Zebrafish pp 13-30 | Cite as

Production of Pseudotyped Retrovirus and the Generation of Proviral Transgenic Zebrafish

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 546)

Summary

This chapter describes a method for generation of the high-titer pseudotyped Moloney murine leukemia virus (MLV) that efficiently infects zebrafish embryos (i.e., more than 25 retroviral copies per cell). Injection techniques are also described for production of the retrovirus-infected mosaic “founder” fish. We describe a quantitative PCR (qPCR)-based assay as a quick way to assess the infectivity after each round of viral production and injection. Most of the required equipment is commercially available and commonly present in most research laboratories.

Key words

Zebrafish Retrovirus Pseudotyped Moloney murine leukemia virus Insertional mutagenesis Injection 

References

  1. 1.
    Driever, W., Solnica-Krezel, L., Schier, A. F., Neuhauss, S. C., Malicki, J., Stemple, D. L., Stainier, D. Y., Zwartkruis, F., Abdelilah, S., Rangini, Z., Belak, J., and Boggs, C. (1996). A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123, 37–46.PubMedGoogle Scholar
  2. 2.
    Haffter, P., Granato, M., Brand, M., Mullins, M. C., Hammerschmidt, M., Kane, D. A., Odenthal, J., van Eeden, F. J., Jiang, Y. J., Heisenberg, C. P., Kelsh, R. N., Furutani-Seiki, M., Vogelsang, E., Beuchle, D., Schach, U., Fabian, C., and Nusslein-Volhard, C. (1996). The identification of genes with unique and essential functions in the development of the zebrafish, Danio rerio. Development 123, 1–36.PubMedGoogle Scholar
  3. 3.
    Lin, S., Gaiano, N., Culp, P., Burns, J. C., Friedmann, T., Yee, J. K., and Hopkins, N. (1994). Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish. Science 265, 666–9.CrossRefPubMedGoogle Scholar
  4. 4.
    Burns, J. C., Friedmann, T., Driever, W., Burrascano, M., and Yee, J. K. (1993). Vesicular stomatitis virus G glycoprotein pseudotyped ­retroviral vectors: concentration to very high titer and efficient gene transfer into mammalian and nonmammalian cells. Proc Natl Acad Sci U S A 90, 8033–7.CrossRefPubMedGoogle Scholar
  5. 5.
    Yee, J. K., Miyanohara, A., LaPorte, P., Bouic, K., Burns, J. C., and Friedmann, T. (1994). A general method for the generation of high-titer, pantropic retroviral vectors: highly efficient infection of primary hepatocytes. Proc Natl Acad Sci U S A 91, 9564–8.CrossRefPubMedGoogle Scholar
  6. 6.
    Amsterdam, A., and Hopkins, N. (2006). Mutagenesis strategies in zebrafish for identifying genes involved in development and disease. Trends Genet 22, 473–8.CrossRefPubMedGoogle Scholar
  7. 7.
    Amsterdam, A., Burgess, S., Golling, G., Chen, W., Sun, Z., Townsend, K., Farrington, S., Haldi, M., and Hopkins, N. (1999). A large-scale insertional mutagenesis screen in zebrafish. Genes Dev 13, 2713–24.CrossRefPubMedGoogle Scholar
  8. 8.
    Amsterdam, A., Nissen, R. M., Sun, Z., Swindell, E. C., Farrington, S., and Hopkins, N. (2004). Identification of 315 genes essential for early zebrafish development. Proc Natl Acad Sci U S A 101, 12792–7.CrossRefPubMedGoogle Scholar
  9. 9.
    Gaiano, N., Amsterdam, A., Kawakami, K., Allende, M., Becker, T., and Hopkins, N. (1996). Insertional mutagenesis and rapid cloning of essential genes in zebrafish. Nature 383, 829–32.CrossRefPubMedGoogle Scholar
  10. 10.
    Golling, G., Amsterdam, A., Sun, Z., Antonelli, M., Maldonado, E., Chen, W., Burgess, S., Haldi, M., Artzt, K., Farrington, S., Lin, S. Y., Nissen, R. M., and Hopkins, N. (2002). Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development. Nat Genet 31, 135–40.CrossRefPubMedGoogle Scholar
  11. 11.
    Ellingsen, S., Laplante, M. A., Konig, M., Kikuta, H., Furmanek, T., Hoivik, E. A., and Becker, T. S. (2005). Large-scale enhancer detection in the zebrafish genome. Development 132, 3799–811.CrossRefPubMedGoogle Scholar
  12. 12.
    Chen, W., Burgess, S., Golling, G., Amsterdam, A., and Hopkins, N. (2002). High-throughput selection of retrovirus producer cell lines leads to markedly improved efficiency of germ line-transmissible insertions in zebra fish. J Virol 76, 2192–8.CrossRefPubMedGoogle Scholar
  13. 13.
    Wang, D., Jao, L. E., Zheng, N., Dolan, K., Ivey, J., Zonies, S., Wu, X., Wu, K., Yang, H., Meng, Q., Zhu, Z., Zhang, B., Lin, S., and Burgess, S. M. (2007). Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions. Proc Natl Acad Sci U S A 104, 12428–33.CrossRefPubMedGoogle Scholar
  14. 14.
    Miyoshi, H., Takahashi, M., Gage, F. H., and Verma, I. M. (1997). Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc Natl Acad Sci U S A 94, 10319–23.CrossRefPubMedGoogle Scholar
  15. 15.
    Yoon, C., Kawakami, K., and Hopkins, N. (1997). Zebrafish vasa homologue RNA is localized to the cleavage planes of 2- and 4-cell-stage embryos and is expressed in the primordial germ cells. Development 124, 3157–65.PubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Cell and Developmental BiologyVanderbilt UniversityNashvilleUSA
  2. 2.Genome Technology Branch, National Human Genome Research InstituteNational Institutes of HealthBethesdaMD

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