A novel Xenopus oocyte expression system based on cytoplasmic coinjection of T7-driven plasmids and purified T7-RNA polymerase.
The Xenopus laevis South African frog oocyte is a well suited and widely used system for protein biochemistry and functional studies. So far, two methods are commonly in use for the expression of exogenous proteins in this system. Investigators have the choice between cytoplasmic injections of in vitro synthesized cRNA or nuclear injections of cDNA. Here, we describe a new method for ion channel expression in oocytes, which consists of a coinjection of T7-driven cDNA and T7-RNA polymerase directly into the cytoplasm. This technique uses very limited amounts of purified enzyme and is also applicable to SP6 polymerase. Commercially available polymerases can also conveniently substitute for self-purified enzymes. The technique can be used for electrophysiological and biochemical analysis. In particular, high level expressions have been achieved for potassium (Shaker B, Kv1.2 and Kv1.3) and sodium (P mu 1.2) channels, and we also demonstrate efficient metabolic labeling of the calcium channel auxiliary beta 3 subunit. The properties of the channels expressed by this technique are indistinguishable from those of the channels expressed by classical methods. Expression of multi-subunit proteins was also achieved illustrating that the technique can be used for structure-function analyses. Moreover, this novel expression technique avoids many drawbacks of the two former techniques. It clearly bypasses the costly and time-consuming step of cRNA synthesis in vitro, prevents delicate cRNA manipulation and is easier to perform and more reliable than nuclear injection. Finally, it does not affect cell survival rate. These data indicate that the T7-RNA polymerase expression technique could be widely used in the future for the expression of exogenous proteins in the Xenopus oocyte system.
Geib
S
S
Laboratoire de Neurobiologie des Canaux Ioniques, INSERM U464, IFR Jean Roche, Faculté de Médecine Nord, Boulevard Pierre Dramard, 13916 Marseille 20, France.
Sandoz
G
G
Carlier
E
E
Cornet
V
V
Cheynet-Sauvion
V
V
De Waard
M
M
D000818:Animals; D017123:Bacteriophage T7; D015220:Calcium Channels / Q000096:biosynthesis / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D005260:Female; D007473:Ion Channels / Q000096:biosynthesis* / Q000235:genetics; D051663:Kv1.2 Potassium Channel; D051664:Kv1.3 Potassium Channel; D008845:Microinjections; D009865:Oocytes / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics*; D015221:Potassium Channels / Q000096:biosynthesis / Q000235:genetics; D024642:Potassium Channels, Voltage-Gated; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis*; D015222:Sodium Channels / Q000096:biosynthesis / Q000235:genetics; D014764:Viral Proteins; D014982:Xenopus laevis
D015220:Calcium Channels; D007473:Ion Channels; D051663:Kv1.2 Potassium Channel; D051664:Kv1.3 Potassium Channel; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011994:Recombinant Proteins; D015222:Sodium Channels; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases