Primary culture of rat taste bud cells that retain molecular markers for taste buds and permit functional expression of foreign genes.
Taste buds are constituted of several kinds of cells which have distinct characteristics and play different roles. In this study, we have established an in vitro culture system by optimizing the method for isolating the cells and by selecting culture media and reagents effective for cell viability and adhesion. As a result, the taste bud cells were adhesive and viable for over 3 days when cultured onto Matrigel-coated dishes in medium based on keratinocyte growth medium. The cells retained molecular markers for both the cytoskeleton and intracellular signaling such as cytokeratin 8 and phospholipase Cbeta2. In addition, three intracellular signaling molecules, gustducin, phospholipase Cbeta2, and inositol 1,4,5-trisphosphate receptor type 3, are expressed in the same correlation as those in vivo, although the ratio of signaling molecule-positive cells vs. total cells was somewhat lower in the culture than in vivo. Next, we tried several methods to introduce foreign genes into the cells, and obtained a greater than 90% efficiency of introduction using an adenovirus vector. Finally, we show that an exogenously expressed myc-tagged alpha1A-adrenoceptor sorts into the plasma membrane, and transduces a ligand-dependent signal resulting in intracellular [Ca(2+)] increase in about half of the infected cells. These results suggest that taste bud cells after 3 days of culture retain characteristic molecular markers, and may prove useful for describing the molecular and physiological features of taste bud cells, and that these cells can be further manipulated by adenovirus-mediated gene introduction.
Kishi
M
M
Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. aka7308@mail.ccu.u-tokyo.ac.jp
Emori
Y
Y
Tsukamoto
Y
Y
Abe
K
K
D000818:Animals; D015415:Biomarkers / Q000737:chemistry; D020013:Calcium Signaling / Q000235:genetics; D002448:Cell Adhesion / Q000187:drug effects / Q000502:physiology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D048429:Cell Size / Q000187:drug effects / Q000502:physiology; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured / Q000166:cytology* / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000502:physiology*; D005822:Genetic Vectors / Q000502:physiology; D049452:Green Fluorescent Proteins; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D007424:Intracellular Fluid / Q000378:metabolism; D007527:Isoenzymes / Q000378:metabolism; D007633:Keratins / Q000378:metabolism; D008164:Luminescent Proteins / Q000493:pharmacokinetics; D008297:Male; D054799:Phospholipase C beta; D051381:Rats; D017208:Rats, Wistar; D018340:Receptors, Adrenergic, alpha-1 / Q000235:genetics / Q000378:metabolism; D013649:Taste / Q000502:physiology*; D013650:Taste Buds / Q000166:cytology* / Q000378:metabolism*; D015293:Transducin / Q000378:metabolism; D014162:Transfection / Q000379:methods; D010738:Type C Phospholipases / Q000378:metabolism
C547715:Adra1a protein, rat; D015415:Biomarkers; D003470:Culture Media; D007202:Indicators and Reagents; D007527:Isoenzymes; D008164:Luminescent Proteins; D018340:Receptors, Adrenergic, alpha-1; D049452:Green Fluorescent Proteins; C075192:gustducin; D007633:Keratins; D010738:Type C Phospholipases; D054799:Phospholipase C beta; D015293:Transducin