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Wnt Signaling pp 119-125 | Cite as

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

  • Zhendong A. Zhong
  • Nicole J. Ethen
  • Bart O. Williams
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1481)

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 (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.

Key words

Calvarial cell Osteogenic differentiation Adenovirus Alkaline phosphatase Alizarin red 

References

  1. 1.
    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
  2. 2.
    Zhong Z, Ethen NJ, Williams BO (2014) WNT signaling in bone development and homeostasis. Wiley Interdiscip Rev Dev Biol 3(6):489–500CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Bonewald LF, Johnson ML (2008) Osteocytes, mechanosensing and Wnt signaling. Bone 42(4):606–615CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    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
  5. 5.
    Riddle RC et al (2013) Lrp5 and Lrp6 exert overlapping functions in osteoblasts during postnatal bone acquisition. PLoS One 8(5):e63323CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Zhong Z et al (2012) Wntless functions in mature osteoblasts to regulate bone mass. Proc Natl Acad Sci U S A 109(33):E2197–E2204CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Holmen SL et al (2005) Essential role of beta-catenin in postnatal bone acquisition. J Biol Chem 280(22):21162–21168CrossRefPubMedGoogle Scholar
  8. 8.
    Sooy K, Sabbagh Y, Demay MB (2005) Osteoblasts lacking the vitamin D receptor display enhanced osteogenic potential in vitro. J Cell Biochem 94(1):81–87CrossRefPubMedGoogle Scholar
  9. 9.
    Muzumdar MD et al (2007) A global double-fluorescent Cre reporter mouse. Genesis 45(9):593–605CrossRefPubMedGoogle Scholar
  10. 10.
    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

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Zhendong A. Zhong
    • 1
  • Nicole J. Ethen
    • 1
  • Bart O. Williams
    • 1
  1. 1.Program for Skeletal Disease and Tumor Microenvironment and Center for Cancer and Cell BiologyVan Andel Research InstituteGrand RapidsUSA

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