T7 mmessage mmachine crna transcription

Oocytes (Stage V–VI Dumont’s classification; 1200–1300 μm in diameter) were removed from X. laevis by surgical laparotomy and defolliculated with 1.5 mg/ml collagenase Type II (Worthington Biochemical Corp., Lakewood, NJ, USA). Defolliculation was done at room temperature (21 –24 °C) for 1–2 h in OR-2 solution containing (in mmol/L) 82.5 NaCl, 2 KCl, 1 MgCl₂, 5 HEPES, pH 7.4.
Plasmid pT7TS constructs of hα3 and hβ4 were linearized with XbaI (NEB, Ipswich, MA, USA) for in vitro T7 mMessage mMachine®-cRNA transcription (AMBION, Foster City, CA, USA). Oocytes were injected with 5 ng of hα3β4 cRNAs at α3 to β4 ratio of 1:1 (concentration confirmed spectrophotometrically and by gel electrophoresis) using glass pipettes as described previously (Arias et al. 2020 (link)).

All procedures were approved by the University of Wollongong Animal Ethics Committees (project number AE2003). Female X. laevis were sourced from Nasco (Fort Atkinson, WI, USA) and a maximum of four frogs were kept in a 15 L aquarium at 20–26°C with a 12 h light/dark cycle. Oocytes (Stage V–VI Dumont’s classification; 1200–1300 μm in diameter) were removed from X. laevis by surgical laparotomy and defolliculated with 1.5 mg/mL collagenase Type II (Worthington Biochemical Corp., Lakewood, NJ, USA). Oocytes were obtained from three frogs. Defolliculation was done at room temperature (21–24 °C) for 1–2 h in OR-2 solution containing (in mmol/L) 82.5 NaCl, 2 KCl, 1 MgCl₂, 5 HEPES, pH 7.4. Plasmid pT7TS constructs of hα3 and hβ4 were linearized with XbaI (NEB, Ipswich, MA, USA) for in vitro T7 mMessage mMachine®-cRNA transcription (AMBION, Foster City, CA, USA). Oocytes were injected with 5 ng of hα3β4 cRNAs at α3 to β4 ratio of 1:1 (concentration confirmed spectrophotometrically and by gel electrophoresis) using glass pipettes as described previously [32 (link)]. Oocytes were incubated at 18 °C in sterile ND96 solution composed of (in mM) 96 NaCl, 2 KCl, 1 CaCl₂, 1 MgCl₂, and 5 HEPES at pH 7.4, supplemented with 5% fetal bovine serum (FBS), 50 mg/L gentamicin (GIBCO, Grand Island, NY, USA) and 10,000 U/mL penicillin-streptomycin (GIBCO).