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A Novel Strategy for a Splice-Variant Selective Gene Ablation: The Example of the Versican V0/V2 Knockout

  • María T. Dours-Zimmermann
  • Dieter R. ZimmermannEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 836)

Abstract

The complete knockout of genes that give rise to alternative splice products can often provide only an integral view of the dominant function(s) of all the isoforms they encode. If one of these isoforms is indispensable for life, a constitutive and complete inactivation may even preclude any in vivo studies of later expressed splice-variants in mice. To explore function of the tissue-restricted versican V2 isoform during central nervous system maturation, for instance, we had to circumvent the early embryonic lethality of the complete knockout by employing a novel splice-variant-specific gene ablation approach. For this purpose, we introduced a preterm translational stop codon preceded by an ER-retention signal (KDEL) into the alternatively spliced exon 7 of the VCAN gene. This way the synthesis of the V2- and the V0-forms of the proteoglycan was entirely abolished in the mutant mice, most likely mediated by a KDEL-promoted intracellular degradation of the mutant fragment and by a nonsense-mediated decay mechanism. The expression of the vitally important V1-isoform and the smallest V3-variant remained, however, unaffected. Here we provide the details of our targeting strategy, the screening procedure, the generation of isoform-specific antibodies, and the transcript analysis and we supply the experimental protocols for the biochemical and immunohistological examinations of the mutant mouse strain Vcantm1.1Dzim.

Key words

Knock-in Alternative splicing Gene inactivation Proteoglycan Versican Hyaluronan Hyaluronidase Antibodies Immunofluorescence PCR 

Notes

Acknowledgments

We thank Reinhard Fässler and Uwe Rauch for crucial inputs for the targeting strategy and invaluable advices for the generation of the mouse strain, Bernhard Odermatt for many helpful hints regarding the immunhistological stainings, and Philipp U. Heitz and Holger Moch for their continuous support of the project. Our work has been financed in part by grants from the Swiss National Science Foundation and the Velux-Foundation to D.R.Z.

References

  1. 1.
    Aszodi, A., Legate, K. R., Nakchbandi, I., and Fassler, R. (2006) What mouse mutants teach us about extracellular matrix function. Annu Rev Cell Dev Biol. 22, 591–621.PubMedCrossRefGoogle Scholar
  2. 2.
    Kuhn, R. and Wurst, W. (2009) Overview on mouse mutagenesis. Methods Mol Biol. 530, 1–12.PubMedCrossRefGoogle Scholar
  3. 3.
    Mjaatvedt, C., Yamamura, H., Capehart, A., Turner, D., and Markwald, R. (1998) The cspg2 gene, disrupted in the hdf mutant, is required for right cardiac chamber and endocardial cushion formation. Dev. Biol. 202, 56–66.PubMedCrossRefGoogle Scholar
  4. 4.
    Zimmermann, D. R. and Dours-Zimmermann, M. T. (2008) Extracellular matrix of the central nervous system: from neglect to challenge. Histochem. Cell Biol. 130, 635–653.PubMedCrossRefGoogle Scholar
  5. 5.
    Dours-Zimmermann, M. T., Maurer, K., Rauch, U., Stoffel, W., Fassler, R., and Zimmermann, D. R. (2009) Versican V2 assembles the extracellular matrix surrounding the nodes of ranvier in the CNS. J Neurosci. 29, 7731–7742.PubMedCrossRefGoogle Scholar
  6. 6.
    Talts, J. F., Brakebusch, C., and Fassler, R. (1999) Integrin gene targeting, Methods Mol. Biol. 129, 153–187.Google Scholar
  7. 7.
    Chomczynski, P. and Sacchi, N. (2006) The single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction: twenty-something years on. Nat. Protoc. 1, 581–585.PubMedCrossRefGoogle Scholar
  8. 8.
    Yamagata, M., Shinomura, T., and Kimata, K. (1993) Tissue variation of two large chondroitin sulfate proteoglycans (PG-M/versican and PG-H/aggrecan) in chick embryos. Anat. Embryol. Berl. 187, 433–444.PubMedCrossRefGoogle Scholar
  9. 9.
    Herrin, D. L. and Schmidt, G. W. (1988) Rapid, reversible staining of Northern blots prior to hybridization. BioTechniques 6, 196.PubMedGoogle Scholar
  10. 10.
    Zimmermann, D. R., Dours-Zimmermann, M. T., Schubert, M., and Bruckner-Tuderman, L. (1994) Versican is expressed in the proliferating zone in the epidermis and in association with the elastic network of the dermis. J. Cell Biol. 124, 817–825.PubMedCrossRefGoogle Scholar
  11. 11.
    Harlow, E. and Lane, D. (1988) Antibodies, a laboratory manual, Cold Spring Harbor Laboratory, Cold Spring Harbor.Google Scholar
  12. 12.
    Reineke, T., Jenni, B., Abdou, M. T., Frigerio, S., Zubler, P., Moch, H., and Tinguely, M. (2006) Ultrasonic decalcification offers new perspectives for rapid FISH, DNA, and RT-PCR analysis in bone marrow trephines. Am J Surg Pathol. 30, 892–896.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • María T. Dours-Zimmermann
    • 1
  • Dieter R. Zimmermann
    • 1
    Email author
  1. 1.Institute of Surgical PathologyUniversity Hospital ZurichZurichSwitzerland

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