Microinjection into Xenopus Oocytes

Abstract

Microinjection of genetic information in the form of complimentary ribonucleic acid (cRNA) into the Xenopus oocyte leads to the functional expression of the encoded proteins. In this novel environment, exogenous proteins may be functionally characterised under well‐defined conditions. Complimentary deoxyribonucleic acid (cDNA) coding for the protein may be mutated, the altered cDNA transcribed and polyadenylated in vitro to the corresponding cRNA and the function of the resulting altered protein is compared with that of the wild‐type protein. The oocyte is a major expression system used in membrane protein structure–function studies. It is especially popular for expression of membrane transport proteins such as carriers and ion channels. In another approach, total messenger RNA (mRNA) may be prepared from a tissue of choice, microinjected into oocytes, the encoded functions characterised and the encoding genetic information isolated.

Key Concepts:

  • Proteins can be ‘transplanted’ into the Xenopus oocyte.

  • The Xenopus oocyte may be used as a test tube for newly introduced protein functions.

  • Genetic information in the form of cRNA may be prepared in vitro from the coding cDNA.

  • The Xenopus oocyte translates foreign microinjected cRNA.

  • The Xenopus oocyte is favourite expression system for structure–function studies.

  • Expression cloning can identify genes purely based on protein function.

Keywords: Xenopus laevis oocyte; protein expression; mRNA; cDNA; expression cloning; microinjection

Figure 1.

Schematic representation of the use of the Xenopus oocyte for the expression of functional proteins after microinjection of the oocyte with genetic information in the form of RNA or DNA. cDNA, complementary DNA and mRNA, messenger RNA.

Figure 2.

Photograph of a portion of a cut open ovary lobe showing oocytes at different stages of maturity. Immature oocytes are small and whitish, whereas mature oocytes are 1–1.2 mm in diameter with a darkly pigmented animal hemisphere and a yellowish‐vegetal hemisphere. Photograph (© Sigel, ) of a portion of a cut open ovary lobe showing oocytes at different stages of maturity.

Figure 3.

Photograph of the microinjection procedure. The follicles containing the oocytes are submersed in medium and lined up on a nylon mesh. A glass capillary with a tip diameter of approximately 15 μm is inserted into the oocyte cytoplasm and the RNA solution is microinjected. Photograph (© Sigel, ) of the microinjection procedure.

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References

Barnard EA, Miledi R and Sumikawa K (1982) Translation of exogenous messenger RNA coding for nicotinic acetylcholine receptors produces functional receptors in Xenopus oocytes. Proceedings of the Royal Society of London. Series B 215: 241–246.

Buhr A, Baur R and Sigel E (1997) Subtle changes in residue 77 of the γ subunit of α1β2γ2 GABAA receptors drastically alter the affinity for ligands of the benzodiazepine binding site. Journal of Biological Chemistry 272: 11799–11804.

Colman A (1984a) Expression of exogenous DNA in Xenopus oocytes. In: Hames DB and Higgins SJ (eds) Transcription and Translation. A Practical Approach, pp. 49–69. Oxford, UK: IRL Press.

Colman A (1984b) Translation of eukaryotic messenger RNA in Xenopus oocytes. In: Hames DB and Higgins SJ (eds) Transcription and Translation. A Practical Approach, pp. 271–302. Oxford, UK: IRL Press.

Dascal N (1987) The use of Xenopus oocytes for the study of ion channels. CRC Critical Reviews in Biochemistry 22: 313–387.

Goldin AL (1992) Maintenance of Xenopus laevis and oocyte injection. Methods in Enzymology 207: 266–279.

Gurdon JB, Lane CD, Woodland HR 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.

Kressmann A, Clarkson S, Pirotta V and Birnstiel M (1978) Transcription of cloned tRNA gene fragments and subfragments injected into the oocyte nucleus of Xenopus laevis. Proceedings of the National Academy of Sciences of the USA 75: 1176–1180.

Mertz JE and Gurdon JB (1977) Purified DNAs are transcribed after microinjection into Xenopus oocytes. Proceedings of the National Academy of Sciences of the USA 74: 1502–1506.

Methfessel C, Witzemann V, Takahashi T et al. (1986) Patch clamp measurements on Xenopus laevis oocytes: currents through endogenous channels and implanted acetylcholine receptor and sodium channels. Pflügers Archiv 407: 577–588.

Nowak MW, Gallivan JP, Silverman SK et al. (1998) In vivo incorporation of unnatural amino acids into ion channels in Xenopus oocyte expression system. Methods in Enzymology 293: 504–529.

Sigel E (1987) Properties of single sodium channels translated by Xenopus oocytes after injection with messenger ribonucleic acid. Journal of Physiology (London) 386: 73–90.

Sigel E (1990) The use of Xenopus oocytes for the functional expression of plasma membrane proteins. Journal of Membrane Biology 117: 201–221.

Sigel E and Minier F (2005) Educational paper: the Xenopus oocyte: system for the study of functional expression and modulation of proteins. Molecular Nutrition and Food Research 49: 228–234.

Soreq H (1985) The biosynthesis of biologically active proteins in mRNA‐microinjected Xenopus oocytes. CRC Critical Reviews in Biochemistry 18: 199–238.

Sumikawa K, Houghton M, Emtage JS, Richards BM and Barnard EA (1981) Active multi‐subunit ACh receptor assembled by translation of heterologous mRNA in Xenopus oocytes. Nature 292: 862–864.

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How to Cite close
Sigel, E(Sep 2010) Microinjection into Xenopus Oocytes. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0002658.pub2]