Summary
The oocytes of the South African clawed frog Xenopus laevis have been widely used as a reliable system for the expression and characterization of different types of proteins, including ion channels and membrane receptors. The large size and resilience of these oocytes make them easy to handle and to microinject with different molecules such as natural mRNAs, cRNAs, and antibodies. A variety of methods can then be used to monitor the expression of the proteins encoded by the microinjected mRNA/cRNA, and to perform a functional characterization of the heterologous polypeptides. In this chapter, after describing the equipment required to maintain X. laevis in the laboratory and to set up a microinjection system, we provide detailed procedures for oocyte isolation, micropipet and cRNA preparation, and oocyte microinjection. A method for the labeling of oocyte-synthesized proteins and for the immunological detection of the heterologous polypeptides is also described.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fabbrini, M. S., Carpani, D., Soria, M. R., and Ceriotti, A. (2000) Cytosolic immunization allows the expression of preATF-saporin chimeric toxin in eukaryotic cells. FASEB J. 14, 391–398.
Ceriotti, A. and Colman, A. (1990) Trimer formation determines the rate of influenza virus haemagglutinin transport in the early stages of secretion in Xenopus oocytes. J. Cell Biol. 111, 409–420.
Blumenstein, Y., Ivanina, T., Shistik, E., Bossi, E., Peres, A., and Dascal, N. (1999) Regulation of cardiac L-type Ca2+ channel by coexpression of Gas in Xenopus oocytes. FEBS Lett. 444, 78–84.
Schultz, T. W. and Dawson, D. A. (2003) Housing and husbandry of Xenopus for oocyte production. Lab. Anim. 32, 34–39.
Bonifacino, J. S. (2000) Protein labelling and immunoprecipitation, in Current Protocols in Cell Biology, Cell Biology and Cytology, (Bonifacino, J. S., ed.), Wiley, Hoboken, NJ.
Green, S. L. (2002) Factors affecting oogenesis in the South African clawed frog (Xenopus laevis). Comp. Med. 52, 307–312.
Godfrey, E. W. and Sanders, G. E. (2004) Effect of water hardness on oocyte quality and embryo development in the African clawed frog (Xenopus laevis). Comp. Med. 54, 170–175.
Hilken, G., Dimigen, J., and Iglauer, F. (1995) Growth of Xenopus laevis under different laboratory rearing conditions. Lab. Anim. 29, 152–162.
Iglauer, F., Willmann, F., Hilken, G., Huisinga, E., and Dimigen, J. (1997) Anthelmintic treatment to eradicate cutaneus capillariasis in a colony of South African clawed frogs. Lab. Anim. Sci. 47, 477–482.
Dumont, J. N. (1972) Oogenesis in Xenopus Laevis (Daudin). Stages of oocyte development in laboratory maintained animals. J. Morphol. 136, 153–179.
Smith, L. D., Weilong, X., and Varnold, R. L. (1991) Oogenesis and oocyte isolation, in Methods in Cell Biology, Vol. 3, Xenopus laevis: Practical Use in Cell and Molecular Biology (Kay, B. K. and Peng, H. B., eds.), Academic Press, San Diego, CA, pp. 45–60.
Dascal, N. and Lotan, I. (1992) Expression of exogenus ion channel and neurotrasmitter receptor in RNA-injected Xenopus oocytes, in Methods in Molecular Biology, Vol. 13, Protocols in Molecular Neurobiology, (Bausch, S. and Prevest, eds.), Humana Press, Totowa, NJ, pp. 205–225.
Drummond, D. R., Armstrong, J., and Colman, A. (1985) The effect of capping and polyadenylation on the stability, movement and translation of synthetic messenger RNAs in Xenopus oocytes. Nucl. Acids Res. 13, 7375–7394.
Furuichi, Y., LaFiandra, A., and Shatkin, A. J. (1977) 5′-Terminal structure and mRNA stability. Nature 266, 235–239.
Krieg, P. A. and Melton, D. A. (1984) Functional messenger RNAs are produced by SP6 in vitro transcription of cloned cDNAs. Nucl. Acids Res. 12, 7057–7070.
Leaf, D. S., Roberts, S. J., Gerhart, J. C., and Moore, H. (1990) The secretory pathway is blocked between the trans-Golgi and the plasma membrane during meiotic maturation in Xenopus oocytes. Dev. Biol. 141, 1–12.
Becq, F., Hamon, Y., Bajetto, A., Gola, M., Verrier, B., and Chimini, G. (1997) ABC1, an ATP binding cassette transporter required for phagocytosis of apoptotic cells, generates a regulated anion flux after expression in Xenopus laevis oocytes. J. Biol. Chem. 272, 2695–2699.
Ludewig, U., von Wiren, N., and Frommer, W. B. (2002) Uniport of NH4 + by the root hair plasma membrane ammonium transporter LeAMTl;1. J. Biol. Chem. 277, 13,548–13,555.
Nowak, M. W., Gallivan, J. P., Silverman, S. K., Labarca, C., Daugherty, D.A., and Lester, H. A. (1998) In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocytes expression system. Methods Enzymol. 293, 504–529.
Buller, A. L. and White, M. M. (1988) Control of Torpedo acethylcoline receptor biosynthesis in Xenopus oocytes. Proc. Natl. Aca. Sci. USA 85, 8717–8721.
Drummond, D. R., McCrae, M. A., and Colman, A. (1985) Stability and movement of mRNAs and their encoded proteins in Xenopus oocytes. J. Cell Biol. 100, 1148–1156.
Ceriotti, A. and Colman, A. (1988) Binding to membrane proteins within the endoplasmic reticulum cannot explain the retention of the glucose-regulated protein GRP78 in Xenopus oocytes. EMBO J. 7, 633–638.
Altschuler, Y. and Galili, G. (1994) Role of conserved cysteines of a wheat gliadin in its transport and assembly into protein bodies in Xenopus oocytes. J. Biol. Chem. 269, 6677–6682.
Tate, S. S., Urade, R., Micanovic, R., Gerber, L., and Udenfriend, S. (1990) Secreted alkaline phosphatase: an internal standard for expression of injected mRNAs in Xenopus oocytes. FASEB J. 4, 227–231.
Evans, J. P. and Kay, B. K. (1991) Biochemical fractionation of oocytes, in Methods in Cell Biology, Vol. 36, Xenopus laevis: Pratical Uses in Cell and Molecular Biology, (Kay, B. K. and Peng, H. B., eds.), Academic Press, San Diego, CA, pp. 133–148.
Turk, E., Kerner, C. J., Lostao, M. P., and Wright, E. M. (1996) Membrane topology of the human Na+/glucose cotransporter SGLT1. J. Biol. Chem. 271, 1925–1934.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Bossi, E., Fabbrini, M.S., Ceriotti, A. (2007). Exogenous Protein Expression in Xenopus Oocytes. In: Grandi, G. (eds) In Vitro Transcription and Translation Protocols. Methods in Molecular Biology™, vol 375. Humana Press. https://doi.org/10.1007/978-1-59745-388-2_6
Download citation
DOI: https://doi.org/10.1007/978-1-59745-388-2_6
Publisher Name: Humana Press
Print ISBN: 978-1-58829-558-3
Online ISBN: 978-1-59745-388-2
eBook Packages: Springer Protocols