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 (Wlsflox/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.
Calvarial cell Osteogenic differentiation Adenovirus Alkaline phosphatase Alizarin red
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
Regard JB et al (2012) Wnt signaling in bone development and disease: making stronger bone with Wnts. Cold Spring Harb Perspect Biol 4(12)Google Scholar
Zhang M et al (2003) Paracrine overexpression of IGFBP-4 in osteoblasts of transgenic mice decreases bone turnover and causes global growth retardation. J Bone Miner Res 18(5):836–843CrossRefPubMedGoogle Scholar
Feng J et al (1977) Determination of L-ascorbic acid levels in culture medium: concentrations in commercial media and maintenance of levels under conditions of organ culture. In Vitro 13(2):91–99CrossRefPubMedGoogle Scholar