title: A PCR shortcut to oocyte expression.
abstract: 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.
authors.affiliation: Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore 21201-1559.
authors.forename: I N
authors.initials: IN
authors.lastname: Cestari
authors.forename: E X
authors.initials: EX
authors.lastname: Albuquerque
authors.forename: D R
authors.initials: DR
authors.lastname: Burt
mesh_terms: D000818:Animals; D001483:Base Sequence; D004247:DNA / Q000235:genetics; D005260:Female; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics*; D011963:Receptors, GABA-A / Q000235:genetics; D014981:Xenopus
chemical_list: D011963:Receptors, GABA-A; D012313:RNA; D004247:DNA