relatively high transfection efficiency coupled with low cellular toxicity in endothelial cells. This chapter will detail an optimized protocol that has been shown to knockdown gene expression using siRNA in primary cultures of human umbilical vein endothelial cells (HUVECs) – perhaps the most widely utilized endothelial cell line for vascular research. While developed for optimal siRNA transfection of HUVECs, aspects of this protocol can be empirically modified to yield efficient siRNA transfection in most other cell lines.