2] - Expression of Neurotransmitter Receptors and Voltage-Activated Channels from Brain mRNA in Xenopus Oocytes

Abstract

This chapter focuses on use of Xenopus oocytes for the study of brain neurotransmitter receptors and ion channels. A protocol is described for the extraction of RNA using phenol-chloroform as a protein denaturant and a means of separating the proteins from the nucleic acids, which consistently provides us with active mRNA coding for many brain neurotransmitter receptors and voltage-activated ion channels. The great variety of receptors and channels expressed by brain mRNA in oocytes have all been induced by poly(A) + mRNA. Female Xenopus laevis is obtained from commercial suppliers and sacrificed by decapitation. After injection, the oocytes are cultured at 16°C in sterile Barth's solution. Gene expression in various cells, including Xenopus oocytes and mammalian cells, can be selectively inhibited by antisense RNA. This approach is useful to examine receptor or ion-channel heterogeneity, blocking expression of one receptor (or ion channel) subtype with a specific oligonucleotide and studying another subtype encoded by a similar, but not identical, mRNA.