The transgenic expression of proteins is an important tool in the arsenal for analysing protein function. The experiments described here indicate that transgenically expressed innexin protein is incorporated into the subcellular structures/domains that normally contain endogenous innexins. In image A (above), myc-tagged Ogre is detected in annular junctions but it can also be found in plaques (not shown). The fact that transgenically supplied innexins can rescue plaque formation involving other innexin family subunits (Rescue of Inx3 plaques in an inx2null mutant, Lehmann et al, 2006) and can rescue loss-of-function phenotypes (Curtin et al, 2002) also confirms that transgenic innexin proteins are produced and can interact with their normal partners whether they be other innexin family members or proteins involved in transport, junction formation or degradation. This approach may consequently prove fruitful in experiments that attempt to alter the stoichiometry of subunits within channels or to incorporate modified innexins and observe their biological effect. Moreover, endogenous innexin protein levels do not appear to be affected by different levels of transgenic expression (graph B). The observations raise some issues:
- - high levels of transgene expression can lead to a build-up of protein in the cytoplasm which could have unanticipated consequences.
- - Transgenic expression of innexins, even in cells that normally express that particular innexin, can lead to developmental defects via unknown mechanisms. (Images: UAS-ogre induces leg joint defects, Endogenous Ogre in leg discs, Perils of Innexin transgene expression)
- - even within cells of the same tissue, some cells express significantly different amounts of transgene (as shown in salivary cells, image A above, when using the GAL4/UAS system (Brand and Perrimon, 1993)). Phenotypic effects could prove highly variable. This lack of consistency could become annoying if the phenotype under observation is labour intensive and quantitative eg.requires dye-coupling or electrophysiological measurements taken from neighbouring pairs of cells.
- - polyclonal antibodies were used to detect endogenous Inx3 protein. This will naturally introduce some variability and could be responsible for the differences in Inx3 staining intensity between cells 1-4 in plot B. But the error bars suggest that the staining is pretty consistent.
- - transgenic Ogre-myc may be incorporated inconsistently into membrane in the golgi apparatus (due to steric impedance caused by the myc epitope); some plaques may then receive more Ogre subunits and some less.
- - Myc-tagged Ogre may be degraded at a different rate than Inx3 in annular junctions.
- - The annular junctions within a single cell will contain an Ogre-myc protein contribution recieved from all of the gap junction-coupled neighbouring cells (Description of annular junctions). Annular junctions produced from neighbours that express transgene strongly will have a much lower ratio of Inx3 to Ogre-myc (red/green ratio) than annular junctions produced from weakly expressing neighbours.