Chem. Senses 27: 779-787,
2002
© Oxford University Press 2002
Virus-mediated Transfer of Foreign DNA into Taste Receptor Cells
1 Department of Biomedical Sciences, Colorado State University, Fort Collins, CO 80523, USA 2 Department of Microbiology, Colorado State University, Fort Collins, CO 80523, USA 3 Rocky Mountain Taste and Smell Center, University of Colorado Health Sciences Center, Denver, CO 80262, USA
Correspondence to be sent to: Leslie Stone, Department of Anatomy and Neurobiology, Colorado State University, Fort Collins, CO 80523, USA. e-mail: Istone{at}lamar.colostate.edu
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Abstract |
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Foreign genes can be transferred into taste cells via adenoviral vectors. The present study was undertaken to characterize the subpopulation of taste cells that are susceptible to adenovirus infection and to determine whether another viral vector, derived from herpes simplex 1 (HSV-1), infects the same subpopulation of taste cells. Using an adenovirus containing the gene for enhanced green fluorescent protein (EGFP) under the control of the human cytomegalovirus (CMV) immediate early promoter, we found that EGFP was present in blood group antigen H immunoreactive (ir) taste cells, but not in gustducin-ir or PGP 9.5-ir cells. Infection of taste buds with an HSV-1 vector containing EGFP also resulted in a subpopulation of EGFP-positive taste cells. However, both gustducin-ir and PGP 9.5-ir taste cells expressed the marker protein. In conclusion, this study shows that both adenoviral and HSV-1 vectors can be used to transfer foreign genes into the cells of isolated rat taste buds and that different viruses can be used to target specific subpopulations of taste cells.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Taste cells are specialized receptor cells that detect a wide range of chemical stimuli, including ions and complex molecules. These stimuli interact with the taste cell via receptors or channels on the cell surface and subsequent intracellular responses vary depending on the tastant [for review see (Gilbertson and Kinnamon, 1996
; Kinnamon and Margolskee,
1996; Lindemann,
1996; Stewart et al.,
1997; Herness and Gilbertson,
1999)]. Simple species, such as protons and Na+,
interact with ion channels located on the plasma membrane, directly leading to
depolarization of the cell. Other tastants, including bitter substances,
sugars and amino acids, bind to protein receptors and activate intracellular
second messenger systems.
Several genes encoding proteins involved in the transduction of chemical
stimuli have been cloned from taste cells. These include genes for putative
receptor proteins: taste-mGluR4, a metabotropic glutamate receptor
(Chaudhari et al.,
2000); two families of G-protein-coupled receptors
(Hoon et al., 1999;
Adler et al., 2000;
Chandrashekar et al.,
2000; Matsunami et
al., 2000; Kitagawa
et al., 2001; Max
et al., 2001;
Montmayeur et al.,
2001; Sainz et al.,
2001); and MDEG1, a proton-gated cation channel
(Ugawa et al., 1998).
In addition, PLCß2 and the G-protein subunits 
-gustducin and
13 (McLaughlin et al.,
1992; Huang et al.,
1999) have been cloned. To determine the roles of specific
components in taste transduction pathways, cloned genes have been
characterized in heterologous systems, including CHO cells and
Xenopus oocytes (Ugawa et
al., 1998; Chandrashekar
et al., 2000;
Chaudhari et al.,
2000). In addition, the G-protein -gustducin has been
studied extensively in cell-free assays
(Hoon et al., 1995;
Ming et al., 1998;
Huang et al., 1999).
Expression of cloned genes in their native environment, the taste cell, could
define the molecular roles of specific proteins even further. For example,
targeting of proteins to specific intracellular or membrane domains could be
studied.
There are several methods available for transferring foreign genes into
cells. These include lipofection, particle-mediated gene transfer (gene gun),
electroporation and viral gene transfer. Preliminary studies in our laboratory
indicate that viral transfer (Stone et al.,
1999,
2001) can be used to transfer
foreign genes into taste cells. In addition, other workers
(Kishi et al., 2001)
have recently reported successful infection of taste cells with adenoviral
vectors. Following infection, a subpopulation of taste cells expresses virally
encoded proteins. The present study was undertaken to examine this
subpopulation of cells and to determine whether a herpes vector infects the
same subpopulation of cells. Preliminary presentations of this work have been
published in abstract form (Stone et al.,
1999,
2001).
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Materials and methods |
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Taste bud isolation
Taste buds were isolated from 6- to 10-week-old male Sprague—Dawley
rats as described in elsewhere (Ruiz
et al., 2001). Whole taste buds were chosen for analysis
rather than isolated taste cells. Taste buds consist of many different cell
types based on ultrastructural and immunocytochemical characteristics and we
wished to maintain both the different cell types and the normal intercellular
interactions between these specialized receptor cells. To obtain taste buds
for culture, tongue segments containing the designated papillae were injected
with 
0.5 ml of an enzyme solution containing collagenase B, dispase II
and trypsin inhibitor (see Solutions and media). Following incubation in
oxygenated Tyrode's solution for 50-60 min, the lingual epithelium was gently
peeled from the underlying connective tissue. The epithelial strip was pinned
out on a Sylgard dish and bathed in calcium-free Tyrode's solution (plus 1 mM
BAPTA) for 20 min. Taste buds were removed with gentle suction using a pipet
(tip diameter 100 µm) and placed into a drop of Tyrode's or culture
medium on a lysine-coated `Biocoat Cell Environments' culture slide (Cat. No.
40632; Becton Dickinson). Prior to plating taste buds, slides were
equilibrated to room temperature for 20-30 min to enhance taste bud adhesion.
After removing buds from a defined region (circumvallate, foliate or fungiform
papillae), the taste buds were allowed to settle for 5-10 min before
additional culture medium was added to each well. Both two- and eight-well
culture slides were used, resulting in media volumes of 500-2000 µl or
200-300 µl, respectively.
Solutions and media
Enzyme solution consisted of: 0.7 mg collagenase B (Roche Diagnostics
Corp., Indianapolis, IN), 3.0 mg dispase II (Roche Diagnostics Corp.) and 1.0
mg trypsin inhibitor (Sigma, St Louis, MO) in 1.0 ml Tyrode's solution (140 mM
NaCl, 5 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10
mM glucose, 10 mM pyruvate, pH 7.4). Taste buds were cultured and infected in
serum-free medium adapted from Pixley (Pixley, 1992;
Ruiz et al., 2001).
This medium consisted of: DMEM (Dulbecco's modified Eagle medium with
D-glucose, L-glutamine and sodium pyruvate; Cat. No. 12800-017; Gibco,
Baltimore, MD); 15 mM KCl; 18 mM HEPES; 10 ml/l MEM NEAA IX (Earle's Salts
with non-essential amino acids; Gibco); 30 µm hypoxanthine; 3 µm
thymidine; 100 U/ml penicillin; 0.1 mg/ml streptomycin; 25 µm
2-mercaptoethanol; ITS + Premix (20 ml/l—6.25 µg/ml transferrin, 6.25
µg/ml selenious acid, 1.25 mg/ml bovine serum albumin (BSA) and 5.35
µg/ml linoleic acid; Collaborative Biomedical Products, Bedford, MA). The
medium was buffered to pH 7.4 with NaOH. Unless otherwise noted, chemicals
were obtained from Sigma Chemical Corp.
Viral vectors
The ability of three viral vectors to express the marker enhanced green
fluorescent protein (EGFP) in isolated taste buds was tested and compared. Two
of the vectors were derived from adenovirus and differed only in the promoter
used to drive expression of the marker. Ad-RSV-EGFP contained the Rous sarcoma
virus (RSV) promoter and Ad-CMV-EGFP contained the human cytomegalovirus (CMV)
immediate early promoter. The third vector was a recombinant herpes simplex
virus type 1 (HSV-1) vector containing a fusion of the native viral protein
ICP4 (infected cell peptide 4) with EGFP driven by the native ICP4 promoter.
Construction of Ad-CMV-EGFP and Ad-RSV-EGFP have been described previously
(Smith et al.,
2000).
Infection of taste cells with adenoviral vectors
Adenoviral vectors were added to the medium bathing isolated taste buds within 6 h of taste bud isolation (n = 16 preparations). The amount of virus used to infect the buds depended on the volume of media. For wells containing 200-300 µl of media, 20 µl of virus was added (2.5 x 107 pfu/ml). For larger wells containing 500-2000 µl, 50 µl of virus was added. Due to the variability encountered with taste bud isolation and survival, it was not possible to determine the multiplicity of infection (amount of infectious virus per cell). After addition of the virus, taste buds were cultured at 35-37°C for 18-24 h.
Infection of taste cells with the herpes vector
The herpes vector was added to the taste bud medium 0-2 h following isolation and infected cultures were incubated for 17.5-21 h at 35-37°C.
Immunocytochemistry
Adenovirus- and HSV-1-infected taste buds were fixed with 4%
paraformaldehyde overnight at 4°C then washed 3 x 10 min with 0.1 M
phosphate buffered saline (PBS, pH 7.2). To decrease non-specific binding,
taste buds were incubated for 2-3 h in blocking solution consisting of 1%
normal goat serum (Jackson Laboratories, West Grove, PA), 1% BSA (Sigma, St
Louis, MO) and 0.3% Triton X-100 (United States Biochemical Corp., Cleveland,
OH) in PBS. Next, anti--gustducin (1:500, raised against amino acids
93-113; No. sc-395; Santa Cruz, Santa Cruz, CA), anti-PGP 9.5 (1:1500; No.
7863-0504; Biogenesis, Kingston, NH), or anti-blood group antigen H antibodies
(1:50; clone 92FR-A2; Dako Corp., Carpinteria, CA) were applied to the taste
buds. Each primary antibody was diluted in blocking solution and buds were
incubated at 4°C for 1-3 days, then washed 3 x 10 min in PBS.
Rhodamine red-X goat anti-rabbit (1:100; No. 111-295-144; Jackson Labs)
secondary antibodies were then applied to -gustducin and PGP 9.5
labeled taste buds and rhodamine red-X goat anti-mouse (No. 115-295-100;
Jackson Labs) secondary antibodies were applied to the blood group antigen H
labeled taste buds. Secondary antibody incubation was carried out at room
temperature for 2-3 h, after which the buds were washed 3 x 10 min in
PBS and coverslipped with fluoromount G (Southern Biotechnologies Inc.,
Birmingham, AL). Coverslipped slides were stored at 4°C.
Imaging
Following incubation with the viral constructs, taste buds were viewed while still in the culture dish with a Nikon Diaphot 200 inverted microscope. Fluorescent and bright-field images were captured with a Cool Snap digital camera and Image Pro software, and then processed using Adobe Photoshop 5.0 software. Confocal images of fixed, anti-body-labeled taste buds were collected using an Olympus FVX-IHRT Fluoview laser scanning confocal microscope equipped with argon, green HeNe and red HeNe lasers. For immunocytochemical analysis of taste buds double-labeled with virally expressed EGFP and rhodamine-tagged secondary antibodies, images were collected sequentially. After each fluorescent image was collected, an accompanying differential interference contrast (DIC) image was taken at the same focal plane to enhance visualization of individual taste cells. Adobe Photoshop 5.0 software was used to process both epifluorescent and confocal images. Processing included adjustments for brightness and contrast as well as application of identifying labels and scale bars.
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Results |
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Infection of taste buds with Ad-RSV-EGFP and Ad-CMV-EGFP
Rat taste buds were isolated from strips of lingual epithelium and infected with adenoviral vectors. Intact taste buds were used for these experiments instead of isolated cells, so that intercellular physiological interactions were retained during infection and to maintain the polarized structure of individual taste cells. Isolated taste cells lose their elongated shape in culture and membrane domains become less distinct. In addition, identification of isolated taste cells in culture is sometimes difficult, since both taste cells and epithelial cells are present in cultures. Two different adenoviral vectors were tested for their ability to infect and express EGFP in cells of isolated rat taste buds. The adenoviral vectors tested in this experiment differed only in the promoter used to drive the EGFP gene; one construct contained the RSV promoter and the other contained the CMV immediate early promoter. Twenty-four hours after virus application, taste buds infected with vectors containing the RSV promoter (n = 3 experiments) showed only rare, light labeling of isolated cells in taste buds, although occasional isolated cells not associated with taste buds showed bright EGFP labeling (data not shown). The isolated labeled cells were found in wells containing non-labeled taste buds. Isolated cells in our taste bud cultures appear to consist of both taste and non-taste cells based on immunocytochemical labeling (personal observations). Because bright EGFP labeling was never seen in intact taste buds, the isolated cells expressing EGFP were probably lingual epithelial cells rather than taste cells.
In contrast to the limited labeling seen with Ad-RSV-EGFP, taste buds
infected with the adenoviral vector containing the CMV promoter exhibited
robust EGFP labeling of many taste buds. The EGFP label appeared to cover most
of the bud when viewed with epifluorescence microscopy
(Figure 1A). Bright-field
images were taken to facilitate identification of taste bud boundaries
(Figure 1B). Although many
cells appeared to be labeled, the EGFP label was brighter in some cells than
others and seemed to be more prevalent in the central, compact regions of the
taste buds. Studies of cultured taste buds indicate that the compact region of
taste buds contains the healthiest cells
(Ruiz et al., 2001).
No difference in the extent or quality of labeling was noted in taste buds
isolated from fungiform, circumvallate and foliate papillae (data not shown).
Following this initial study, subsequent adenovirus experiments were carried
out using the vector with the CMV promoter driving expression of EGFP.
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Immunocytochemical characterization of cell types expressing EGFP following adenoviral infection
Confocal imaging showed that EGFP expression following infection with
Ad-CMV-EGFP was limited to a subset of cells (data not shown). To characterize
the EGFP+ sub-population of taste cells, immunocytochemical analysis was
carried out. Three antibodies were tested to determine the usefulness of
adenoviral vectors as investigational tools for studying certain types of
taste cells. The antibodies were: (i) an antibody to PGP 9.5, an ubiquitin
carboxyl-terminal hydrolase found in some type II and III taste cells
(Wilkinson et al.,
1989; Iwanaga et al.,
1992; Kanazawa and Yoshie,
1996; Yee et al.,
2001)—four preparations, 13 taste buds analyzed; (ii) an
antibody to -gustducin, a G-protein found in a different subset of type
II cells (Boughter et al.,
1997; Yang et al.,
2000)—10 preparations, 50 taste buds analyzed; and (iii) an
antibody to blood group antigen H, a cell surface carbohydrate that is present
on a large subset of taste cells and identifies the type I or dark cells in
rat taste buds (Pumplin et al.,
1997; Smith et al.,
1994)—two preparations, 12 taste buds analyzed.
-Gustducin appears to be involved in the transduction of some bitter,
sweet and umami stimuli (Ruiz-Avila et
al., 1995; Wong et
al., 1996). The functions of PGP 9.5 and antigen H in taste
cells are unknown. We used antibodies against these proteins as markers for
cell type, rather than to imply function.
Ad-CMV-EGFP infection followed by immunocyto-chemical lableling showed that
EGFP was not present in -gustducin-ir
(Figure 2A,B) or PGP 9.5-ir
(Figure 2C,D) cells. In
contrast, some blood group antigen H-ir cells expressed EGFP
(Figure 2E,F). The marker
protein was not present in all antigen H-ir cells, however, and EGFP was
present in some cells that lacked visible blood group antigen H
immunoreactivity.
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Infection of taste buds with HSV-1-(ICP4)-EGFP
Because Ad-CMV-EGFP infected only a subpopulation of taste cells, we examined EGFP expression in taste buds following infection with a recombinant HSV-1 vector containing EGFP under the control of the herpes ICP4 promoter. As with adenoviruses, HSV-1 infects both dividing and differentiated cells and infects a variety of cell types. Following incubation with HSV-1-(ICP4)-EGFP, taste buds exhibited robust EGFP expression when viewed with epifluorescence microscopy (Figure 3). The nuclear EGFP signal appeared to be in many taste cells. Similar to cultures infected with the adenoviral vector, EGFP expressed from the HSV-1 vector was found in most taste buds and appeared to be present in numerous cells within each bud.
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Immunocytochemical analysis of HSV-1-(ICP4)-EGFP infected buds revealed
that the herpes vector was successful in transferring EGFP into
-gustducin- and PGP 9.5-ir cells
(Figure 4). Whether antigen H
immunoreactive cells expressed EGFP following herpes infection was unclear,
due to the nuclear localization of the EGFP marker and the cell surface
labeling with this antibody.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The current study shows that both adenoviral and herpes vectors are capable of transferring foreign genes into cells of isolated rat taste buds. Interestingly, the two types of vectors infect different, although possibly overlapping, subsets of taste cells. Ad-CMV-EGFP infects a subpopulation of taste cells including some blood group antigen H-ir cells, but not
-gustducin-ir or PGP 9.5-ir cells. In addition to transferring the
marker gene into blood group antigen H-ir cells, Ad-CMV-EGFP infection
resulted in EGFP expression in an unidentified subpopulation of taste cells.
The herpes vector HSV-1 (ICP4)-EGFP also infects a subpopulation of taste
cells, including taste cells immunoreactive for -gustducin and PGP
9.5.
The cells that make up a rat taste bud are a heterogeneous population.
Based on ultrastructural characteristics, the elongated taste cells in a bud
can be classified into three types: type I (dark) cells; type II (light)
cells; and type III cells (Lindemann,
1996; Finger and Simon,
2000). Type I cells have electron-dense cytoplasm, their nuclei
may be invaginated and many type I cells wrap around other cells. Type II
cells are electron-lucent, have a round or oval nucleus and short apical
microvilli. Type III cells have electron-lucent cytoplasm, nuclei that are
intermediate in appearance between type I and type II cells, and have a single
large microvillus. In addition to ultrastructural criteria, immunocytochemical
markers can be used to classify taste cells. For example, a subpopulation of
taste cells is immunoreactive for several components known to be involved in
bitter taste transduction: IP3R3, PLCß2 and 13
(Clapp et al., 2001).
A portion of taste cells expressing these proteins also expresses
-gustducin. In some cases the ultrastructural and immunocytochemical
subpopulations overlap, so that antibodies can be used to roughly identify
ultrastructural taste cell types. The antibodies used in this study were
raised to -gustducin, PGP 9.5 and blood group antigen H.
-Gustducin is a G-protein found primarily in some type II taste cells
(Boughter et al.,
1997), PGP 9.5 is found in some type II and type III cells
(Yee et al., 2001)
and blood group antigen H is found on the cell surface of most type I cells
(Pumplin et al.,
1997).
Our results indicate that adenoviral vectors are capable of transferring
genes into type I cells, since we found cells double-labeled with virally
expressed EGFP and antigen H immunoreactivity. The specific functions of type
I taste cells are unclear, although both supportive and sensory roles have
been suggested (Kinnamon et al.,
1985,
1988;
McPheeters et al.,
1994). Type I cells wrap around nerve fibers and other taste
cells, suggesting that, as with glial cells, type I cells may have supportive
functions including control of extra-cellular ions and transmitters, nutrition
and phagocytosis (Lindemann,
1996). The presence of the glutamate—aspartate transporter
(GLAST) in rat type I taste cells supports this idea
(Lawton et al.,
2000). GLAST is found in glial cells and likely is involved in
taking up glutamate from the extracellular space, thus regulating the
availability of this neurotransmitter. Additional evidence for taste cells
with glia-like functions comes from electrophysiological studies that
identified a subpopulation of taste cells with a leakage conductance highly
permeable to K+ (Bigiani,
2001). The identity of the `leaky' cells is unclear, although
Bigiani speculates that they may be type I cells. In addition to a supportive
role, ultrastructural analyses of murine taste buds and associated nerve
fibers indicate that some type I cells may have synaptic contacts with nerve
fibers, suggesting a chemosensory function for these cells (Kinnamon et
al., 1985,
1988).
A recent study (Kishi et al.,
2001) has reported successful transfer of foreign genes into
isolated rat taste buds using an adenoviral vector. They reported that GFP
expression was present in >90% of the cells in their cultures following
infection with an adenoviral vector. In our studies, the subpopulation of
EGFP-expressing cells following Ad-CMV-EGFP excludes gustducin-ir cells and
PGP 9.5-ir cells, thus EGFP expression in our infected cultures is <90%.
PGP 9.5-ir cells in rat constitute10% of the taste bud population
(Kanazawa and Yoshie, 1996)
and -gustducin-ir cells also comprise at least 10% of the circumvallate
or foliate taste bud population (Boughter
et al., 1997). These two taste bud populations are
non-overlapping (Yee et al.,
2001) and therefore in our cultures no more than 80% of taste bud
cells express EGFP following Ad-CMV-EGFP infection. Furthermore, qualitative
analysis of our confocal images indicates that <50% of cells in our taste
bud cultures express EGFP following infection. Both our vector and that used
by Kishi et al. were constructed with Ad5 sequences, so the cause of
the difference in infection efficiency is unclear. Taste bud cultures contain
both taste cells and non-sensory epithelial cells, so some of the
EGFP-expressing cells in both our studies and those done by Kishi et
al. probably were not taste cells. A difference in the number of
non-taste cells in the cultures may have contributed to the difference in EGFP
expression.
The HSV-1 vector used in the present study was able to transfer EGFP into
type II and possibly type III cells. Cells double-labeled with virally
expressed EGFP and either -gustducin-ir or PGP 9.5-ir were evident.
-Gustducin is present in some type II taste cells
(Tabata et al., 1995;
Boughter et al., 1997;
Yang et al., 2000)
and PGP 9.5 is present in some type II cells and some type III cells
(Yee et al., 2001).
Both type II and type III cells are likely to have chemosensory roles.
Components of the bitter transduction pathway, including -gustducin,
PLCß2, IP3R3 and 13, have been identified in type II
cells (Asano-Miyoshi et al.,
2000; Clapp et al.,
2001) and type III cells exhibit synapses in several species,
including mouse and rabbit (Delay et
al., 1986; Royer and
Kinnamon, 1991).
The present study indicates that adenoviral vectors can be used for
transferring genes into type I taste cells and that herpes vectors can be used
for transferring genes into type II and possibly type III cells. The exact
functions of each of the taste cell types are unclear. All three might have
sensory functions, or type I cells may have a supportive, glia-like role, with
type II and type III cells responsible for taste transduction. Gene transfer
via viral vectors may help define cellular roles by providing a tool to
interfere with, or enhance, specific proteins in a subpopulation of cells. For
example, modification of the GLAST in type I cells, or the G-protein
-gustducin in type II cells may affect taste bud physiology. Infection
of taste buds with viral vectors containing apically located, tagged proteins
may help determine how taste transduction machinery gets targeted to the small
apical membrane of taste receptor cells. Cell lineage studies utilizing
adenoviral vectors also are possible. Although recent studies suggest that
taste cells expressing serotonin constitute a separate cell population
(Stone et al., 2002),
it is possible that other markers indicate specific stages in the lifetime of
a taste cell. For example, type I cells may give rise to type II cells
(Delay et al., 1986).
Infection of type I cells with an adenoviral vector expressing EGFP, followed
by an incubation period of several days, may reveal whether blood group
antigen H (type I) cells, mature into a cell type expressing other markers. In
addition to examination of the roles of specific taste cells, molecular
functions of proteins within taste cells could be studied by modification or
over-expression of specific proteins followed by analysis of the effects on
the expressed protein and its associated molecules. In conclusion, both
adenoviral and herpes vectors will provide tools for examination of the roles
of specific taste cells and taste molecules.
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Acknowledgments |
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We would like to thank Drs David Pumplin and David Smith for advice concerning blood group antigen antibodies and Drs Tom Finger, Linda Barlow and Kathryn Medler for critical reading of the manuscript. This study was supported by NIH grants P01 DC00244 and P20 DC01439 to S.C.K., Public Health Service grant NS29046 to C.L.W. and NRSA grant 5 F32 DC00287 to L.M.S.
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Accepted July 30, 2002
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