This chapter describes a complementation test based on the expression of foreign messenger RNA (mRNA) in channel-deficient cells, for the study of biogenesis of cell–cell channels and analysis of its protein components. The mRNA was isolated from uterine smooth muscle cells that were actively involved in new channel formation in response to estrogen treatment. As a vehicle for transporting the mRNA into recipient cells, “artificial viruses” were used. These were obtained by encapsulating mRNA in liposomes which, like viruses, interact with cell membranes by fusion and probably also to some extent by endocytosis. It is assumed that the formation of new gap junctions in myometrium is because of the de novo synthesis of channel proteins. An alternative hypothesis would be that estrogen induces the synthesis of mRNA coding for a regulatory protein. The observed induction of cell–cell coupling in CL-1D cells appears to be mediated by mRNA. This conclusion is supported by a series of control experiments including ribonuclease (RNase) treatment of the mRNA, inhibition by cycloheximide, and study of concentration dependence.
ASJC Scopus subject areas
- Molecular Biology