A secreted fluorescent reporter targeted to pituitary growth hormone cells in transgenic mice.
In stable transfection experiments in the GH-producing GC cell line, a construct containing the entire signal peptide and the first 22 residues of human GH linked in frame with enhanced green fluorescent protein (eGFP), produced brightly fluorescent cells with a granular distribution of eGFP. This eGFP reporter was then inserted into a 40-kb cosmid transgene containing the locus control region for the hGH gene and used to generate transgenic mice. 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 cell sorting. Confocal scanning microscopy of pituitary GH cells from GH-eGFP transgenic mice showed a markedly granular appearance of fluorescence. Immunogold electron microscopy and RIA confirmed that the eGFP product was packaged in the dense cored secretory vesicles of somatotrophs and was secreted in parallel with GH in response to stimulation by GRF. Using eGFP fluorescence, it was possible to identify clusters of GH cells in acute pituitary slices and to observe spontaneous transient rises in their intracellular Ca2+ concentrations after loading with Ca2+ sensitive dyes. This transgenic approach opens the way to direct visualization of spontaneous and secretagogue-induced secretory mechanisms in identified GH cells.
Magoulas
C
C
Division of Neurophysiology, National Institute for Medical Research Mill Hill, London, United Kingdom.
McGuinness
L
L
Balthasar
N
N
Carmignac
D F
DF
Sesay
A K
AK
Mathers
K E
KE
Christian
H
H
Candeil
L
L
Bonnefont
X
X
Mollard
P
P
Robinson
I C
IC
D000818:Animals; D002118:Calcium / Q000032:analysis; D003360:Cosmids; D003594:Cytoplasmic Granules / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry; D005434:Flow Cytometry; D015870:Gene Expression; D049452:Green Fluorescent Proteins; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology; D019382:Human Growth Hormone / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D008164:Luminescent Proteins / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D010903:Pituitary Gland, Anterior / Q000378:metabolism* / Q000648:ultrastructure
D008164:Luminescent Proteins; D019382:Human Growth Hormone; D049452:Green Fluorescent Proteins; D013007:Growth Hormone-Releasing Hormone; D002118:Calcium