The ability to study the role of specific genes in endothelial cell biology is made possible by our ability to modulate their expression through siRNA or knockout technologies. However, many in vitro protocols, particularly those of a biochemical nature, require large numbers of endothelial cells. These types of analyses are encumbered by the need to repeatedly produce and characterize primary endothelial cell cultures and can be greatly facilitated by the use of immortalized microvascular endothelial cells. However, we have found that the manipulation of gene expression in these cells is not always straight forward. Here we describe how we alter gene expression in polyoma middle T antigen immortalized microvascular endothelial cells isolated from wild-type and genetically modified mice to study the role of cell adhesion molecules in downstream assays.