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.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
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.
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.
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.
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.
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.
Amsterdam, A., and Hopkins, N. (2006). Mutagenesis strategies in zebrafish for identifying genes involved in development and disease. Trends Genet 22, 473–8.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Acknowledgements
This research was supported by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Jao, LE., Burgess, S.M. (2009). Production of Pseudotyped Retrovirus and the Generation of Proviral Transgenic Zebrafish. In: Lieschke, G., Oates, A., Kawakami, K. (eds) Zebrafish. Methods in Molecular Biology, vol 546. Humana Press. https://doi.org/10.1007/978-1-60327-977-2_2
Download citation
DOI: https://doi.org/10.1007/978-1-60327-977-2_2
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-976-5
Online ISBN: 978-1-60327-977-2
eBook Packages: Springer Protocols