Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Creating transgenic Drosophila by microinjecting the site-specific φC31 integrase mRNA and a transgene-containing donor plasmid

Nature Protocols volume 2, pages 2325–2331 (2007)Cite this article

Abstract

We describe a microinjection-based φC31 integrase mRNA-mediated method for creating transgenic Drosophila strains. This approach is more efficient than traditional methods and ensures that the transgene is targeted to a precise genomic position. The method involves targeting integration of an exogenous plasmid (containing the transgene and sequences to facilitate integration) to a preplaced recipient site in the Drosophila genome. The plasmid is coinjected into embryos with mRNA encoding the φC31 integrase, the enzyme that catalyzes the integration reaction. Using the protocol described here, transgenic lines can be established from, on average, 46% of fertile adults obtained after injection, and all integrations should be targeted to the chosen genomic insertion site. The whole procedure, from injection to established transgenic stocks, can be completed in three generations (approximately 1 month) and can be adapted for other types of transgenesis and mRNA injections in Drosophila.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: A schematic outlining φC31-mediated integration.
Figure 2: Physical map of the plasmid pUASTattB used in this protocol.
Figure 3: Breaking an injection needle.
Figure 4: Injecting embryos.

Similar content being viewed by others

References

  1. Rubin, G.M. & Spradling, A.C. Genetic transformation of Drosophila with transposable element vectors. Science 218, 348–353 (1982).

    Article  CAS  PubMed Central  Google Scholar 

  2. Brand, A.H. & Perrimon, N. Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401–415 (1993).

    CAS  Google Scholar 

  3. Xu, T. & Rubin, G.M. Analysis of genetic mosaics in developing and adult Drosophila tissues. Development 117, 1223–1237 (1993).

    CAS  Google Scholar 

  4. Groth, A.C., Olivares, E.C., Thyagarajan, B. & Calos, M.P. A phage integrase directs efficient site-specific integration in human cells. Proc. Natl. Acad. Sci. USA 97, 5995–6000 (2000).

    Article  CAS  PubMed Central  Google Scholar 

  5. Allen, B.G. & Weeks, D.L. Transgenic Xenopus laevis embryos can be generated using φC31 integrase. Nat. Methods 2, 975–979 (2005).

    Article  CAS  PubMed Central  Google Scholar 

  6. Nimmo, D.D., Alphey, L., Meredith, J.M. & Eggleston, P. High efficiency site-specific genetic engineering of the mosquito genome. Insect Mol. Biol. 15, 129–136 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  7. Groth, A.C., Fish, M., Nusse, R. & Calos, M.P. Construction of transgenic Drosophila by using the site-specific integrase from phage φC31. Genetics 166, 1775–1782 (2004).

    Article  CAS  PubMed Central  Google Scholar 

  8. Bateman, J.R., Lee, A.M. & Wu, C.T. Site-specific transformation of Drosophila via φC31 integrase-mediated cassette exchange. Genetics 173, 769–777 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  9. Venken, K.J.T., He, Y., Hoskins, R.A. & Bellen, H.J. P[acman]: a BAC transgenic platform for targeted insertion of large DNA fragments in D. melanogaster. Science 314, 1747–1751 (2006).

    Article  CAS  PubMed Central  Google Scholar 

  10. Bischof, J., Maeda, R.K., Hediger, M., Karch, F. & Basler, K. An optimized transgenesis system for Drosophila using germ-line-specific φC31 integrases. Proc. Natl. Acad. Sci. USA 104, 3312–3317 (2007).

    Article  CAS  PubMed Central  Google Scholar 

  11. Hollis, R.P. et al. Phage integrases for the construction and manipulation of transgenic animals. Reprod. Biol. Endocrinol. 1, 79 (2003).

    Article  PubMed Central  Google Scholar 

  12. Miller, D.F., Holtzman, S.L. & Kaufman, T.C. Customized microinjection glass capillary needles for P-element transformations in Drosophila melanogaster. Biotechniques 33, 366–372 (2002).

    Article  CAS  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work is dedicated to the memory of my son Lance Raymond Fish (July 30, 2004 to April 30, 2007).

Author information

Authors and Affiliations

  1. Department of Developmental Biology and Howard Hughes Medical Institute, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, 94305-5120, California, USA

    Matthew P Fish & Roel Nusse

  2. Department of Genetics, Stanford University School of Medicine, 300 Pasteur Drive, Stanford, 94305-5120, California, USA

    Amy C Groth & Michele P Calos

Authors
  1. Matthew P Fish
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Amy C Groth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Michele P Calos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Roel Nusse
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Matthew P Fish.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fish, M., Groth, A., Calos, M. et al. Creating transgenic Drosophila by microinjecting the site-specific φC31 integrase mRNA and a transgene-containing donor plasmid. Nat Protoc 2, 2325–2331 (2007). https://doi.org/10.1038/nprot.2007.328

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.328

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing