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.

Item Type:

Journal Article (Further Contribution)

Division/Institute:

04 Faculty of Medicine > Pre-clinic Human Medicine > Institute of Biochemistry and Molecular Medicine

UniBE Contributor:

Maldifassi, Maria Constanza; Wongsamitkul, Nisa; Baur, Roland and Sigel, Erwin

Subjects:

500 Science > 570 Life sciences; biology
600 Technology > 610 Medicine & health

ISSN:

1940-087X

Publisher:

MYJoVE Corporation

Language:

English

Submitter:

Barbara Järmann-Bangerter

Date Deposited:

23 May 2017 15:34

Last Modified:

04 Oct 2017 18:29

Publisher DOI:

10.3791/55034

PubMed ID:

28117773

BORIS DOI:

10.7892/boris.99596

URI:

https://boris.unibe.ch/id/eprint/99596

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