Cestari_1993_Biotechniques_14_404

We describe a new method, RNA amplification with oocyte expression, for efficient expression of proteins in the Xenopus oocyte after PCR amplification of cDNA coding regions, using as examples mouse GABAA receptor alpha 1 and beta 2 subunits. RNA is reverse-transcribed and the cDNAs are amplified using 5' primers containing a T7 RNA polymerase promoter and a consensus ribosome binding site and 3' primers giving a short poly(A) tail. This is followed by direct in vitro transcription of the PCR products and injection of the resulting mRNAs into Xenopus oocytes. The method gave abundant GABA-gated chloride channels in the oocyte membrane, as measured by recording agonist-induced currents. It promises to be a rapid route to expression of cloned proteins in oocytes, without requiring actual clones, and is free of possible variations in efficiency from untranslated regions.