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

Summary

Optimized procedures for the isolation of single follicles, cytoplasmic RNA microinjections, the removal of surrounding cell layers, and protein expression in Xenopus oocytes are described. In addition, a simple method for fast solution changes in electrophysiological experiments with ligand-gated ion channels is presented.

Abstract

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 channels⁴. This expression system has proven useful for the characterization of many proteins, among them ligand-gated ion channels.

The expression of GABA_A 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 GABA_A receptors^10-12, human recombinant chloride channels¹³, Trypanosome potassium channels¹⁴, 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.

Introduction

Xenopus oocytes are widely used as an expression system (References 2 - 3, and references therein). They are able to properly assemble and incorporate functionally active multisubunit proteins into their plasma membranes. Using this system, it is possible to functionally investigate membrane proteins alone or in combination with other proteins, in order to study the properties of mutated, chimeric, or concatenated proteins, and to screen potential drugs.

Advantages of using oocytes over other heterologous expression systems include the simple handling of the giant cells, the high proportion of cells expressing foreign genetic infor....

Protocol

Animal experiments have been approved by the local committee of the Canton Bern Kantonstierarzt, Kantonaler Veterinärdienst Bern (BE85/15).

1. Preparation of Xenopus Oocytes

1. Maintain frogs (Xenopus laevis) on a 12 h/12 h light/dark cycle in water that is strictly kept at 20 °C.
2. Remove the lobes of the ovaries from female frogs⁹.
   NOTE: The removal of the lobes is a stimulus for regeneration, and often the quality of the oocytes improves with surgery. The oocyte is covered by a vitelline layer, a layer of follicle cells, and connective tissue containing the blood vessels^....

Results

Xenopus oocytes were mechanically singled out using a platinum loop (Figure 1). The oocytes were microinjected with mRNA coding for the GABA_A receptor subunits α₄, β₂, δ, 0.5:0.5:2.5 fmol/oocyte (Figure 2). After 4 d, follicular cell layers were removed (Figure 3). Oocytes were voltage clamped at -80 mV and exposed to increasing concentrations of γ-aminobutyric acid (GABA) in the presence of 1 µM .......

Discussion

The methods described in this article deviate from those used traditionally^7-9. It is standard to expose the lobes of the ovary to a 1 to 2 h collagenase treatment⁸; isolate undamaged, denuded oocytes; and inject them with mRNA using commercial injection devices. This classical procedure has the following drawbacks: 1) Oocytes are likely to be damaged by the long exposure to high concentrations of collagenase. 2) The unstable denuded oocytes must be stored until the experiment. 3) Denuded oocytes ar.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	Sigma	71380
KCl	Sigma	P-9541
NaHCO3	Sigma	S6014
MgSO4	Sigma	M-1880
CaCl2	Sigma	223560
Ca(NO3)2	Sigma	C1396
HEPES	Sigma	H3375
Penicilin/streptomycin	Gibco	15140-148	100 μg penicillin/mL and 100 μg streptomycin/mL
Platinum wire loop	home-made
Micropipette puller	Zeitz-Instruments GmBH	DMZ
Hamilton syringe	Hamilton	80300	10 μL, Type 701N
Thick walled polytetrafluoroethylene tubing	Labmarket GmBH		1.0 mm OD
Paraffin oil	Sigma	18512
Nylon net, G: 0.8 mm	ZBF Züricher Beuteltuchfabrik AG
Borosilicate glass tube	Corning	99445-12	PYREX
Collagenase NB Standard Grade	SERVA	17454
Trypsin inhibitor type I-S	Sigma	T-9003
EGTA	Sigma	E3389
Glass capillary	Jencons (Scientific ) LTD.	H15/10	1.35 ID mm (for perfusion), alternative company: Harvard Apparatus Limited
Borosilicate glass capillary	Harvard Apparatus Limited	30-0019	1.0 OD x  0.58 ID x 100 Length mm (for microinjection)
Borosilicate glass capillary	Harvard Apparatus Limited	30-0044	1.2 OD x 0.69 ID x 100 Length mm (for two-electrode voltage clamp)
γ-Aminobutyric acid (GABA)	Sigma	A2129
3α,21-Dihydroxy-5α-pregnan-20-one (THDOC)	Sigma	P2016
grill motor	Faulhaber		DC micromotor Type 2230 with gear Type 22/2
micrometer screw	Kiener-Wittlin	10400	TESA, AR 02.11201
Sterile plastic transfer pipettes	Saint-Amand  Mfg.	222-20S