Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments.

Maldifassi, Maria Constanza; Wongsamitkul, Nisa; Baur, Roland; Sigel, Erwin (2016). Xenopus Oocytes: Optimized Methods for Microinjection, Removal of Follicular Cell Layers, and Fast Solution Changes in Electrophysiological Experiments. Journal of visualized experiments(118) MYJoVE Corporation 10.3791/55034

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The Xenopus oocyte as a heterologous expression system for proteins, was first described by Gurdon et al.(1) and 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 channels(4). 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 laboratory(5,6) and deviate from the ones routinely used(7-9). 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 receptors(10-12), human recombinant chloride channels(13), Trypanosome potassium channels(14), and a myo-inositol transporter(15, 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.

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