Skip to main content

Advertisement

SpringerLink
Log in

Generation of bicistronic Dmp1-Cre knock-in mice using a self-cleaving 2A peptide

  • Original Article
  • Published:
Journal of Bone and Mineral Metabolism Aims and scope Submit manuscript

Abstract

Introduction

The conditional manipulation of genes using the Cre recombinase-locus of crossover in P1 (Cre/loxP) system is an important tool for revealing gene functions and cell lineages in vivo. The outcome of this method is dependent on the performance of Cre-driver mouse strains. In most cases, Cre knock-in mice show better specificity than randomly inserted Cre transgenic mice. However, following knock-in, the expression of the original gene replaced by Cre is lost.

Materials and methods

We generated a new differentiated osteoblast- and osteocyte-specific Cre knock-in mouse line that carries the viral T2A sequence encoding a 2A self-cleaving peptide at the end of the coding region of the dentin matrix protein 1 (Dmp1) gene accompanied by the Cre gene.

Results

We confirmed that Dmp1-T2A-Cre mice showed high Cre expression in osteoblasts, osteocytes, odontoblasts, and periodontal ligament cells and that the 2A self-cleaving peptide efficiently produced both Dmp1 and Cre proteins. Furthermore, unlike the Dmp1 knockout mice, homozygous Dmp1-T2A-Cre mice showed no skeletal abnormalities. Analysis using the Cre reporter strain confirmed differentiated osteoblast- and osteocyte-specific Cre-mediated recombination in the skeleton. Furthermore, recombination was also detected in some nuclei of skeletal muscle cells, spermatocytes, and intestinal cells.

Conclusion

2A-Cre functions effectively in vivo, and Dmp1-T2A-Cre knock-in mice are a useful tool for studying the functioning of various genes in hard tissues.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Robling AG, Bonewald LF (2020) The osteocyte: new insights. Annu Rev Physiol 82:485–506. https://doi.org/10.1146/annurev-physiol-021119-034332

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Qin L, Liu W, Cao H, Xiao G (2020) Molecular mechanosensors in osteocytes. Bone Res 8:1–24. https://doi.org/10.1038/s41413-020-0099-y

    Article  CAS  Google Scholar 

  3. Fisher LW, Torchia DA, Fohr B et al (2001) Flexible structures of SIBLING proteins, bone sialoprotein, and osteopontin. Biochem Biophys Res Commun 280:460–465. https://doi.org/10.1006/bbrc.2000.4146

    Article  CAS  PubMed  Google Scholar 

  4. Feng JQ, Huang H, Lu Y et al (2003) The dentin matrix protein 1 (Dmp1) is specifically expressed in mineralized, but not soft, tissues during development. J Dent Res 82:776–780. https://doi.org/10.1177/154405910308201003

    Article  CAS  PubMed  Google Scholar 

  5. Oya K, Ishida K, Nishida T et al (2017) Immunohistochemical analysis of dentin matrix protein 1 (Dmp1) phosphorylation by Fam20C in bone: Implications for the induction of biomineralization. Histochem Cell Biol 147:341–351. https://doi.org/10.1007/s00418-016-1490-z

    Article  CAS  PubMed  Google Scholar 

  6. Fukumoto S, Martin TJ (2009) Bone as an endocrine organ. Trends Endocrinol Metab 20:230–236. https://doi.org/10.1016/j.tem.2009.02.001

    Article  CAS  PubMed  Google Scholar 

  7. Martin A, Liu S, David V et al (2011) Bone proteins PHEX and DMP1 regulate fibroblastic growth factor Fgf23 expression in osteocytes through a common pathway involving FGF receptor (FGFR) signaling. FASEB J 25:2551–2562. https://doi.org/10.1096/fj.10-177816

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Ye L, Mishina Y, Chen D et al (2005) Dmp1-deficient mice display severe defects in cartilage formation responsible for a chondrodysplasia-like phenotype. J Biol Chem 280:6197–6203. https://doi.org/10.1074/jbc.M412911200

    Article  CAS  PubMed  Google Scholar 

  9. Feng JQ, Ward LM, Liu S et al (2006) Loss of DMP1 causes rickets and osteomalacia and identifies a role for osteocytes in mineral metabolism. Nat Genet 38:1310–1315. https://doi.org/10.1038/ng1905

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Li X, Zhang Y, Kang H et al (2005) Sclerostin binds to LRP5/6 and antagonizes canonical Wnt signaling. J Biol Chem 280:19883–19887. https://doi.org/10.1074/jbc.M413274200

    Article  CAS  PubMed  Google Scholar 

  11. Xiong J, Onal M, Jilka RL et al (2011) Matrix-embedded cells control osteoclast formation. Nat Med 17:1235–1241. https://doi.org/10.1038/nm.2448

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Nakashima T, Hayashi M, Fukunaga T et al (2011) Evidence for osteocyte regulation of bone homeostasis through RANKL expression. Nat Med 17:1231–1234. https://doi.org/10.1038/nm.2452

    Article  CAS  PubMed  Google Scholar 

  13. Bélanger LF (1969) Osteocytic osteolysis. Calcif Tissue Res 4:1–12. https://doi.org/10.1007/BF02279101

    Article  PubMed  Google Scholar 

  14. Nango N, Kubota S, Hasegawa T et al (2016) Osteocyte-directed bone demineralization along canaliculi. Bone 84:279–288. https://doi.org/10.1016/j.bone.2015.12.006

    Article  CAS  PubMed  Google Scholar 

  15. Kato Y, Windle JJ, Koop BA et al (1997) Establishment of an osteocyte-like cell line, MLO-Y4. J Bone Miner Res 12:2014–2023. https://doi.org/10.1359/jbmr.1997.12.12.2014

    Article  CAS  PubMed  Google Scholar 

  16. Woo SM, Rosser J, Dusevich V et al (2011) Cell line IDG-SW3 replicates osteoblast-to-late-osteocyte differentiation in vitro and accelerates bone formation in vivo. J Bone Miner Res 26:2634–2646. https://doi.org/10.1002/jbmr.465

    Article  CAS  PubMed  Google Scholar 

  17. Kalajzic I, Matthews BG, Torreggiani E et al (2013) In vitro and in vivo approaches to study osteocyte biology. Bone 54:296–306. https://doi.org/10.1016/j.bone.2012.09.040

    Article  CAS  PubMed  Google Scholar 

  18. Schmidt-Supprian M, Rajewsky K (2007) Vagaries of conditional gene targeting. Nat Immunol 8:665–668. https://doi.org/10.1038/ni0707-665

    Article  CAS  PubMed  Google Scholar 

  19. Dallas SL, Xie Y, Shiflett LA, Ueki Y (2018) Mouse Cre models for the study of bone diseases. Curr Osteoporos Rep 16:466–477. https://doi.org/10.1007/s11914-018-0455-7

    Article  PubMed  PubMed Central  Google Scholar 

  20. Lu Y, Xie Y, Zhang S et al (2007) DMP1-Targeted Cre expression in odontoblasts and osteocytes. J Dent Res 86:320–325. https://doi.org/10.1177/154405910708600404

    Article  CAS  PubMed  Google Scholar 

  21. Bivi N, Condon KW, Allen MR et al (2012) Cell autonomous requirement of connexin 43 for osteocyte survival: Consequences for endocortical resorption and periosteal bone formation. J Bone Miner Res 27:374–389. https://doi.org/10.1002/jbmr.548

    Article  CAS  PubMed  Google Scholar 

  22. Song AJ, Palmiter RD (2018) Detecting and avoiding problems when using the Cre–lox system. Trends Genet 34:333–340. https://doi.org/10.1016/j.tig.2017.12.008

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Cui Y, Niziolek PJ, MacDonald BT et al (2011) Lrp5 functions in bone to regulate bone mass. Nat Med 17:684–691. https://doi.org/10.1038/nm.2388

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Lim J, Burclaff J, He G et al (2017) Unintended targeting of Dmp1-Cre reveals a critical role for Bmpr1a signaling in the gastrointestinal mesenchyme of adult mice. Bone Res 5:1–8. https://doi.org/10.1038/boneres.2016.49

    Article  CAS  Google Scholar 

  25. Becher B, Waisman A, Lu LF (2018) Conditional gene-targeting in mice: problems and solutions. Immunity 48:835–836. https://doi.org/10.1016/j.immuni.2018.05.002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Maeda K, Kobayashi Y, Udagawa N et al (2012) Wnt5a-Ror2 signaling between osteoblast-lineage cells and osteoclast precursors enhances osteoclastogenesis. Nat Med 18:405–412. https://doi.org/10.1038/nm.2653

    Article  CAS  PubMed  Google Scholar 

  27. Akiyama H, Kim JE, Nakashima K et al (2005) Osteo-chondroprogenitor cells are derived from Sox9 expressing precursors. Proc Natl Acad Sci USA 102:14665–14670. https://doi.org/10.1073/pnas.0504750102

    Article  CAS  PubMed  Google Scholar 

  28. Mizuguchi H, Xu Z, Ishii-Watabe A et al (2000) IRES-dependent second gene expression is significantly lower than cap-dependent first gene expression in a bicistronic vector. Mol Ther 1:376–382. https://doi.org/10.1006/mthe.2000.0050

    Article  CAS  PubMed  Google Scholar 

  29. Ryan MD, Flint M (1997) Virus-encoded proteinases of the picornavirus super-group. J Gen Virol 78:699–723. https://doi.org/10.1099/0022-1317-78-4-699

    Article  CAS  PubMed  Google Scholar 

  30. Szymczak AL, Workman CJ, Wang Y et al (2004) Correction of multi-gene deficiency in vivo using a single “self-cleaving” 2A peptide-based retroviral vector. Nat Biotechnol 22:589–594. https://doi.org/10.1038/nbt957

    Article  CAS  PubMed  Google Scholar 

  31. Madisen L, Zwingman TA, Sunkin SM et al (2010) A robust and high-throughput Cre reporting and characterization system for the whole mouse brain. Nat Neurosci 13:133–140. https://doi.org/10.1038/nn.2467

    Article  CAS  PubMed  Google Scholar 

  32. Nakamura T, Nakamura-Takahashi A, Kasahara M et al (2020) Tissue-nonspecific alkaline phosphatase promotes the osteogenic differentiation of osteoprogenitor cells. Biochem Biophys Res Commun 524:702–709. https://doi.org/10.1016/j.bbrc.2020.01.136

    Article  CAS  PubMed  Google Scholar 

  33. Kawamoto T, Shimizu M (2000) A method for preparing 2-to 50-μm-thick fresh-frozen sections of large samples and undecalcified hard tissues. Histochem Cell Biol 113:331–339. https://doi.org/10.1007/s004180000149

    Article  CAS  PubMed  Google Scholar 

  34. Yagi T, Tokunaga T, Furuta Y et al (1993) A novel ES cell line, TT2, with high germline-differentiating potency. Anal Biochem 214:70–76

    Article  CAS  PubMed  Google Scholar 

  35. Terasawa M, Shimokawa R, Terashima T et al (2004) Expression of dentin matrix protein 1 (DMP1) in nonmineralized tissues. J Bone Miner Metab 22:430–438. https://doi.org/10.1007/s00774-004-0504-4

    Article  CAS  PubMed  Google Scholar 

  36. Wilson MJ, Liaw L, Koopman P (2005) Osteopontin and related SIBLING glycoprotein genes are expressed by sertoli cells during mouse testis development. Dev Dyn 233:1488–1495. https://doi.org/10.1002/dvdy.20456

    Article  CAS  PubMed  Google Scholar 

  37. Feng JQ, Zhang J, Dallas SL et al (2002) Dentin matrix protein 1, a target molecule for Cbfa1 in bone, is a unique bone marker gene. J Bone Miner Res 17:1822–1831. https://doi.org/10.1359/jbmr.2002.17.10.1822

    Article  CAS  Google Scholar 

  38. Torreggiani E, Matthews BG, Pejda S et al (2013) Preosteocytes/osteocytes have the potential to dedifferentiate becoming a source of osteoblasts. PLoS One. https://doi.org/10.1371/journal.pone.0075204

    Article  PubMed  PubMed Central  Google Scholar 

  39. Terada M, Lan YB, Kawano F et al (2010) Myonucleus-related properties in soleus muscle fibers of mdx mice. Cells Tissues Organs 191:248–259. https://doi.org/10.1159/000240245

    Article  PubMed  Google Scholar 

  40. Nakamura T, Imai Y, Matsumoto T et al (2007) Estrogen prevents bone loss via estrogen receptor α and induction of fas ligand in osteoclasts. Cell 130:811–823. https://doi.org/10.1016/j.cell.2007.07.025

    Article  CAS  PubMed  Google Scholar 

  41. Debnath S, Yallowitz AR, McCormick J et al (2018) Discovery of a periosteal stem cell mediating intramembranous bone formation. Nature 562:133–139. https://doi.org/10.1038/s41586-018-0554-8

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Tsukasaki M, Komatsu N, Negishi-Koga T et al (2022) Periosteal stem cells control growth plate stem cells during postnatal skeletal growth. Nat Commun 13:4166. https://doi.org/10.1038/s41467-022-31592-x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Dzamukova M, Brunner TM, Miotla-Zarebska J et al (2022) Mechanical forces couple bone matrix mineralization with inhibition of angiogenesis to limit adolescent bone growth. Nat Commun 13:1–12. https://doi.org/10.1038/s41467-022-30618-8

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank Drs. Yuko Nakamichi, Yasuharu Kurokawa, Takahito Okudaira, Naoko Irie, Yasunari Takada, Tsukasa Oikawa and Mr. Gen Sakai for technical help and discussion; Dr. Shinichi Aizawa for the DT-ApA/conditional KO FW plasmid and TT2 ES cells; Dr. Neal Copeland for the PL451 plasmid; Dr. Akira Yamaguchi for continued support.

Funding

This work was supported by the Suematsu Gas Biology Project, Exploratory Research for Advanced Technology, from the Japan Science and Technology Agency and by grants from the Grants-in-Aid for Young Scientists A (23689067), Challenging Exploratory Research (26670673), and Scientific Research C (21K09214) from the Japan Society for the Promotion of Science (JSPS).

Author information

Authors and Affiliations

  1. Department of Biochemistry, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan

    Takashi Nakamura, Sayako Honda, Takehiro Yamamoto, Yasuaki Kabe & Makoto Suematsu

  2. Suematsu Gas Biology Project, Exploratory Research for Advanced Technology, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi-shi, Saitama, 332-0012, Japan

    Takashi Nakamura, Takehiro Yamamoto, Yasuaki Kabe & Makoto Suematsu

  3. Department of Biochemistry, Tokyo Dental College, 2-9-18 Kanda-Misakicho, Chiyoda-ku, Tokyo, 101-0061, Japan

    Takashi Nakamura

  4. Oral Health Science Center, Tokyo Dental College, 2-9-18 Kanda-Misakicho, Chiyoda-ku, Tokyo, 101-0061, Japan

    Takashi Nakamura & Toshihide Mizoguchi

  5. Department of Pharmacology, Tokyo Dental College, 2-9-18 Kanda-Misakicho, Chiyoda-ku, Tokyo, 101-0061, Japan

    Shinichirou Ito & Masataka Kasahara

  6. Laboratory of Cell and Tissue Biology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan

    Koichi Matsuo

  7. WPI-Bio2Q and AMED Moonshot Project, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo, 160-8582, Japan

    Makoto Suematsu

Authors
  1. Takashi Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sayako Honda
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shinichirou Ito
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Toshihide Mizoguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Takehiro Yamamoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Masataka Kasahara
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Yasuaki Kabe
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Koichi Matsuo
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Makoto Suematsu
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

TN designed and performed most experiments, interpreted the results, and wrote the manuscript. SH, TY, and YK contributed to the generation and characterization of the Dmp1-T2A-Cre mice and data interpretation. SI, TM, and MK contributed to the characterization of the Dmp1-T2A-Cre mice and data interpretation. KM and MS supervised the project and wrote the manuscript.

Corresponding authors

Correspondence to Takashi Nakamura or Makoto Suematsu.

Ethics declarations

Conflict of interest

All authors have no conflicts of interest.

Ethical approval

Prior to the study, all experiments were reviewed and approved by the Animal Care and Use Committee of Keio University (No. 12039) and Tokyo Dental College (No. 300402, No. 300706). This study is reported in accordance with the ARRIVE guidelines.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Nakamura, T., Honda, S., Ito, S. et al. Generation of bicistronic Dmp1-Cre knock-in mice using a self-cleaving 2A peptide. J Bone Miner Metab 41, 470–480 (2023). https://doi.org/10.1007/s00774-023-01425-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00774-023-01425-y

Keywords

Search

Navigation