Sp6 rna polymerase mmessage mmachine kit

Defollicularized oocytes from Xenopus laevis were obtained from the FAU Institute of Cellular and Molecular Physiology. The cDNA of human α1and β2-GABA A R subunits were subcloned into the pcDNA 3.1 vector. The DNA for each subunit was linearised by digestion with Bgl II. Then the cRNA for each subunit was synthesised from the linearised DNA using the mMessage mMachine T7 RNA polymerase kit (Ambion, Austin, TX, USA). The cRNA of human GABA A R α1and β2-subunits were mixed in a ratio of 1:1 and 17-28 ng of the mixture was microinjected into the oocytes to express the human GABA A R. The cDNA of the α1subunit of human GlyR was subcloned into the pNKS 2 vector. cRNA was performed on HindII linearised plasmid DNA using the SP6 RNA polymerase mMessage mMachine kit (Ambion, Austin, TX, USA). Approximately 0.5 pg of cRNA was injected into the oocytes to express human GlyR. After injection, the oocytes were kept in ND96 medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 5 mM HEPES, pH 7.4) with gentamycin at 18 °C for 24-72 h.