title: Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments.
abstract: The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.1and has been widely used since its discovery (References 2 - 3, and references therein). A characteristic that makes the oocyte attractive for foreign channel expression is the poor abundance of endogenous ion channels4. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels. The expression of GABAA receptors in Xenopus oocytes and their functional characterization is described here, including the isolation of oocytes, microinjections with cRNA, the removal of follicular cell layers, and fast solution changes in electrophysiological experiments. The procedures were optimized in this laboratory5,6and deviate from the ones routinely used7-9. Traditionally, denuded oocytes are prepared with a prolonged collagenase treatment of ovary lobes at RT, and these denuded oocytes are microinjected with mRNA. Using the optimized methods, diverse membrane proteins have been expressed and studied with this system, such as recombinant GABAA receptors10-12, human recombinant chloride channels13, Trypanosome potassium channels14, and a myo-inositol transporter15, 16. The methods detailed here may be applied to the expression of any protein of choice in Xenopus oocytes, and the rapid solution change can be used to study other ligand-gated ion channels.
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern.
authors.forename: Maria C
authors.initials: MC
authors.lastname: Maldifassi
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern.
authors.forename: Nisa
authors.initials: N
authors.lastname: Wongsamitkul
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern.
authors.forename: Roland
authors.initials: R
authors.lastname: Baur
authors.affiliation: Institute of Biochemistry and Molecular Medicine, University of Bern; sigel@ibmm.unibe.ch.
authors.forename: Erwin
authors.initials: E
authors.lastname: Sigel
mesh_terms: D000818:Animals; D055724:Electrophysiological Phenomena; D004594:Electrophysiology / Q000379:methods*; D005260:Female; D007473:Ion Channels / Q000378:metabolism; D008565:Membrane Proteins / Q000032:analysis; D008845:Microinjections / Q000379:methods*; D009865:Oocytes / Q000166:cytology*; D012996:Solutions / Q000737:chemistry; D014982:Xenopus laevis
chemical_list: D007473:Ion Channels; D008565:Membrane Proteins; D012996:Solutions