Xenopus oocyte preparation and injection

Oocytes isolated from mature female Xenopus laevis were supplied by the European Xenopus Resource Centre, University of Portsmouth, UK. Oocytes were treated with collagenase (0.5 mg/ml, Sigma type 1 A) in Ca²⁺-free solution (96 mM NaCl, 2 mM KCl, 1 mM MgCl₂, 5 mM HEPES, 2.5 mM Na-pyruvate, 100 U/mL penicillin, 0.1 mg/mL streptomycin, pH 7.5) with shaking at 19 °C to defolliculate and remove the connective tissue surrounding the cells. After separation, oocytes were washed 7 times with modified Barth’s solution (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl₂, 1 mM MgCl₂, 5 mM HEPES, 2.5 mM Na-pyruvate, 0.5 mM theophylline, 50 μg/mL gentamicin, pH 7.5) and kept at 19 °C in the same solution.

Healthy oocytes were injected with cRNA using a Nano-liter Injector (World Precision Instruments Inc, USA). Mixtures of nAChR subunit cRNAs were injected as follows; for heteromeric rat neuronal receptors a 1:1 ratio of α:β at 200 ng/μL; for mouse embryonic muscle a 1:1:1:1 ratio of α:β:γ:δ at 25 ng/μL; human α7 at 100 ng/μL was mixed with RIC-3 at 30 ng/μL. Each oocyte was injected with 50 nL of RNA solution. Injected oocytes were saved in Barth’s solution at 19 °C for two to three days for expression of the target protein. During this time oocytes were regularly checked to remove unhealthy ones.