Skip to main content

Preparation and Testing of Synthetic mRNA for Microinjection

  • Protocol
Molecular Methods in Developmental Biology

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 127))

Abstract

The misexpression and overexpression of proteins by injection of synthetic mRNA into Xenopus oocytes and, more particularly, embryos has provided the experimental basis for a considerable portion of our current knowledge of early vertebrate development (1, and references therein). The original experiments performed were designed to test the function of proteins in early development by expressing large quantities of the protein itself both in the cells and at the times when it would normally be expressed (overexpression) and in cells or at times when it would not (ectopic expression). These experiments was used to test the roles of both transcription factors and signaling molecules in Xenopus embryos.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Heasman, J. (1997) Patterning the Xenopus Blastula. Development 124, 4179–4191.

    PubMed  CAS  Google Scholar 

  2. Hopwood, N. D. and Gurdon, J. B. (1990) Activation of muscle genes without myogenesis by ectiopic expression of MyoD in frog embryo cells. Nature 347, 197–200.

    Article  PubMed  CAS  Google Scholar 

  3. Amaya, E., Musci, T. J., and Kirschner, M. W. (1991) Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos. Cell 66, 257–270.

    Article  PubMed  CAS  Google Scholar 

  4. Suzuki, A., Kaneko, E., Ueno, N., and Hemmati-Brivanlou, A. (1997) Regulation of epidermal induction by BMP2 and BMP7 signaling. Dev. Biol. 189, 112–122.

    Article  PubMed  Google Scholar 

  5. Joseph, E. M. and Melton, D. A. (1998) Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos. Development 125, 2677–2685.

    PubMed  CAS  Google Scholar 

  6. Piccolo, S., Agius, E., Lu, B., Goodman, S., Dale, L., and DeRobertis, E. (1997) Cleavage of chordin by Xolloid metalloprotease suggests a role for proteolytic processing in the regulation of Spemann organizer activity. Cell 91, 407–416.

    Article  PubMed  CAS  Google Scholar 

  7. Conlon, F. L., Sedgwick, S. G., Weston, K. M., and Smith, J. C. (1996) Inhibition of Xbra transcription activation causes defects in mespdermal patterning and reveals autoregulation of Xbra in dorsal mesoderm. Development 122, 2427–2435.

    PubMed  CAS  Google Scholar 

  8. Lagna, G. and Hemmati-Brivanlou, A. (1998) Use of dominant negative constructs to modulate gene expression. Curr. Topics Dev. Biol. 36, 75–98.

    Article  CAS  Google Scholar 

  9. Ullrich, A. and Schlessinger, J. (1990) Signal transduction by receptors with tyrosine kinase activity. Cell 61, 203–212.

    Article  PubMed  CAS  Google Scholar 

  10. Smith, D. P., Mason, C. S., Jones, E. and Old, R. (1994) Expression of a dominant-negative retinoic acid receptor-gamma in Xenopus embryos leads to partial resistance to retinoic acid. Rouxs Arch. Dev. Biol. 203, 254–265.

    Article  CAS  Google Scholar 

  11. Suzuki, A., Thies, R. S., Yamaji, N., Song, J. J., Wozney, J. M., Murakami, K., et al. (1994) A truncated bone morphogenetic protein-receptor affects dorsal-ventral patterning in the early Xenopus embryo. Proc. Natl. Acad. Sci. USA 91, 10,255–10,259.

    Article  PubMed  CAS  Google Scholar 

  12. Ataliotis, P., Symes, K., Chou, M. M., Ho, L., and Mercola, M. (1995) PDGF Signaling is required for gastrulation of Xenopus-Laevis. Development 121, 3099–3110.

    PubMed  CAS  Google Scholar 

  13. Hawley, S. H. B., Wunnenbergstapleton, K., Hashimoto, C., Laurent, M. N., Watabe, T., Blumberg, B. W., et al. (1995) Disruption of BMP signals in embryonic Xenopus ectoderm leads to direct neural induction. Genes Dev. 9, 2923–2935.

    Article  PubMed  CAS  Google Scholar 

  14. Xu, Q. L., Alldus, G., Holder, N., and Wilkinson, D. G. (1995) Expression of truncated Sek-1 receptor tyrosine kinase disrupts the segmental restriction of gene-expression in the Xenopus and zebrafish hindbrain. Development 121, 4005–4016.

    PubMed  CAS  Google Scholar 

  15. Ulisse, S., Esslemont, G., Baker, B. S., Chatterjee, V. K. K., and Tata, J. R. (1996) Dominant-negative mutant thyroid-hormone receptors prevent transcription from the Xenopus thyroid-hormone receptor-beta gene promoter in response to thyroid-hormone in Xenopus tadpoles in-vivo. Proc. Natl. Acad. Sci. USA 93, 1205–1209.

    Article  PubMed  CAS  Google Scholar 

  16. Aoki, S., Takahashi, K., Matsumoto, K., and Nakamura, T. (1997) Activation of Met tyrosine kinase by hepatocyte growth factor is essential for internal organo-genesis in Xenopus embryos. Biochem. Biophys. Res. Commun. 234, 8–14.

    Article  PubMed  CAS  Google Scholar 

  17. Glinka, A., Wu, W., Onichtchouk, D., Blumenstock, C., and Niehrs, C. (1997) Head induction by simultaneous repression of Bmp and Wnt signalling in Xenopus. Nature 389, 517–519.

    Article  PubMed  CAS  Google Scholar 

  18. Frisch, A. and Wright, C. V. E. (1998) XBMPRII, a novel Xenopus type II receptor mediating BMP signaling in embryonic tissues. Development 125, 431–442.

    PubMed  CAS  Google Scholar 

  19. Hemmati-Brivanlou, A. and Melton, D. A. (1992) A truncated activin receptor inhibits mesoderm induction and formation of axial structures in Xenopus embryos. Nature 359, 609–614.

    Article  PubMed  CAS  Google Scholar 

  20. Han, K. and Manley, J. (1993) Functional domains of the Drosophila engrailed protein. EMBO J 12, 2723–2733.

    PubMed  CAS  Google Scholar 

  21. Badiani, P., Corbella, P., Kioussis, D., Marvel, J., and Weston, K. (1994) Dominant interfering alleles define a role for c-myb in T-cell development. Genes Dev. 8, 770–782.

    Article  PubMed  CAS  Google Scholar 

  22. Fisher, A. L. and Caudy, M. (1998) Groucho proteins: transcriptional corepressors for specific subsets of DNA-binding transcription factors in vertebrates and invertebrates. Genes Dev. 12, 1931–1940.

    Article  PubMed  CAS  Google Scholar 

  23. Kim, J., Lin, J. J., Xu, R. H., and Kung, H. F. (1998) Mesoderm induction by heterodimeric AP-1 (c-Jun and c-Fos) and its involvement in mesoderm formation through the embryonic fibroblast growth factor Xbra autocatalytic loop during the early development of Xenopus embryos. J. Biol. Chem. 273, 1542–1550.

    Article  PubMed  CAS  Google Scholar 

  24. Beck, C. W., Sutherland, D. J., and Woodland, H. R. (1998) Involvement of NF-kappa B associated proteins in FGF-mediated mesoderm induction. Int. J. Dev. Biol. 42, 67–77.

    PubMed  CAS  Google Scholar 

  25. Kessler, D. S. (1997) Siamois is required for formation of Spemann’s organizer. Proc. Natl. Acad. Sci. USA 94, 13,017–13,022.

    Article  PubMed  CAS  Google Scholar 

  26. Snape, A. M. and Smith, J. C. (1996) Regulation of embryonic-cell division by a Xenopus gastrula-specific protein kinase. EMBO J. 15, 4556–4565.

    PubMed  CAS  Google Scholar 

  27. Kolm, P. and Sive, H. (1995) Efficient hormone-inducible protein function in Xenopus laevis. Dev. Biol. 171, 267–272.

    Article  PubMed  CAS  Google Scholar 

  28. Tada, M., OReilly, M. A. J., and Smith, J. C. (1997) Analysis of competence and of Brachyury autoinduction by use of hormone-inducible Xbra. Development 124, 2225–2234.

    PubMed  CAS  Google Scholar 

  29. Ferreiro, B., Artinger, M., Cho, K. W. Y., and Niehrs, C. (1998) Antimorphic goosecoids. Development 125, 1347–1359.

    PubMed  CAS  Google Scholar 

  30. Krieg, P. A. and Melton, D. A. (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucleic Acids Res. 12, 7057–7070.

    Article  PubMed  CAS  Google Scholar 

  31. Maniatis, T., Fritsch, E. F. and Sambrook, J. (1982) Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY.

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 1999 Humana Press Inc.

About this protocol

Cite this protocol

Moore, W., Guille, M. (1999). Preparation and Testing of Synthetic mRNA for Microinjection. In: Guille, M. (eds) Molecular Methods in Developmental Biology. Methods in Molecular Biology™, vol 127. Humana Press. https://doi.org/10.1385/1-59259-678-9:99

Download citation

  • DOI: https://doi.org/10.1385/1-59259-678-9:99

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-790-8

  • Online ISBN: 978-1-59259-678-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics