Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Regard JB et al (2012) Wnt signaling in bone development and disease: making stronger bone with Wnts. Cold Spring Harb Perspect Biol 4(12)
Zhong Z, Ethen NJ, Williams BO (2014) WNT signaling in bone development and homeostasis. Wiley Interdiscip Rev Dev Biol 3(6):489–500
Bonewald LF, Johnson ML (2008) Osteocytes, mechanosensing and Wnt signaling. Bone 42(4):606–615
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):836–843
Riddle RC et al (2013) Lrp5 and Lrp6 exert overlapping functions in osteoblasts during postnatal bone acquisition. PLoS One 8(5):e63323
Zhong Z et al (2012) Wntless functions in mature osteoblasts to regulate bone mass. Proc Natl Acad Sci U S A 109(33):E2197–E2204
Holmen SL et al (2005) Essential role of beta-catenin in postnatal bone acquisition. J Biol Chem 280(22):21162–21168
Sooy K, Sabbagh Y, Demay MB (2005) Osteoblasts lacking the vitamin D receptor display enhanced osteogenic potential in vitro. J Cell Biochem 94(1):81–87
Muzumdar MD et al (2007) A global double-fluorescent Cre reporter mouse. Genesis 45(9):593–605
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):91–99
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Zhong, Z.A., Ethen, N.J., Williams, B.O. (2016). Use of Primary Calvarial Osteoblasts to Evaluate the Function of Wnt Signaling in Osteogenesis. In: Barrett, Q., Lum, L. (eds) Wnt Signaling. Methods in Molecular Biology, vol 1481. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6393-5_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6393-5_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6391-1
Online ISBN: 978-1-4939-6393-5
eBook Packages: Springer Protocols