A PCR shortcut to oocyte expression.
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.
Cestari
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Department of Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore 21201-1559.
Albuquerque
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Burt
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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
D011963:Receptors, GABA-A; D012313:RNA; D004247:DNA