A secreted fluorescent reporter targeted to pituitary growth hormone cellsin transgenic mice

In stable transfection experiments in the GI-I-producing GC cell Line, a co nstruct containing the entire signal peptide and the first 22 residues of h uman GH linked in frame with enhanced green fluorescent protein (eGFP), pro duced brightly fluorescent cells with a granular distribution of eGFP. This eGFP reporter was then inserted into a 40-kb cosmid transgene containing t he locus control region for the hGH gene and used to generate transgenic mi ce. Anterior pituitaries from these GH-eGFP transgenic mice showed numerous clusters of strongly fluorescent cells, which were also immunopositive for GH, and which could be isolated and enriched by fluorescence-activated cel l sorting. Confocal scanning microscopy of pituitary GH cells from GH-eGFP transgenic mice showed a markedly granular appearance of fluorescence. Immu nogold electron microscopy and RIA confirmed that the eGFP product was pack aged in the dense cored secretory vesicles of somatotrophs and was secreted in parallel with GH in response to stimulation by GRF. Using eGFP fluoresc ence, it was possible to identify clusters of GH cells in acute pituitary s lices and to observe spontaneous transient rises in their intracellular Ca2 + concentrations after loading with Ca2+ sensitive dyes. This transgenic ap proach opens the way to direct visualization of spontaneous and secretagogu e-induced secretory mechanisms in identified GH cells.