Xenopus laevis oocyte expression

The cDNAs coding for the Drosophila melanogaster receptors Gr21a or Gr63a were cloned into the expression vector pcDNA3 using standard PCR methods. cRNAs were prepared using the Ampli Cap-Max^™ T7 High Yield Message Maker Kit (CellScript, Inc.) following the manufacturer’s protocol. Xenopus laevis oocytes were kindly provided by the laboratory of Prof. Dr. M. Hollmann in the department of biochemistry I—receptor biochemistry of the Ruhr-University Bochum, Germany. As previously described, oocytes were obtained from female Xenopus laevis frogs that were anesthetized with 0.06% (w/v) ethyl-2-aminobenzoic acid (methansulfonate salt; Sigma-Aldrich, Taufkirchen, Germany) for 30 minutes [23]. Ovary tissue was removed and placed in Barth’s solution (88 mM NaCl₂, 1 mM KCl, 0.82 MgSO₄, 0.33 mM Ca(NO₃)₂, 0.42 mM CaCl₂, 2.4 mM NaHCO₃, 5 mM Tris-HCl, pH 7.4, 100 U/ml penicillin, and 50 μg/ml streptomycin). Afterwards, oocytes were treated with collagenase (2 mg/ml Type II) in Ca²⁺-free Barth’s solution for 1.5–2 h at room temperature. Healthy stadium IV-VI oocytes were selected for cytoplasmic cRNA injection. Each oocyte was injected with 5–20 ng of receptor coding cRNA using the nanoliter injector 2000 (WPI, Berlin, Germany). Injected oocytes were placed in fresh Barth’s solution and incubated at 18 °C. Oocytes were measured 2–4 days after the injection.