JP2012154661A - Method for searching taste bud cell marker molecule using skn-1a/i knockout mouse - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for searching I-, II-, and III-type taste bud cell marker molecules, a use of gene/protein involved in receiving salty taste and gene/protein for identifying salty-taste substitutes or salty-taste enhancing substances, and a use of knockout mice or cultured cells for screening the salty-taste substitutes or the salty-taste enhancing substances/salty-taste receptors.SOLUTION: A screening method of taste substances includes, allowing Skn-la/i knockout mice to ingest candidate substances of the taste substances, and performing a behaviour analysis of the mice. The taste substances are selected from a group comprising salty substances, sweet substances, delicious substances, bitter substances and sour substances.

Description

  The present invention relates to a basic 5 taste (salt, sour, sweet, umami, and bitter) screening method, a basic 5 taste classification method, a search method for a miso cell marker molecule that recognizes basic 5 taste, and basic 5 The present invention relates to a marker molecule for taste bud cells involved in taste.

  Taste substances taken into the oral cavity are received by taste cells in tissues called taste buds distributed on the surface of the tongue, pharynx, and larynx. On the tongue, the miso is present at a site called the nipple. Sponge-like nipples are scattered in the front half of the tongue, the foliate nipples on the posterior side of the tongue, and the circumscribed nipples on the posterior center of the tongue. There is one taste bud on the fungiform nipple, and many taste buds on the foliate nipple and the circumvallate nipple. When the taste substance is received by a taste receptor present in the taste cell, the signal transmission substance is released to the taste nerve projected onto the taste cell via intracellular signal transmission. The information transmitted to the taste nerve finally reaches the brain and is recognized as a taste. In recent years, receptors for sweet taste, umami taste, bitter taste, and sour taste, and many factors involved in intracellular signal transduction have been identified, and the molecular mechanism of taste substance acceptance and taste intracellular signal transmission in miso is becoming clear.

  Each taste bud is composed of about 50 to 100 cells, and is present in spindle-shaped type I, type II, type III cells and the basal part based on the electron morphology and intracellular microstructure. It is classified as type IV cell. From the expression patterns of taste receptors and signal transduction factors, it is clear that type II cells are cells that recognize sweetness, umami, and bitterness, and type III cells are cells that recognize sourness.

  As a receptor for sweet and umami substances, a molecular species of the T1R (also called TAS1R) family of receptors (GPCRs) coupled to G proteins is functioning. It consists of three molecular species T1R1, T1R2 and T1R3.The T1R1 + T1R3 heteromer is an L-amino acid containing umami substances, and the T1R2 + T1R3 heteromer is a sugar, D-amino acid, artificial sweetener and sweet protein. The non-patent literature 1 accepts sweet substances such as. Bitter substances are also accepted by molecular species of the T2R (also called TAS2R) family, which is a kind of GPCR, and 25 T2R molecular species have been reported in humans. With respect to sour substances, it has been reported that the heteromolecules of ion channels PKD1L3 and PKD2L1 are candidate molecules responsible for their acceptance (Non-patent Document 2).

  In expression analysis of taste receptors and signaling factors, taste cells expressing T1R1 or T1R2 co-express T1R3, T1R1 and T1R2 are expressed in different taste cells, and T1Rs and T2Rs show exclusive expression patterns It has become clear. It has also been reported that taste cells expressing PKD2L1 are different from taste cells expressing signaling factors PLC-β2 and TRPM5 expressed in T1Rs and T2Rs expressing cells. From these, it can be said that sweet taste / umami taste / bitter taste / acidity receptors are expressed in different taste cells in miso, that is, sweet taste / umami taste / bitter taste / acidity are accepted by different taste cells. This is not only an expression analysis, but also an experimental result that mice in which PKD2L1-expressing cells were eliminated using diphtheria toxin lost the neural response to acidity only, and responses to other tastes remained (Non-Patent Document 2). The T1R1-KO, T1R2-KO, and T2R-KO mice lost the neural responses to umami, sweetness, and bitter taste stimuli, but the neural responses to other taste stimuli were not changed (Non-patent Document 1). It is proved from the experimental results. From these facts, it is reasonably expected that cells involved in salty taste reception are different from taste cells receiving sweetness, umami, bitterness, and sourness, that is, salty taste recognition is performed by type I taste bud cells. It is.

  An analysis using a knockout mouse as a candidate salty taste receptor has reported that ENaC (Epithelial Sodium Channel) functions as a sensor for low-concentration NaCl (Non-patent Document 3). However, the effect of ENaC knockout mice on high salt taste preference has not been found, and since ENaC is a sodium channel, how chloride ions in salt (NaCl) affect salt taste preference It is not completely clear about the acceptance of salty taste in miso.

  Patent Document 1 discloses a salty taste receptor, but the effect of the receptor has not been demonstrated in Examples.

Special table 2009-539377

Nature Vol.444, November 16, 2006 288-294 Nature Vol.442, August 24, 2006 934-938 Nature Vol.464, March 11, 2010, 297-301

  The present invention is a method for classifying basic five tastes (salt taste, sour taste, sweet taste, umami taste, bitter taste), a basic five taste substance screening method, a search method for type I, II and III miso cell marker molecules, and salty taste acceptance. For screening genes / proteins involved, use of said genes / proteins for identification of salty taste substitutes or potentiators, receptor-expressing cultured cells and amphibian oocytes, and salty taste substitutes or potentiators / salt taste receptors The object is to provide the use of knockout animals or receptor-expressing cultured cells and amphibian oocytes.

  Since there are many taste buds in the nipple papilla in the epithelium of the tongue epithelium layer, the search for genes expressed in the taste bud is performed by the fold papillary epithelium and non-papillary lingual epithelium (non-papillary epithelium). In many cases, comparative analysis is performed using. However, these comparative analyzes yield many genes that are specifically expressed in the papillary epithelium other than miso, and it is not easy to identify genes that reflect the characteristics of miso. However, it was more difficult to identify genes expressed in specific cells in miso. Therefore, by producing mutant mice with different proportions of cells that constitute miso, comparative analysis of gene expression in the nipple with wild-type mice (hereinafter referred to as `` WT mice '') This is considered to be a very effective technique for identifying genes expressed in specific cells.

  As a method for changing the cell structure of miso, the present inventor first searched for a transcriptional regulatory factor that is specifically expressed in miso. Among them, there was a Skn-1a / i gene, and it was confirmed that this gene was expressed specifically in sweet taste / umami taste / bitter taste receptor cells. This Skn-1a / i knockout mouse has already been prepared (Genes & Development, 11: 1873-1884, 1997), and a Skn-1a / i knockout mouse (hereinafter referred to as “S-KO mouse”) prepared in the same manner. ''), The sensitivity to sweetness, umami, and bitterness has disappeared, and in situ hybridization (ISH) analysis shows that the marker molecules for cells that accept sweetness, umami, and bitterness are completely intact. Had disappeared. On the other hand, in in situ hybridization (ISH) analysis, it was found that the expression of marker molecules (PKD2L1 and PKD1L3) that accept sour taste increased from WT mice. Nerve response analysis to salty taste showed no difference between S-KO and WT mice. The above results indicate that S-KO mice lose taste cells involved in accepting sweetness, umami, and bitterness, and the number of taste cells that accept sourness increases, and sweetness, umami, bitterness, and sourness. The proportion of cells other than taste cells to be received (mostly thought to be type I cells) suggests that there is no difference between WT and S-KO mice.

  Therefore, the present inventor has compared the gene expression level in the circumscribed papillary epithelial tissue of the S-KO mouse and the WT mouse, and by comparing the circumscribed papillary epithelial tissue and the non-papillary epithelial tissue of the WT mouse. Genes that are specifically expressed in cells (including those that are involved in salty taste reception) were found efficiently.

In particular,
(i) Extraction of genes (S-KO / WT is selected from 0.7 to 1.5) whose ratio of gene expression in the circumpapillary epithelium of S-KO and WT mice is not large
(ii) Extraction of genes that are specifically expressed in the circumpapillary papillae (a gene whose expression level in the circumpapillary epithelial tissue is more than 5 times greater than that of the nonpapillary epithelial tissue)
(iii) Using in situ hybridization (ISH), we examined gene expression based on three criteria: extraction of genes that expressed expression levels that could be confirmed in miso (expression levels in WT mice> 100). 395 genes were selected.

  Furthermore, among these genes, those having multiple transmembrane regions were selected, and then duplicated genes were excluded and 34 genes were selected (Table 1).

  Among these, as a molecule that is expressed on the membrane surface of type I miso cells and may be involved in the reception of salty taste, due to gene expression patterns and gene functions in miso by in situ hybridization (ISH) , Ano1, Kcnk16, Mfsd3, Tmc5, Trpc1, Gpr63, Gpr161, Gpr177, Slc16a7, Slc25a14, Slc35a1, Slc9a7, Sec61a1, Hsd3b7, Mal, Ptch1, and Tm4sf1. Of these, Ano1, Hsd3b7, Slc35a1, Gpr63, Kcnk16, Mal, Ptch1, Tm4sf1, and Sec61a1 are more preferable (expression confirmed in miso by ISH), and among these, some cells of miso (I Ano1 is considered to be most preferred as a molecule involved in salty taste reception, which is strongly expressed specifically in type Miso cells) and has a function expected to be involved in the salty taste receptor called a chloride ion channel. .

  The above is a method to find genes that are specifically expressed in type I cells. By changing the ratio of gene expression in the circumpapillary epithelium of S-KO and WT mice, it is specific to type II cells. Genes that are specifically expressed and genes that are specifically expressed in type II cells can be found in the same manner.

The present invention includes the following five basic taste (salt taste, sour taste, sweet taste, umami taste, bitter taste) classification method, basic five taste substance screening method, I, II and III type Miso cell marker molecule search method, salty taste Screening genes / proteins involved in reception, use of the genes / proteins for identification of salty taste substitutes or potentiators, receptor-expressing cultured cells and amphibian oocytes, and salty taste substitutes or potentiators / salt taste receptors For the use of knockout animals or receptor-expressing cultured cells and amphibian oocytes.
Item 1. Skn-1a / i knockout mice are ingested with candidate substances of taste substances, and the behavior analysis of the mice is performed, wherein the taste substances are selected from the group consisting of salty substances, sweet substances, umami substances, bitter substances and sour substances A method for screening a tastant, which is selected.
Item 2. Item 2. The screening method according to Item 1, wherein the taste substance is a salty substance.
Item 3. Item 2. The screening method according to Item 1, wherein the taste substance is a sweet substance or an umami substance.
Item 4. Item 2. The screening method according to Item 1, wherein the taste substance is a bitter substance.
Item 5. Item 2. The screening method according to Item 1, wherein the taste substance is a sour substance.
Item 6. Item 6. The screening method according to Item 1, wherein a wild type mouse is further ingested with a candidate substance of a taste substance together with the Skn-1a / i knockout mouse, and behavior analysis of both mice is performed.
Item 7. Skn-1a / i knockout mouse use for screening taste substances, wherein the taste substances are selected from the group consisting of salty substances, sweet substances, umami substances, bitter substances and sour substances Use for.
Item 8. Item 8. The use for screening according to Item 7, wherein the taste substance is a salty substance.
Item 9. Item 8. The use for screening according to Item 7, wherein the taste substance is a sweet substance or an umami substance.
Item 10. Item 8. The use for screening according to Item 7, wherein the taste substance is a bitter substance.
Item 11. Item 8. The use for screening according to Item 7, wherein the taste substance is a sour substance.
Item 12. A method of classifying a test substance into salty taste, sweet taste or umami taste, bitter taste, or sour taste, wherein a Skn-1a / i knockout mouse and, if necessary, a wild type mouse ingest the test substance, A method comprising performing behavior analysis on a mouse.
Item 13. A method for searching marker molecules of taste bud cells using Skn-1a / i knockout mice.
Item 14. Item 13 determines whether the miso cell is a type I miso cell recognizing salty taste, a type II miso cell recognizing sweet, umami, or bitter taste, or a type III miso cell recognizing acidity. The marker molecule search method described.
Item 15. A comparative analysis of genes expressed in the circumpapillary epithelial cells of Skn-1a / i knockout mice and wild-type mice, and the marker molecule expressed only in wild-type mice is determined to be a marker molecule of type II taste bud cells 15. A method for searching for a marker molecule according to 13 or 14.
Item 16. Comparing the amount of gene expressed in the circumscribed papillary epithelial cells of Skn-1a / i knockout mice and wild type mice, comparing the amount of expressed genes in the circumscribed papillary epithelial tissue and non-papillary epithelial tissue. Item 15. The method for searching for a marker molecule according to Item 13 or 14, wherein the marker molecule is determined to be a marker molecule of a type III taste bud cell or a type III taste bud cell.
Item 17. (i) Gene extraction process that does not significantly change the expression level in circumscribed papillary epithelial cells of Skn-1a / i knockout mice and wild type mice
(ii) Extraction process of the gene specifically expressed in the circumvallate nipple or both the circumvallate nipple and epithelium papillary epithelium
(iii) The method for searching a marker molecule of type I taste bud cells according to item 16, comprising a step of extracting a gene capable of confirming expression in miso by in situ hybridization (ISH).
Item 18. (i) Extraction process of gene with high expression level in circumscribed papillary epithelial cells of Skn-1a / i knockout mice and wild type mice
(ii) Extraction process of the gene specifically expressed in the circumvallate nipple or both the circumvallate nipple and epithelium papillary epithelium
(iii) The method for searching a marker molecule of a type III taste bud cell according to item 16, comprising a step of extracting a gene capable of confirming expression in miso by in situ hybridization (ISH).
Item 19. Item 19. The method for searching for a marker molecule according to any one of Items 13 to 18, wherein a gene having a transmembrane region multiple times is selected.
Item 20. Ano1, Kcnk16, Mfsd3, Tmc5, Trpc1, Gpr63, Gpr161, Gpr177, Slc16a7, Slc25a14, Slc35a1, Slc9a7, Sec61a1, Hsd3b7, Mal, Ptch1, Tm4sf1 Use as.
Item 21. Salty taste receptor of at least one gene selected from Ano1, Kcnk16, Mfsd3, Tmc5, Trpc1, Gpr63, Gpr161, Gpr177, Slc16a7, Slc25a14, Slc35a1, Slc9a7, Sec61a1, Hsd3b7, Mal, Ptch1, Tm4sf1 Use for alternative or potentiator screening.
Item 22. At least one foreign expression gene selected from Ano1, Kcnk16, Mfsd3, Tmc5, Trpc1, Gpr63, Gpr161, Gpr177, Slc16a7, Slc25a14, Slc35a1, Slc9a7, Sec61a1, Hsd3b7, Mal, Ptch1, and Tm4sf1 for salty substance screening Cultured cells or amphibian oocytes.
Item 23. Item 22. A screening method for a salty taste substitute or salty taste enhancing substance by observing a cellular response by causing a salty taste substitute or salty taste enhancing substance to act on the cultured cell or amphibian oocyte according to Item 22.
Item 24. The cellular response of the cell salty taste substitute or salty taste enhancing substance according to Item 22 is monitored using a patch clamp method, measurement of total cell current, radiolabeled ion flux assay, membrane potential sensitive dye and ion concentration sensitive dye, Item 22. The screening method according to Item 21.

  Since the gene or protein involved in human salty taste acceptance has been clarified by the present invention, it becomes possible to search for a completely new salty taste substitute or enhancer.

  Furthermore, according to the present invention, it has been found that S-KO mice lose sensitivity to sweetness, umami, and bitter taste, and marker molecules (PKD2L1 and PKD1L3) that accept sour taste have increased expression compared to WT mice. These can be used to screen basic five taste substances. In addition, by identifying genes that are specifically expressed at type I, type II, and type III receptors, any of the five basic taste substances can be identified using cells into which the gene has been introduced. .

Licking test with WT mice using an inhibitor of chloride ion channel Inhibitors were added to NaCl, KCl or NH ₄ Cl solutions at various concentrations and subjected to a licking test. The inhibitor concentration was fixed at 500 μM, and DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid) and NFA (Niflumic acid, Niflumic acid) and amiloride as an inhibitor of ENaC. Gene expression analysis in taste buds of WT mice (+ / +) and S-KO mice (Skn-1 ^{− / −} ). a: Expression analysis of sweetness / umami taste / bitter taste receptor cell marker molecules (T1R1, T1R2, T1R3, T2R5, Ggust, PLC-β2, TRPM5). b: Expression analysis of sour taste receptor cell marker molecules (PKD2L1, PKD1L3). It shows that type II miso cells responsible for sweet taste, umami taste, and bitter taste disappeared in S-KO mice, and type III cells responsible for sour taste recognition increased instead. Chorionic nerve analysis (A) and glossopharyngeal nerve analysis (B) (S-) using WT mice (WT; + / +) and Skn-1a / i knockout mice (S-KO; Skn-1 ^-/- ) In KO mice, each neural response to sweet, umami, and bitter taste stimuli is significantly suppressed, and the neural responses to sour and salty stimuli are not significantly different from WT). Two-bottle selection test (behavioral analysis) using WT mice (+ / +) and S-KO mice (Skn-1 ^-/- ) (S-KO mice lose their preference for sweet, umami, and bitter substances, The preference for sour and salty substances is not significantly different from WT). Expression analysis of genes specifically expressed in type I taste bud cells. The gene expression pattern in the circumpapillary epithelial layer of each gene is shown. Left: WT mouse circumpapillary, right: S-KO mouse circumpapillary. Expression analysis of genes specifically expressed in type I taste bud cells. The gene expression pattern in the circumpapillary epithelial layer of each gene is shown. Left: WT mouse circumpapillary, right: S-KO mouse circumpapillary. Expression analysis of genes specifically expressed in type I taste bud cells. The gene expression pattern in the circumpapillary epithelial layer of each gene is shown. Left: WT mouse circumpapillary, right: S-KO mouse circumpapillary. The construction of S-KO mice is shown.

  Based on the previous reports of neural response analysis using mice lacking each taste receptor and the results of behavioral analysis, neural response analysis and gene expression analysis of the S-KO mice described above, the cells that receive salty taste are sweet and umami.・ It is thought that it is a group of cells that do not express bitter and sour taste receptors, that is, type I taste bud cells (part of it), and molecular species involved in salt taste reception are thought to be expressed in type I taste bud cells .

  In the behavioral analysis for salty taste, the mice prefer the low saltiness and the high saltiness is repellent. As a salty receptor candidate molecule, ENaC has been reported as a molecule deeply involved in accepting salty taste at low concentrations. ENaC is an amiloride-sensitive sodium channel that functions only when all three subunits, α, β, and γ are present. Cells expressing both of the three subunits are different from sweet, umami, bitter, and sour receptor cells and are consistent with the above predictions.

On the other hand, KCl, NH ₄ Cl, CH ₃ COONa and GluNa (MSG) are recognized as bitterness, astringency, acidity, umami, and other tastes in addition to salty taste. It is believed that both, not a single taste, are necessary to bring out a salty taste. However, at present, there is no knowledge of molecules involved in accepting a high concentration of salty taste or molecules sensing chloride ions in salt.

When the inventor conducted a licking test using WT mice for three of NaCl, KCl, and NH ₄ Cl, amiloride (inhibitor of DIDS, NFA, or ENaC, which is an inhibitor of chloride ion channel) Amiloride) alone could not alleviate the repellent behavior for high concentrations of NaCl above 300 mM. On the other hand, we found that the combined use of DIDS and amiloride can significantly suppress repellent behavior against high concentrations of NaCl (Fig. 1). From this, it can be said that both sodium ions and chloride ions are involved in accepting a high concentration of salty taste. On the other hand, KCl, avoidance behavior of the high salt concentration by NH ₄ Cl has been possible to suppress a chloride ion channel inhibitors, since it could not be suppressed in amiloride, the repellency of high concentrations of KCl or NH ₄ Cl This suggests that the chloride ion is greatly involved.

  As described above, the present inventor paid attention to the knockout mouse (S-KO mouse) of the Skn-1a / i gene, and analyzed its behavioral analysis, neural response analysis, and expression analysis of taste-related genes. It was found that sweetness / umami taste / bitter taste receptor cells disappeared and sweet taste / umami taste / bitter taste could not be perceived, but acidity and saltiness could be perceived (FIGS. 2-5, Tables 2-3).

  Subsequently, genes expressed in type I taste bud cells were extracted by DNA microarray analysis of WT and S-KO mice. Specifically, (i) the ratio of gene expression levels in the circumpapillary epithelium of S-KO and WT mice is not large (select S-KO / WT from 0.7 to 1.5), and (ii) It is expressed specifically in the papillary epithelium (a gene whose expression level in the circumscribed papillary epithelial tissue is more than 5 times greater than that in the non-papillary epithelial tissue), and (iii) expression in miso by in situ hybridization (ISH) 395 genes were selected by extracting genes (expression level in WT> 100) that showed an expression level assumed to be confirmed. Furthermore, since all the ion channels known in the past have a plurality of transmembrane regions, among these genes, by eliminating the duplicated genes that have multiple transmembrane regions, 34 genes were selected (Table 1).

Using these 34 genes as targets, we observed expression in miso by in situ hybridization (ISH). From the results (Fig. 5), a gene that is strongly and specifically expressed in miso cells is involved in salt taste reception. The following genes were considered promising from the results of ISH analysis:
Chloride ion channel: Ano1
Cation channel (candidate): Kcnk16, Mfsd3, Tmc5, Trpc1
GPCR: Gpr63, Gpr161, Gpr177
Ion transporters (candidates): Slc16a7, Slc25a14, Slc35a1, Slc9a7
What was expressed in miso
Specific expression in type I taste bud cells: Ano1, Sec61a1
Highly expressed in cells of whole miso: Hsd3b7, Slc35a1, Gpr63, Kcnk16, Mal, Ptch1, Tm4sf1
Of these genes, Ano1 is one of the most likely molecules for salty taste recognition, even if it is expressed specifically in type I taste bud cells and has the function of a chloride ion channel. .

  Genes likely to be involved in the recognition of salty taste described above can be used for screening for salty taste substitutes or enhancers by creating knockout, knockin or transgenic non-human mammals of at least one of these genes. it can. Alternatively, by co-expressing reporter genes such as luciferase, fluorescent protein, alkaline phosphatase, beta galactosidase, diaphorase, and peroxidase in cultured cells expressing these genes, and measuring the expression level of these reporter genes, the salty taste receptor A compound (a salty (enhancement) substance) that increases the expression level or sensitivity of can be screened. Alternatively, the expression products of these genes, that is, substances that specifically bind to or interact with proteins, substances that regulate the function of these genes (for example, substances that inhibit or enhance the signal response by sodium ions or chloride ions, these genes) By searching for a substance that binds), a salty (enhanced) substance) can be screened.

  As used herein, “taste cell” includes all type I, II, III, and IV taste bud cells.

  As used herein, “cultured cells” refers to mammalian cells such as humans, monkeys, pigs, cows, mice, rats, rabbits and dogs, vertebrate cells such as chicken cells such as chickens, amphibian cells, reptile cells and insect cells. Including animal cells such as yeast.

  “Salt taste substance” means a salt taste substitute substance and / or a salt taste enhance substance. A salt taste substitute substance is a substance having a taste similar to or similar to sodium chloride, and a salt taste enhance substance itself has a salty taste. Means a substance that enhances the salty taste of salt or salty taste substitutes, which may or may not have.

  “Sour substance” means a sour substitute substance and / or a sour suppressant, and the sour substitute substance is a substance having a taste quality similar to or similar to a sour substance represented by citric acid or acetic acid. Means a substance that suppresses the acidity of a sour substance or a sour substitute substance.

  “Sweet substance” means a sweet taste substitute substance and / or a sweet taste enhancing substance, and the sweet taste substitute substance has a taste equivalent to or similar to a sweet substance represented by sugar, D-amino acid, or artificial sweetener. A substance and a sweetness-enhancing substance means a substance that enhances the sweetness of a sweetening substance or sweetness-substituting substance as described above, which may or may not have sweetness.

  “Umami substance” means an umami substitute substance and / or an umami enhance substance, and the umami substitute substance is a substance having a taste quality equivalent to or similar to an umami substance represented by MSG, etc. Although it may or may not have umami itself, it means a substance that enhances the umami of the umami substance or the umami substitute substance as described above.

  “Bitter substance” means a bitter substance and / or a bitter substance, and the bitter substance is a substance having a taste quality similar to or similar to a bitter substance typified by a bitter peptide or catechin. The substance means a substance that suppresses the bitter taste of the bitter taste substance or the bitter taste substitute substance as described above.

  In addition, the “marker molecule” means a molecule that is specifically expressed in each type of taste bud cell, which makes it possible to easily determine which type of taste bud cell each “taste bud cell” is. For example, Ano1, Kcnk16, Mfsd3, Tmc5, Trpc1, Gpr63, Gpr161, Gpr177, Slc16a7, Slc25a14, Slc35a1, Slc9a7, Sec61a1, Hsd3b7, Mal, Ptch1, Tm4sf1 and Tm4sf1 found by the marker molecule search method of the present invention It is a marker molecule for type I taste bud cells that are thought to recognize salty taste.

  In S-KO mice, taste cells recognizing sweetness, umami, and bitterness disappear, but cells recognizing sourness and saltiness remain, so that sweetness, umami, and bitterness cannot be recognized, while acidity and saltiness can be perceived. Focusing on this characteristic, those that do not change the taste with water by behavioral analysis such as the 2-bottle selection test and licking test using S-KO mice are sweet, umami, bitter, or tasteless materials If it is, the taste quality of a test substance can be judged largely. Furthermore, by combining behavioral analysis using WT mice, whether the test substance shows sweetness or umami or bitterness by observing whether the repellent or palatability behavior is caused by ingestion of the test substance. It is also possible to determine whether or not On the other hand, since a sour substance has a low pH, it can be determined whether or not the material exhibits acidity by measuring the pH of the material. In this way, combining the behavioral analysis of S-KO mice and WT mice makes it possible to classify the taste quality of the test substance without actually performing sensory evaluation. It can be a very useful tool for evaluating the taste quality of foods and natural products.

Moreover, as a problem when searching for a salty taste substitute or an enhanced material by sensory evaluation, the effect of the material cannot be accurately judged due to the taste of the evaluation substance itself. In particular, sweetness and umami generally tend to mask the taste. In addition, since KCl and MgCl ₂ that are currently used as salty substitutes have a bitter and astringent taste, not only salty taste receptors but also bitter taste receptors are included in the materials that can be originally salty taste substitutes. There may be many things with the property to activate. From this point of view, screening for salty taste substitutes or salty taste enhancing substances using laboratory animals that cannot sense these taste qualities is very useful. Since it is known that the preference for the salty taste of mice varies depending on the concentration (preference at a low concentration, avoidance at a high concentration), a material showing a similar behavior pattern can be a candidate for a salty substitute. As described above, since S-KO mice cannot perceive sweetness, umami, and bitterness, it is necessary to consider the influence of the sweetness, umami, and bitterness on repellent and palatability of the material. Absent. Based on the above, a method for ingesting a test substance into S-KO mice and analyzing the behavior was not able to be found by a method for searching for candidate substances for salty taste substitutes or salty taste enhancing substances using WT mice. This is a new screening method that makes it possible to find materials.

  The salty taste receptor candidate molecule specified in the present invention is useful for screening for a salty taste substitute substance or a salty taste enhancing substance. For example, if a salty taste substitute or salty taste enhancing substance is allowed to act on cultured cells in which at least one of these genes (foreign gene) has been introduced into the cell, and a cell response similar to NaCl stimulation is observed, the salty taste substitute or salty taste It is considered as a candidate substance for the enhancement substance. Examples of the salty taste enhancing substance include a substance having an effect of feeling a salty taste that could not be felt by coexisting with a salty substance having a threshold value or less, or a substance having an effect of inducing a strong salty taste when added. Refers to that.

  Functional analysis in an in vitro system using cultured cells, amphibian oocytes, etc. includes, for example, patch clamp method, measurement of total cell current, radiolabeled ion flux assay, and membrane potential sensitive dyes (eg, Vestergard-Bogind et al. J. Membrane Biol. 88: 67-75 (1988); Daniel et al., J. Pharmacol. Meth. 25: 185-193 (1991); Hoevinsky et al., J. Membrane Biol. 137: 59-70 (1994). ) And an ion-sensitive dye. For example, a molecule encoding an ion channel can be expressed in Xenopus oocytes, and then the activity of the channel can be evaluated by measuring changes in membrane current due to test substance stimulation. One means for obtaining an electrophysiological measurement value may be a method of measuring current using a patch clamp method, such as a “cell attach” method, an “inside out” method, and a “whole cell” method (for example, Ackerman et al., New Engl. J. Med. 336: 1575-1595, 1997). Total cell currents are described in Hamil et al., Pflugers. Archiv. 391: 185 (1981).

  In addition to the electrophysiological experimental methods described above, screening for salty taste substitutes or salty taste enhancing substances using cultured cells into which a salty taste receptor candidate molecule such as Ano1 has been introduced is not limited to functional analysis using membrane potential sensitive dyes. It is considered feasible. After loading a membrane potential sensitive dye on cultured cells expressing a salty taste receptor candidate molecule, if a salty taste substitute or salty taste enhancing substance is allowed to act, if the effect is equal to or greater than that caused by NaCl stimulation, then the material It is strongly expected to be used as a salty taste substitute or salty taste enhancer. In one embodiment, the dye may be used at a final concentration of about 2 μM to about 5 μM. In addition, optimal dye loading times are performed at room temperature to 37 ° C. in the range of about 30 to about 60 minutes for most cells. A preferred embodiment is a method using Molecular Devices (Catalog No. R8034) as a membrane potential sensitive dye. In other embodiments, suitable dyes include single wavelength dyes such as DiBAC, DiSBAC (Molecular Devices), and Di-4-ANEPPS (Biotium), or DiSBAC2, DiSBAC3, and CC-2-DMPE (Aurora Dual wavelength FRET dyes such as Biosciences) are included. [Chemical name: Di-4-ANEPPS (pyridinium, 4- (2- (6- (dibutylamino) -2-naphthalenyl) ethenyl) -1- (3-sulfopropyl)-, hydroxide, internal salt), DiSBAC4 (2) (bis- (1,2-dibarbituric acid) -trimethine oxanol), DiSBAC4 (3) (bis- (1,3-dibarbituric acid) trimethine oxanol), CC-2-DMPE (Pacific Blue ™ 1,2-ditetradecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt) and SBFI-AM (1,3-benzenedicarboxylic acid, 4,4 ′-[1,4, 10-trioxa-7,13-diazacyclopentadecane-7,13-diylbis (5-methoxy-6,12-benzofurandyl) )] Bis-, tetra-kis [(acetyloxy) methyl] ester].

  In one embodiment, receptor-expressing cells loaded with a dye are stimulated with a test compound (or control) and changes in membrane potential are measured using a fluorescence analyzer such as VIPR or FLIPR®. When NaCl or a receptor activator (that is, a salty taste substitute) activates a receptor expressed in cultured cells, a change in membrane potential is induced. (For example, FLIPR) or a potential intensity plate reader (for example, VIPR). Thus, by observing the response to a test substance stimulus for cultured cells expressing a salty taste receptor candidate molecule such as Ano1, it becomes possible to screen for a salty taste substitute or a salty taste-regulating compound. For example, when a test compound is allowed to act together with NaCl, a salty blocker can be a salty taste blocker when the fluorescence intensity is reduced compared to a salt compound alone, whereas a salty taste enhancing substance can be obtained when the fluorescence is increased.

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1: Preparation of Skn-1a / i gene homo-deficient mice (S-KO mice; Skn-1a / i-/-) Chimeric mice having cells deficient in Skn-1a / i gene by the following procedure ( S-KO mice) were prepared.

(1) Production of chimeric mice
In order to delete the Skn-1a / i gene in ES cells derived from 129SvEv mice, a targeting vector was first prepared. An approximately 4 kbp region including exon 7 to exon 9 of the mouse Skn-1a / i gene was removed, and a construct for introducing a neomycin resistance gene was prepared instead. In other words, a plasmid vector in which BAC DNA (RP22-526M22) containing Skn-1a / i gene is used and ligated to 5 ′ side 2 kbp of exon 7, TK-neo gene and 3 ′ side 6 kbp of exon 9 Was made. A diphtheria toxin gene was introduced on the 5 ′ side of the targeting vector (FIG. 6). This targeting vector was introduced into ES cells by electroporation. After ES cells were selected with G418 (neomycin selection), ES cells that had undergone homologous recombination were selected by Southern blot analysis. Chimeric mice were prepared by introducing positive clones (ES cells) with homologous recombination into blastocysts and transplanting them into temporary parents.

(2) Production of hetero-deficient (Skn-1a / i +/-) mice F1 mice were produced by crossing the chimeric mice obtained in (1) above with C57BL6 strain mice. By analyzing the genotype of F1 mice by PCR, introduction of mutations into the germ line was confirmed, and hetero-deficient mice (Skn-1a / i +/− mice) were produced.

(3) Preparation of Skn-1a / i gene homo-deficient mice
Skn-1a / i +/− F1 mice were crossed to produce Skn-1a / i gene homo-deficient mice (S-KO mice). The genotype of the obtained F2 mice almost followed Mendelian rules. Analysis of mouse genotypes was performed by PCR using DNA extracted from the tail. For detection of a wild type gene, a combination of primer1 (5'-CATGCTGAGATTTGTCCCAAGCTG-3 '; SEQ ID NO: 1) and primer2 (5'-ATCTGCGCACAGCCTTGTATCTTC-3'; SEQ ID NO: 2) is used. PCR was performed using the combination of primer3 (5'-ACAAGATGGATTGCACGCAGGTTC-3 '; SEQ ID NO: 3) and primer4 (5'-GTCCAGATCATCCTGATCGACAAG-3'; SEQ ID NO: 4) using the genomic DNA as a template. However, 0.47 kbp of DNA was amplified in each mutant. The PCR reaction was performed by repeating a cycle of 94 ° C. for 20 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 45 seconds 30 times. The obtained DNA product was detected by staining with ethidium bromide after electrophoresis on a 1% agarose gel prepared with TAE buffer.

Example 2: Functional analysis of S-KO mice
Males of wild-type C57BL / 6J mice (purchased from Saitama Experimental Animal Supply Station) were used for the circumpapillary slices used for ISH. S-KO mice used 6-week-old males that were self-bred by crossing homo-deficient mice. The boxed papillae were removed from fresh mouse tongues, embedded in cryomold 1 (Sakura) with OCT compound (Sakura), and frozen with liquid nitrogen. The frozen block was sliced into 7 μm using a cryostat HM500-OM (Leica) and attached to MAS-coated slide glass (Matsunami Glass). The prepared sections were stored at -80 ° C.
The cDNA fragment derived from the mouse gene was obtained by RT-PCR using total RNA of the circumscribed papillary epithelium, brain and testis. The cloned gene names and the primer sequences used for acquisition are shown in Table 3. The amplified cDNA fragment was cloned into pBlueScript II SK-vector (Stratagene), and the nucleotide sequence was confirmed using an automatic DNA sequencer 310A (Applied Biosystems).

The probe used in ISH is an antisense RNA probe labeled with digoxigenin or fluorescein, the former using DIG RNA Labeling Mix (Roche Diagnostics), the latter using Fluorescein RNA Labeling Mix (Roche Diagnostics), and T3 or T7 RNA polymerase (Stratagene) was subjected to in vitro transcription and synthesized. If the probe length exceeds 1000 bases, perform fragmentation at 60 ° C in a sizing solution (42 mM NaHCO ₃ , 63 mM Na ₂ CO ₃ , 5 mM dithiothreitol (DTT)) to a length of about 500 bases. It was.
Fresh frozen sections of mouse circumpapillary nipples were fixed in a 4% paraformaldehyde (PFA) / PBS solution at room temperature for 10 minutes, then treated with 0.1% diethyl pyrocarbonate (DEPC) / PBS for 15 minutes each to 5x SSC. Replaced buffer. Thereafter, prehybridization was performed at 58 ° C. for 2 hours with a prehybridization solution (40 μg / ml salmon sperm DNA, 5 × SSC, 50% formamide solution). Hybridization was performed using a hybridization solution (5 × SSC, 50% formamide solution, 5 × Denhardt's solution, 500 μg / ml Salmon sperm DNA, 250 μg / ml yeast tRNA, 1 mM DTT, 20-200 ng / ml antisense RNA probe) was placed on the section and incubated at 58 ° C. or 65 ° C. for 2 nights. After hybridization, each was kept at 58 ° C. or 65 ° C., 5 × SSC was washed twice for 5 minutes, and 0.2 × SSC was washed twice for 30 minutes. After further washing with 0.2 × SSC at room temperature for 5 minutes, the buffer was replaced with TBS. Blocking was performed with a blocking solution (0.5% blocking reagent (Roche Diagnostics) / TBS) for 1 hour.

Alkaline phosphatase-conjugated goat anti-digoxigenin antibody Fab fragment (AP-anti-DIG Ab, Roche Diagnostics) was diluted 1000 times with a blocking solution, and antigen-antibody reaction was performed at room temperature for 1 hour. Washing with TBS for 15 minutes was performed 3 times at room temperature, and the buffer was replaced with alkaline phosphatase buffer (100 mM Tris / HCl (pH 9.5), 50 mM MgCl ₂ , 100 mM NaCl). Color was developed by reacting with a substrate solution (200 μg / ml NBT, 50 μg / ml BCIP / alkaline phosphatase buffer) at room temperature. The reaction time was 3 to 16 hours, depending on the probe used.

  The result of ISH is shown in FIG.

c) Two-bottle selection test taste solution was prepared by dissolving the taste substance in deionized water and then autoclaved. In addition, each WT mouse and S-KO mouse were individually bred, and the food was fed with solid feed (Lab MR Breeder, Saitama Experimental Animal Supply Station) and used for the test.

  Each mouse was presented with two 15 ml drinking bottles, one with autoclaved deionized water and the other with a taste solution and allowed to freely ingest for 48 hours. To remove the bias due to position, the positions of the two drinking bottles were changed after 24 hours. After 48 hours, the amount of deionized water and the amount of taste solution consumed were measured, and the preference ratio of the taste solution was determined as the ratio of the taste solution to the total amount of water consumed.

(preference ratio) = (tastant solution consumed) / (total liquid consumed)
When this value is 0.5, water and palatability do not change, and the higher the taste, the lower the taste.

  Student's t-test was performed between the WT and S-KO groups for the obtained preference ratio value to examine whether the difference was statistically significant.

  The results are shown in FIG.

d) Neural response analysis Under Nembutal anesthesia, nerve fibers were exposed, platinum electrodes were connected, and nerve potential changes were measured when taste stimulation was performed on the tongue. Data acquisition and analysis were performed using an electrophysiological analysis system clampex (Molecular Devices). For taste stimulation, sucrose (300 mM), sodium glutamate (MSG 30 mM (+ IMP 0.5 mM)), denatonium (Denatonium, 1 mM), citric acid (Citric acid, 30 mM), NaCl (100 mM) were used.

Responses were measured for each of the WT and S-KO mice for the five taste qualities of sweet, umami, sour, bitter and salty. A taste solution prepared by dissolving a taste substance in deionized water and then sterilized by autoclave was used. Each taste stimulation was performed 3 times, and the average was calculated as a rough response value. A 300 mM NH ₄ Cl aqueous solution was used as a control, and the response intensity obtained by dividing the crude response value of each taste solution by the control response value was used. With respect to the obtained values, Student's t-test was performed between each group of WT mice and S-KO mice for each taste solution to examine whether the difference was statistically significant.

  The results are shown in FIG.

Example 3: DNA microarray analysis
DNA microarray experiments were performed using wild-type C57BL / 6J mice (purchased from Saitama Experimental Animal Supply Station) (WT) and 5 S-KO mice each. The dissected papillary epithelial tissue and non-papillary epithelial tissue of WT mice and the circumscribed papillary epithelial tissue of S-KO mice were removed. Using 10 ng of total RNA, biotinylated cRNA was synthesized using Two-Cycle Target Labeling and Control Reagents Kit (Affymetrix). The cRNA was fragmented with an alkaline solution and hybridized with a DNA microarray (Mouse Genome 430 2.0, Affymetrix) at 45 ° C. for 16 hours according to Expression Analysis Technical Manual (Affymetrix). Washing, phycoerythrin labeling, and reading of the fluorescence signal were performed using the Affymetrix Gene Chip System. The read data was analyzed with Gene Chip Operation Software (Affymetrix). Statistical processing of data was performed using statistical analysis software R. Table 1 lists the genes identified by the following analysis.

First, genes were identified and classified into taste-specific genes that satisfy the following criteria.
Robust multichip algorithm (RMA) normalized data is used Wild-type crypt papillary epithelium vs. tongue epithelial cell expression rate> 5 times Wild-type crypt papillary epithelium vs. tongue epithelial cell expression rate FDR value <0.1
Crude expression value of circumvallate papillary epithelial cells> 100
Next, genes were identified as candidates for genes that are not significantly different in gene expression levels between S-KO mice and WT mice, ie, specific genes for type I taste bud cells, using the specific inclusion criteria described below.

Inclusion criteria:
Using RMA normalized data Wild type circumvallate papillary epithelium vs. skn-1 KO envelopment papillary epithelium 0.7 to 1.5 times Wild type circumvallate papillary epithelium vs. skn-1 KO circumcision papillary epithelium FDR value> 0.1
395 genes (exactly probe sets) identified

  The genes were then identified as those encoding proteins with transmembrane domains using the specific incorporation criteria described below.

62 probe sets (53 genes) identified as encoding proteins with transmembrane domains or predicted to have multiple transmembrane domains by the transmembrane domain prediction program SOSUI are identified. Except for 19 genes described in non-patent papers, 34 genes shown in Table 1 were selected.

Example 4: in situ hydridyzation
The wild-type C57BL / 6J mouse (purchased from the Saitama Experimental Animal Supply Station) (WT) male was used for the circumpapillary slice used for ISH. S-KO mice used were self-bred by crossing homo-deficient mice. The boxed papillae were removed from fresh mouse tongues, embedded in cryomold 1 (Sakura) with OCT compound (Sakura), and frozen with liquid nitrogen. The frozen block was sliced into 7 μm using a cryostat HM500-OM (Leica) and attached to MAS-coated slide glass (Matsunami Glass). The prepared sections were stored at -80 ° C.

  The cDNA fragment derived from the mouse gene was obtained by RT-PCR using total RNA of the circumscribed papillary epithelium, brain and testis. Table 4 shows the names of the cloned genes and the sequences of the primers used for acquisition. The amplified cDNA fragment was cloned into pBlueScript IISK-vector (Stratagene), and the base sequence was confirmed using an automatic DNA sequencer 310A (Applied Biosystems).

The probe used in ISH is an antisense RNA probe labeled with digoxigenin or fluorescein, the former using DIG RNA Labeling Mix (Roche Diagnostics), the latter using Fluorescein RNA Labeling Mix (Roche Diagnostics), and T3 or T7 RNA polymerase (Stratagene) was subjected to in vitro transcription and synthesized. If the probe length exceeds 1000 bases, perform fragmentation in a sizing solution (42 mM NaHCO ₃ , 63 mM Na ₂ CO ₃ , 5 mM dithiothreitol (DTT)) at 60 ° C to a length of about 500 bases. It was.

  Fresh frozen sections of mouse circumpapillary nipples were fixed in a 4% paraformaldehyde (PFA) / PBS solution at room temperature for 10 minutes, then treated with 0.1% diethyl pyrocarbonate (DEPC) / PBS for 15 minutes each to 5x SSC. Replaced buffer. Thereafter, prehybridization was performed at 58 ° C. for 2 hours with a prehybridization solution (40 μg / ml salmon sperm DNA, 5 × SSC, 50% formamide solution). For hybridization, sections of hybridization solution (5 × SSC, 50% formamide solution, 5 × Denhardt's solution, 500 μg / ml salmon sperm DNA, 250 μg / ml yeast tRNA, 1 mM DTT, 20-200 ng / ml antisense RNA probe) Placed on top and performed at 58 ° C or 65 ° C for 2 nights. After hybridization, each was kept at 58 ° C. or 65 ° C., 5 × SSC was washed twice for 5 minutes, and 0.2 × SSC was washed twice for 30 minutes. After further washing with 0.2 × SSC at room temperature for 5 minutes, the buffer was replaced with TBS. Blocking was performed with a blocking solution (0.5% blocking reagent (Roche Diagnostics) / TBS) for 1 hour.

Alkaline phosphatase-conjugated goat anti-digoxigenin antibody Fab fragment (AP-anti-DIG Ab, Roche Diagnostics) was diluted 1000 times with a blocking solution, and antigen-antibody reaction was performed at room temperature for 1 hour. Washing with TBS for 15 minutes was performed 3 times at room temperature, and the buffer was replaced with alkaline phosphatase buffer (100 mM Tris / HCl (pH 9.5), 50 mM MgCl ₂ , 100 mM NaCl). Color was developed by reacting with a substrate solution (200 μg / ml NBT, 50 μg / ml BCIP / alkaline phosphatase buffer) at room temperature for 16 hours.

  The result of ISH is shown in FIG.

Example 5: Inhibitor test (licking test)
It is an example of a licking test (brief access test: palatability evaluation test by ingesting a tasting substance for a short time) in which the influence of various channel inhibitors on the taste of mice is investigated by a behavioral method. WT mice (n = 10, male) were used. Mice were individually raised, and the food was freely fed solid feed (Lab MR Breeder, Saitama Experimental Animal Supply Station). The taste solution of each concentration was used after sterilization by autoclaving. DIDS (diisothiocyanostilbene disulfonic acid; SIGMA), NFA (niflumic acid; SIGMA): inhibitors of Cl ion channel, amiloride (amylolide; SIGMA): an inhibitor of ENaC. The inhibitor was dissolved in dimethyl sulfoxide (DMSO; Kanto Chemical) and added to the taste solution to a final concentration of 500 μM immediately before use, and it was confirmed that the inhibitor and DMSO alone were not repelled.

  Each taste solution was presented to a mouse that had been water-absorbed for 24 hours in advance, and the number of times of licking in 5 seconds was measured using an optical licometer (a device that optically detects the behavior of the Coubourn Instruments mouse licking the solution).

  The preference ratio of the taste solution was determined as the ratio of the number of times the taste solution was licked to the number of times the water was licked. When this value is 1, the taste does not change with water, and the lower the value, the dislike the taste solution.

(Preference rate) = (Number of times of licking taste solution) / (Number of times of licking water)
Student's t-test was performed on the obtained preference rate value, and the statistical significance of the difference due to the presence or absence of the inhibitor was verified. The results are shown in FIG.

Claims (24)

Skn-1a / i knockout mice are ingested with candidate substances of taste substances, and the behavior analysis of the mice is performed, wherein the taste substances are selected from the group consisting of salty substances, sweet substances, umami substances, bitter substances and sour substances A method for screening a tastant, which is selected. The screening method according to claim 1, wherein the taste substance is a salty substance. The screening method according to claim 1, wherein the taste substance is a sweet substance or an umami substance. The screening method according to claim 1, wherein the taste substance is a bitter substance. The screening method according to claim 1, wherein the taste substance is a sour substance. The screening method according to claim 1, wherein a wild-type mouse is further ingested with a candidate substance of a taste substance together with the Skn-1a / i knockout mouse, and behavioral analysis of both mice is performed. Skn-1a / i knockout mouse use for screening taste substances, wherein the taste substances are selected from the group consisting of salty substances, sweet substances, umami substances, bitter substances and sour substances Use for. The use for screening according to claim 7, wherein the taste substance is a salty substance. The use for screening according to claim 7, wherein the taste substance is a sweet substance or an umami substance. The use for screening according to claim 7, wherein the taste substance is a bitter substance. The use for screening according to claim 7, wherein the taste substance is a sour substance. A method of classifying a test substance into salty taste, sweet taste or umami taste, bitter taste, or sour taste, wherein a Skn-1a / i knockout mouse and, if necessary, a wild type mouse ingest the test substance, A method comprising performing behavior analysis on a mouse. A method for searching marker molecules of taste bud cells using Skn-1a / i knockout mice. 14. It is determined whether the miso cell is a type I miso cell recognizing salty taste, a type II miso cell recognizing sweet, umami, or bitter taste, or a type III miso cell recognizing acidity. A method for searching for a marker molecule described in 1. A comparative analysis of genes expressed in the circumpapillary epithelial cells of Skn-1a / i knockout mice and wild-type mice, and it is determined that the marker molecule expressed only in wild-type mice is a marker molecule of type II taste bud cells Item 15. A method for searching for a marker molecule according to Item 13 or 14. Comparing the amount of gene expressed in the circumscribed papillary epithelial cells of Skn-1a / i knockout mice and wild type mice, comparing the amount of expressed genes in the circumscribed papillary epithelial tissue and non-papillary epithelial tissue. The method for searching for a marker molecule according to claim 13 or 14, wherein the marker molecule is determined to be a marker molecule of type 12 taste bud cells or type III taste bud cells. (i) Gene extraction process that does not significantly change the expression level in circumscribed papillary epithelial cells of Skn-1a / i knockout mice and wild type mice
(ii) Extraction process of the gene specifically expressed in the circumvallate nipple or both the circumvallate nipple and epithelium papillary epithelium
(iii) The method for searching a marker molecule of type I taste bud cells according to claim 16, comprising a step of extracting a gene capable of confirming expression in miso by in situ hybridization (ISH). (i) Extraction process of gene with high expression level in circumscribed papillary epithelial cells of Skn-1a / i knockout mice and wild type mice
(ii) Extraction process of the gene specifically expressed in the circumvallate nipple or both the circumvallate nipple and epithelium papillary epithelium
(iii) The method for searching a marker molecule of a type III taste bud cell according to claim 16, comprising a step of extracting a gene capable of confirming expression in miso by in situ hybridization (ISH). The method for searching for a marker molecule according to any one of claims 13 to 18, wherein a gene having a plurality of transmembrane regions is selected. Ano1, Kcnk16, Mfsd3, Tmc5, Trpc1, Gpr63, Gpr161, Gpr177, Slc16a7, Slc25a14, Slc35a1, Slc9a7, Sec61a1, Hsd3b7, Mal, Ptch1, Tm4sf1 Use as. Salty taste receptor of at least one gene selected from Ano1, Kcnk16, Mfsd3, Tmc5, Trpc1, Gpr63, Gpr161, Gpr177, Slc16a7, Slc25a14, Slc35a1, Slc9a7, Sec61a1, Hsd3b7, Mal, Ptch1, Tm4sf1 Use for alternative or potentiator screening. At least one foreign expression gene selected from Ano1, Kcnk16, Mfsd3, Tmc5, Trpc1, Gpr63, Gpr161, Gpr177, Slc16a7, Slc25a14, Slc35a1, Slc9a7, Sec61a1, Hsd3b7, Mal, Ptch1, and Tm4sf1 for salty substance screening Cultured cells or amphibian oocytes. A method for screening a salty taste substitute or salty taste enhancing substance by observing a cellular response by causing a salty taste substitute or salty taste enhancing substance to act on the cultured cells or amphibian oocytes according to claim 22. The cell response of the cell salty taste substitute or salty taste enhancing substance according to claim 22 is monitored using a patch clamp method, measurement of total cell current, radiolabeled ion flux assay, membrane potential sensitive dye and ion concentration sensitive dye. The screening method according to claim 21.

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