Use of Primary Calvarial Osteoblasts to Evaluate the Function of Wnt Signaling in Osteogenesis.

: In vitro culture and genetic manipulation of primary calvarial cell cultures is a convenient and robust system to investigate gene function in osteoblast differentiation. We have used this system to study the functions of many genes in the Wnt signaling pathway within osteoblasts. Here, we describe a detailed protocol outlining the establishment and characterization of primary calvarial cells from mice carrying a conditionally inactivatable allele of the Wntless (Wls) gene (Wls (flox/flox)). We previously used this approach to delete the Wntless gene by infecting with a Cre-expressing adenovirus, and to evaluate the effects of Wnt signaling loss on osteogenic potential in osteogenic medium with ascorbic acid. This detailed protocol is adaptable to use with any floxed allele.