SpringerProtocols: Abstract: Oocyte Expression With Injection of Purified T7 RNA Polymerase

Oocyte Expression With Injection of Purified T7 RNA Polymerase

By: Xavier Altafaj², Nathalie Joux², Michel Ronjat², Michel De Waard²

Abstract

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The Xenopus oocyte is a widely used system for protein expression. Investigators have had the choice between two different techniques: injection into the cytoplasm of in vitro transcribed complementary RNA (cRNA) or injection into the nucleus of complementary DNA (cDNA). We report on a third expression technique that is based on the combined injection of cDNA and purified T7 RNA polymerase directly into the cytoplasm of oocytes.

Affiliation(s): (2) Départment Réponse et Dynamique Cellulaire, Inserm U607, Canaux Calciques, Foncitons et Pathologies, CEA, Grenoble, France

Book Title: Xenopus Protocols: Cell Biology and Signal Transduction

Series: Methods in Molecular Biology | Volume: 322 | Pub. Date: Dec-13-2005 | Page Range: 55-67 | DOI: 10.1007/978-1-59745-000-3_5

Subject: Cell Biology

Key Words: Exogenous protein - expression technique - injection - T7 RNA polymerase - Xenopus oocyte

References

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1.	Dascal, N. (1987) The use of Xenopus oocytes for the study of ion channels. CRC Crit. Rev. Biochem. 22, 317–387.

2.	Schmitt, B. M., and Koepsell, H. (2002) An improved method for real-time monitoring of membrane capacitance in Xenopus laevis oocytes. Biophys. J. 82, 1345–1357.

3.	Scheuner, D., Logsdon, C. D., and Holz, R. W. (1992) Bovine chromaffin granule membranes undergo Ca²⁺-regulated exocytosis in frog oocytes. J. Cell Biol. 116, 359–365.

4.	Jaunin, P., Horisberger, J. D., Richter, K., Good, P. J., Rossier, B.C., and Geering, K. (1992) Processing, intracellular transport, and functional expression of endogenous and exogenous α-β 3 Na,K-ATPase complexes in Xenopus oocytes. J. Biol. Chem. 267, 577–585.

5.	Buller, A. L. and White, M. M. (1992) Ligand-binding assays in Xenopus oocytes. Methods Enzymol. 207, 368–375.

6.	Lee, C. L., Linton, J., Soughayer, J. S., Sims, C. E., and Allbritton, N. L. (1999) Localized measurement of kinase activation in oocytes of Xenopus laevis. Nature Biotech. 17, 759–762.

7.	Zagotta, W. N., Hoshi, T., and Aldrich, R. W. (1990) Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB. Science 250, 568–571.

8.	Geib, S., Sandoz, G., Mabrouk, K., et al. (1992) Use of a purified and functional recombinant calcium-channel µ₄ subunit in surface-plasmon resonance studies. Biochem. J. 364,285–292.

9.	Miledi, R., Duenas, Z., Martinez-Torres, A., Kawas, C. H., and Eusebi, F. (2004) Microtransplantation of functional receptors and channels from the Alzheimer’s brain to frog oocytes. Proc. Natl. Acad. Sci. USA 101, 1760–1763.

10.	Marsal, J., Tigyi, G., and Miledi, R. (1995) Incorporation of acetylcholine receptors and Cl⁻ channels in Xenopus oocytes injected with Torpedo electroplaque membranes. Proc. Natl. Acad. Sci. U. S. A. 92, 5224–5228.

11.	Wall, D. A. and Patel, S. (1989) Isolation of plasma membrane complexes from Xenopus oocytes. J. Membr. Biol. 107, 189–201.

12.	Kamsteeg, E. J. and Deen, P. M. T. (2001) Detection of aquaporin-2 in the plasma membranes of oocytes: a novel isolation method with improved yield and purity. Biochem. Biophys. Res. Comm. 282, 683–690.

13.	Schillers, H., Danker, T., Schnittler, H. J., Lang, F., and Oberleithner, H. (2000) Plasma membrane plasticity of Xenopus laevis oocyte imaged with atomic force microscopy. Cell. Physiol. Biochem. 10, 99–107.

14.	Perez, G., Lagrutta, A., Adelman, J. P., and Toro, L. (1994) Reconstitution of expressed KCa channels from Xenopus oocytes to lipid bilayers. Biophys. J. 66, 1022–1027.

15.	Paine, P. L., Johnson, M. E., Lau, Y. T., Tluczek, L. J., and Miller, D. S. (1992) The oocyte nucleus isolated in oil retains in vivo structure and functions. Biotechniques 13, 238–246.

16.	Lehman, C. W. and Carroll, D. (1993) Isolation of large quantities of functional, cytoplasm-free Xenopus laevis oocyte nuclei. Anal. Biochem. 211, 311–319.

17.	Ford, C. C. and Gurdon, J. B. (1977) A method for enucleating oocytes of Xenopus laevis. J. Embryol. Exp. Morphol. 37, 203–209.

18.	Stefani, E. and Bezanilla, F. (1998) Cut-open oocyte voltage-clamp technique. Methods Enzymol. 293, 300–318.

19.	Cucu, D., Simaels, J., Jans, D., and Van Driessche, W. (2004) The transoocyte voltage clamp: a noninvasive technique for electrophysiological experiments with Xenopus laevis oocytes. Pflügers Arch. 447, 934–942.

20.	Choe, H. and Sackin, H. (1997) Improved preparation of Xenopus oocytes for patch-clamp recordings. Pflügers Arch. 433, 648–652.

21.	Hering, S. (1998) Small-volume and rapid extracellular solution exchange around Xenopus oocytes during voltage-clamp recordings. Pflügers Arch. 436, 303–307.

22.	Dascal, N., Chilcott, G., and Lester, H. A. (1991) Recording of voltage and Ca²⁺-dependent currents in Xenopus oocytes using an intracellular perfusion method. J. Neurosci. Methods 39, 29–38.

23.	Nowak, M. W., Gallivan, J. P., Silverman, S. K., Labarca, C. G., Dougherty, D. A., and Lester, H. A. (1998) In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system. Methods Enzymol. 293, 504–529.

24.	Freeh, G. C. and Joho, R. H. (1992) Isolation of ion channel genes by expression cloning in Xenopus oocytes. Methods Enzymol. 207, 592–604.

25.	Gurdon, J. B., Lane, C. D., Woodland, H. R., and Marbaix, G. (1971) Use of frog eggs and oocytes for the study of messenger RNA and its translation in living cells. Nature 233, 177–182.

26.	Swick, A. G., Janicot, M., Cheneval-Kastelic, T., McLenithan, J. C, and Lane, D. (1992) Promoter-cDNA-directed heterologous protein expression in Xenopus laevis oocytes. Proc. Natl. Acad. Sci. U. S. A. 89, 1812–1816.

27.	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.

28.	Geib, S., Sandoz, G., Carlier, E., Cornet, V., Cheynet-Sauvion, V., and De Waard, M. (2001) A novel Xenopus oocyte expression system based on cytoplasmic coinjection of T7-driven plasmids and purified T7-RNA polymerase. Receptors Channels 7, 331–343.

29.	Arnaud, N., Cheynet, V., Oriol, G., Mandrand, B., and Mallet, F. (1997) Construction and expression of a modular gene encoding bacteriophage T7 RNA polymerase. Gene 199, 149–156.

30.	Kozak, M. (1986) Point mutations define a sequence flanking the AUG initiator codon that modulates translation by eukaryotic ribosomes. Cell 44, 283–292.

31.	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. Nut: Acids Res. 13, 7375–7394.

32.	Lehrach, H., Diamond, D., Wozney, J. M., and Boedtker, H. (1977) RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry 16, 4743–4751.

33.	De Waard, M. and Campbell, K. P. (1995) Subunit regulation of the neuronal α_1A Ca²⁺ channel expressed in Xenopus oocytes. J. Physiol. 485, 619–634.

34.	Eppig, J. J. and Dumont, J. N. (1976) Defined nutrient medium for the in vitro maintenance of Xenopus laevis oocytes. In Vitro 12, 418–427.

35.	Wyllie, A. H., Laskey, R. A., Finch, J., and Gurdon, J. B. (1978) Selective DNA conservation and chromatin assembly after injection of SV40 DNA into Xenopus oocytes. Dev. Biol. 64, 178–188.

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