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Generation and Analysis of Cartilage-Specific CCN2 Overexpression in Transgenic Mice

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CCN Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1489))

Abstract

Recent progress in gene-editing technology has provided a strong impact for improved our understanding of molecular functions in living organisms. Here we describe our method to generate transgene-overexpressing mouse models, which method involves the use of tissue-specific promoters for analyzing a certain molecule (s) in special tissues. The protocol described in this chapter uses the Col2a1 promoter-enhancer, which is known for driving specific and strong transgene expression in cartilage and is based on several of our studies showing a positive role of the connective tissue growth factor (CCN2) in cartilage-bone development and maintenance of articular cartilage. These mice show strongly accelerated endochondral ossification resulting in enhanced bone elongation, as well as resistance to age-related articular degeneration. This protocol also describes how to analyze the molecular mechanisms of these phenomena by use of chondrocytes isolated from CCN2-overexpressing cartilage.

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References

  1. Nakanishi T, Nishida T, Shimo T, Kobayashi K, Kubo T, Tamatani T, Tezuka K, Takigawa M (2000) Effects of CTGF/Hcs24, a product of a hypertrophic chondrocyte-specific gene, on the proliferation and differentiation of chondrocytes in culture. Endocrinology 141:264–273

    CAS  PubMed  Google Scholar 

  2. Nishida T, Nakanishi T, Asano M, Shimo T, Takigawa M (2000) Effects of CTGF/Hcs24, a hypertrophic chondrocyte-specific gene product, on the proliferation and differentiation of osteoblastic cells in vitro. J Cell Physiol 184:197–206

    Article  CAS  PubMed  Google Scholar 

  3. Shimo T, Nakanishi T, Kimura Y, Nishida T, Ishizeki K, Matsumura T, Takigawa M (1998) Inhibition of endogenous expression of connective tissue growth factor by its antisense oligonucleotide and antisense RNA suppresses proliferation and migration of vascular endothelial cells. J Biochem 124:130–140

    Article  CAS  PubMed  Google Scholar 

  4. Ivkovic S, Yoon BS, Popoff SN, Safadi FF, Libuda DE, Stephenson RC, Daluiski A, Lyons KM (2003) Connective tissue growth factor coordinates chondrogenesis and angiogenesis during skeletal development. Development (Cambridge) 130:2779–2791

    Article  CAS  Google Scholar 

  5. Kim Y, Murao H, Yamamoto K, Deng JM, Behringer RR, Nakamura T, Akiyama H (2011) Generation of transgenic mice for conditional overexpression of Sox9. J Bone Miner Metab 29:123–129

    Article  CAS  PubMed  Google Scholar 

  6. Lee HH, Behringer RR (2007) Conditional expression of Wnt4 during chondrogenesis leads to dwarfism in mice. PLoS One 2, e450

    Article  PubMed  PubMed Central  Google Scholar 

  7. Murakami S, Balmes G, McKinney S, Zhang Z, Givol D, de Crombrugghe B (2004) Constitutive activation of MEK1 in chondrocytes causes Stat1-independent achondroplasia-like dwarfism and rescues the Fgfr3-deficient mouse phenotype. Genes Dev 18:290–305

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhang R, Murakami S, Coustry F, Wang Y, de Crombrugghe B (2006) Constitutive activation of MKK6 in chondrocytes of transgenic mice inhibits proliferation and delays endochondral bone formation. Proc Natl Acad Sci U S A 103:365–370

    Article  CAS  PubMed  Google Scholar 

  9. Itoh S, Hattori T, Tomita N, Aoyama E, Yutani Y, Yamashiro T, Takigawa M (2013) CCN family member 2/connective tissue growth factor (CCN2/CTGF) has anti-aging effects that protect articular cartilage from age-related degenerative changes. PLoS One 8, e71156

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tomita N, Hattori T, Itoh S, Aoyama E, Yao M, Yamashiro T, Takigawa M (2013) Cartilage-specific over-expression of CCN family member 2/connective tissue growth factor (CCN2/CTGF) stimulates insulin-like growth factor expression and bone growth. PLoS One 8, e59226

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Cheah KS, Lau ET, Au PK, Tam PP (1991) Expression of the mouse alpha 1(II) collagen gene is not restricted to cartilage during development. Development 111:945–953

    CAS  PubMed  Google Scholar 

  12. Mukhopadhyay K, Lefebvre V, Zhou G, Garofalo S, Kimura JH, de Crombrugghe B (1995) Use of a new rat chondrosarcoma cell line to delineate a 119-base pair chondrocyte-specific enhancer element and to define active promoter segments in the mouse pro-alpha 1(II) collagen gene. J Biol Chem 270:27711–27719

    Article  CAS  PubMed  Google Scholar 

  13. Zhou G, Garofalo S, Mukhopadhyay K, Lefebvre V, Smith CN, Eberspaecher H, de Crombrugghe B (1995) A 182 bp fragment of the mouse pro alpha 1(II) collagen gene is sufficient to direct chondrocyte expression in transgenic mice. J Cell Sci 108(Pt 12):3677–3684

    CAS  PubMed  Google Scholar 

  14. Lefebvre V, Mukhopadhyay K, Zhou G, Garofalo S, Smith C, Eberspaecher H, Kimura JH, de Crombrugghe B (1996) A 47-bp sequence of the first intron of the mouse pro alpha 1(II) collagen gene is sufficient to direct chondrocyte Expression. Ann N Y Acad Sci 785:284–287

    Article  CAS  PubMed  Google Scholar 

  15. Lefebvre V, Zhou G, Mukhopadhyay K, Smith CN, Zhang Z, Eberspaecher H, Zhou X, Sinha S, Maity SN, de Crombrugghe B (1996) An 18-base-pair sequence in the mouse proalpha1(II) collagen gene is sufficient for expression in cartilage and binds nuclear proteins that are selectively expressed in chondrocytes. Mol Cell Biol 16:4512–4523

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Bridgewater LC, Lefebvre V, de Crombrugghe B (1998) Chondrocyte-specific enhancer elements in the Col11a2 gene resemble the Col2a1 tissue-specific enhancer. J Biol Chem 273:14998–15006

    Article  CAS  PubMed  Google Scholar 

  17. Oh CD, Maity SN, Lu JF, Zhang J, Liang S, Coustry F, de Crombrugghe B, Yasuda H (2010) Identification of SOX9 interaction sites in the genome of chondrocytes. PLoS One 5:e10113

    Article  PubMed  PubMed Central  Google Scholar 

  18. Ovchinnikov DA, Deng JM, Ogunrinu G, Behringer RR (2000) Col2a1-directed expression of Cre recombinase in differentiating chondrocytes in transgenic mice. Genesis 26:145–146

    Article  CAS  PubMed  Google Scholar 

  19. Chen M, Lichtler AC, Sheu TJ, Xie C, Zhang X, O'Keefe RJ, Chen D (2007) Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase. Genesis 45:44–50

    Article  PubMed  PubMed Central  Google Scholar 

  20. Zhu M, Chen M, Lichtler AC, O'Keefe RJ, Chen D (2008) Tamoxifen-inducible Cre-recombination in articular chondrocytes of adult Col2a1-CreER(T2) transgenic mice. Osteoarthr Cartil 16:129–130

    Article  CAS  PubMed  Google Scholar 

  21. Henry SP, Jang CW, Deng JM, Zhang Z, Behringer RR, de Crombrugghe B (2009) Generation of aggrecan-CreERT2 knockin mice for inducible Cre activity in adult cartilage. Genesis 47:805–814

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Mao X, Fujiwara Y, Orkin SH (1999) Improved reporter strain for monitoring Cre recombinase-mediated DNA excisions in mice. Proc Natl Acad Sci U S A 96:5037–5042

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Soriano P (1999) Generalized lacZ expression with the ROSA26 Cre reporter strain. Nat Genet 21:70–71

    Article  CAS  PubMed  Google Scholar 

  24. Park J, Gebhardt M, Golovchenko S, Perez-Branguli F, Hattori T, Hartmann C, Zhou X, deCrombrugghe B, Stock M, Schneider H, von der Mark K (2015) Dual pathways to endochondral osteoblasts: a novel chondrocyte-derived osteoprogenitor cell identified in hypertrophic cartilage. Biol Open 4:608–621

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Zhou X, von der Mark K, Henry S, Norton W, Adams H, de Crombrugghe B (2015) Chondrocytes transdifferentiate into osteoblasts in endochondral bone during development, postnatal growth and fracture healing in mice. PLoS Genet 10, e1004820

    Article  Google Scholar 

  26. Tamatani T, Kobayashi H, Tezuka K, Sakamoto S, Suzuki K, Nakanishi T, Takigawa M, Miyano T (1998) Establishment of the enzyme-linked immunosorbent assay for connective tissue growth factor (CTGF) and its detection in the sera of biliary atresia. Biochem Biophys Res Commun 251:748–752

    Article  CAS  PubMed  Google Scholar 

  27. Hofker MH, Breuer M (1998) Generation of transgenic mice. Meth Molecular Biol (Clifton, NJ) 110:63–78

    CAS  Google Scholar 

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Acknowledgements

We thank Drs. Benoit de Crombrugghe and Shunichi Murakami, UTMD Anderson Cancer Center for kindly supplying the 3 kb–3 kb Col2a1/pBS M2 and IRES-LacZ-SV40pA/pBS vectors. The authors also thank Drs. Satoshi Kubota and Takashi Nishida for useful suggestions, as well as Ms. Yoshiko Miyake and Ayako Ogo for technical and secretarial assistance. This study was supported by grants from the program Grants-in-aid for Scientific Research (B) [#JP24390415] and (B) [#JP15H05014] to M.T. and (C) (#JP254628889) to T.H. and a grant from the Japan Society and Foundation for Growth Science to T.H.

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Authors and Affiliations

  1. Department of Biochemistry & Molecular Dentistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan

    Takako Hattori Ph.D.

  2. Department of Orthodontics and Dentofacial Orthopedics, Osaka University Graduate School of Dentistry, Osaka, Japan

    Shinsuke Itoh

  3. Advanced Research Center for Oraland Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan

    Masaharu Takigawa D.D.S., Ph.D.

Authors
  1. Takako Hattori Ph.D.
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  2. Shinsuke Itoh
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  3. Masaharu Takigawa D.D.S., Ph.D.
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Corresponding authors

Correspondence to Takako Hattori Ph.D. or Masaharu Takigawa D.D.S., Ph.D. .

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Editors and Affiliations

  1. Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine Dentistry and Pharmaceutical Sciences, Okayama, Japan

    Masaharu Takigawa

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Hattori, T., Itoh, S., Takigawa, M. (2017). Generation and Analysis of Cartilage-Specific CCN2 Overexpression in Transgenic Mice. In: Takigawa, M. (eds) CCN Proteins. Methods in Molecular Biology, vol 1489. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6430-7_32

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  • DOI: https://doi.org/10.1007/978-1-4939-6430-7_32

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6428-4

  • Online ISBN: 978-1-4939-6430-7

  • eBook Packages: Springer Protocols

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