40] Isolation of ion channel genes by expression cloning in Xenopus oocytes

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This chapter describes the approach of expression cloning, which combines directional cDNA cloning in a transcription-competent vector with a functional electrophysiological assay in cRNA-injected Xenopus oocytes. By this approach, three cDNA clones encoding two members of a class of voltage-gated ion channels and one member of a class of ligand-gated ion channels (a slowly activating voltage-gated potassium channel, a delayed rectifier-type potassium channel, and a kainate receptor) are successfully isolated. If the RNA for the ion channel of interest does not give large currents in the oocyte, enrichment is of particular importance, to limit the number of pools screened. The successful application of the method described in this chapter depends on the length of the mRNA or, which is the distance from the initiation codon of the coding region to the poly(A) tail. In mammalian genes, NotI recognition sites (5'-GCGGCCGC-Y) occur, on average, only once in 10 6 base pairs owing to the significant underrepresentation of CG dinucleotides. This rare occurrence of the NotI recognition sequence makes it the best-suited restriction enzyme recognition sequence in the primer/linker used to initiate cDNA synthesis.