[18] In Vitro synthesis of RNA for expression of ion channels in Xenopus oocytes

Xenopus oocytes are widely used for the heterologous expression of cDNA clones encoding ion channels, and the widespread applicability of this system is because of the ability of the oocyte to translate RNAs encoding channels from a variety of sources. This chapter describes the methods for in vitro synthesis of RNAs suitable for expression in Xenopus oocytes. In vitro synthesis of RNA is most easily carried out by cloning a cDNA encoding the channel into a vector containing a bacteriophage promoter and then specifically transcribing that sequence by using an RNA polymerase that recognizes the phage promoter. Direct in vitro transcription of ion channel cDNAs synthesized using the polymerase chain reaction (PCR) enables the rapid amplification and expression of ion channels encoded by low abundance mRNAs. However, functional expression of a channel cannot be detected in oocytes, even though the RNA is properly sized and readily translated in vitro.