Alpha-Gustducin is a G protein alpha subunit that plays a key role in signal transduction of compounds humans consider either sweet or bitter. The gustducin heterotrimer is thought to transduce responses from a family of bitter responsive receptors to activation of phosphodiesterase and phospholipase Cbeta2. Alpha-Gustducin knockout mice have reduced behavioral and electrophysiological responses to certain bitter and sweet compounds. Alpha-Gustducin is highly expressed in taste receptor cells, but is also expressed in the brush cells of the stomach, intestine and pancreatic ducts. Alpha-Gustducin expressing cells of the digestive tract are suspected to be the chemosensory cells of the gut that may be involved in regulating resorption, secretion of digestive hormones and intestinal motility. To study alpha-gustducin expressing cells we have produced transgenic mice expressing green fluorescent protein (GFP) under the control of 7.7 kb of the 5' flanking region of the alpha-gustducin gene. GFP was expressed in a subset of taste receptor cells, but co-localization of alpha-gustducin and GFP was found in 80-95% of the cells, depending on the particular transgenic line. Moreover GFP was not expressed in the alpha-gustducin positive cells of the gut, and it was ectopically expressed in alpha-gustducin negative cells of the retina. To address these shortfalls of the transgenic mice, we propose to produce knock-in mice in which red fluorescent protein (RFP) and a truncated human CD4 are introduced into the alpha-gustducin locus such that they are faithfully expressed under the control of the cis-acting elements that control endogenous alpha-gustducin expression RFP will be used as a marker of alpha-gustducin-expressing cells. The truncated CD4 will be used as a surface marker to purify these cells after labeling them with an anti-CD4 antibody linked to a magnetic bead, using magnetic cell sorting. The knock-in mice and the purified cells will provide important reagents with which to determine the genes that are co-expressed with alpha-gustducin, to produce cDNA libraries from alpha-gustducin expressing taste and gut cells, and to study the response of the putative chemoreceptor cells of the gut to various compounds, using calcium imaging and electrophysiology.
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