Expression of ion channels and receptors in Xenopus oocytes using vaccinia virus.
The cytoplasmic injection of mRNA synthesized in vitro into Xenopus oocytes is widely used for heterologous expression of ion channels and neurotransmitter receptors. We report two new methods for expression of ion channels and receptors in oocytes using vaccinia virus (VV). 1) A recombinant VV carrying the Shaker H4 K+ channel cDNA driven by the VV P7.5 early promoter was injected into oocytes. 2) A recombinant VV containing the bacteriophage T7 RNA polymerase driven by the P7.5 promoter was coinjected along with plasmids containing a T7 promoter and cDNAs for channels and receptors. The functionally expressed proteins include a) voltage-gated ion channels: the Shaker H4 K+ channel and the rat brain IIA Na+ channel, b) a ligand-gated ion channel: the mouse muscle nicotinic acetylcholine receptor (AChR), and c) a G protein-coupled receptor: the rat brain 5HT1C receptor. After virus/cDNA injection into oocytes, these channels and receptors generally showed characteristics and expression levels similar to those observed in mRNA-injected oocytes. However, the AChR expressed at lower levels in virus/cDNA-injected oocytes than in mRNA-injected oocytes. Because our methods bypass mRNA synthesis, they are more rapid and convenient than the mRNA injection method. Potential applications to structure-function studies and expression cloning are discussed.
Yang
X C
XC
Division of Biology, California Institute of Technology, Pasadena 91125.
Karschin
A
A
Labarca
C
C
Elroy-Stein
O
O
Moss
B
B
Davidson
N
N
Lester
H A
HA
D000818:Animals; D012321:DNA-Directed RNA Polymerases; D015870:Gene Expression; D007473:Ion Channels; D011978:Receptors, Nicotinic / Q000096:biosynthesis; D014162:Transfection; D014616:Vaccinia virus / Q000235:genetics*; D014981:Xenopus / Q000235:genetics*
D007473:Ion Channels; D011978:Receptors, Nicotinic; D012321:DNA-Directed RNA Polymerases