Lentivirus delivery of shRNA constructs into osteoblasts.

Osteoblasts are the sole cell responsible for bone formation in vivo (1). Although genetic techniques have been extremely valuable to study the functions of certain genes in these cells in vivo, this approach is time consuming and expensive. An alternative loss-of-function approach that has been validated in many mammalian systems is shRNA-mediated gene silencing. This chapter describes methodology designed to introduce shRNA constructs into primary murine osteoblasts ex vivo in order to quickly assess the function of genes in osteoblast differentiation and extra cellular matrix mineralization. Both the production of shRNA-expressing lentiviruses and the infection of calvarial osteoblasts with these lentiviruses are detailed.