Cells derived from insects of the family Arctiidae

Abstract

To provide cells having excellent refrigeration resistance that can be used in a chemical substance sensor.SOLUTION: The present invention provides cells derived from an insect of Arctiidae that include an exogenous polynucleotide including a coding sequence of a sensor protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to cells derived from insects of the family Arctiidae. [Background technology]

[0002] Because groups of odor molecules that characterize specific human diseases and mental states have been identified and have high value as diagnostic markers, the development of various odor sensors targeting these molecules is thriving. Since biological olfactory receptors possess superior characteristics in terms of diversity, sensitivity, and selectivity that are not found in conventional odor sensor elements such as semiconductors, the development of new odor sensors using olfactory receptors as sensor elements is highly anticipated.

[0003] Patent Document 1 discloses the use of cells expressing modified olfactory receptors and lipid bilayers equipped with modified olfactory receptors as odor sensors. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2022 / 024902 [Overview of the Initiative] [Problems that the invention aims to solve]

[0005] Odor sensors, which involve the artificial preparation of lipid bilayers containing sensor proteins such as olfactory receptors, do not always have sufficient manufacturing efficiency, and there is a need for further improvements in the manufacturing efficiency of odor sensors. Therefore, we focused on utilizing cells that express sensor proteins.

[0006] When using cells as chemical substance sensors such as odor sensors, from the perspective of ease of use, it is preferable to prepare cells in advance in containers or other containers and use them when needed, rather than culturing and preparing the cells each time. In the latter case, the cells need to be able to be stored, and refrigeration tolerance is particularly important from the perspective of not letting the cells dry out.

[0007] The objective of this disclosure is to provide cells with excellent refrigeration resistance that can be used in chemical substance sensors. [Means for solving the problem]

[0008] In view of the above problems, the inventors diligently conducted research and found that cells derived from Arctiidae insects have excellent refrigeration resistance and can be used as chemical substance sensors. Based on this finding, the inventors furthered their research and completed the invention of this disclosure. That is, this disclosure includes the following aspects.

[0009] Item 1. Cells derived from Arctiidae insects containing exogenous polynucleotides that include the coding sequence of a sensor protein.

[0010] Item 2. The cell described in Item 1, wherein the sensor protein is an olfactory receptor protein.

[0011] Item 3. The cell according to item 1 or 2, wherein the sensor protein is an insect olfactory receptor protein.

[0012] Item 4. The cell according to any one of items 1 to 3, wherein the exogenous polynucleotide comprises a coding sequence for an olfactory receptor coreceptor protein and a coding sequence for a chromogenic or luminescent protein.

[0013] Item 5. The cell according to any one of items 1 to 4, wherein the exogenous polynucleotide contains a coding sequence for a drug resistance gene.

[0014] Item 6. The cell according to any one of Items 1 to 5, wherein the exogenous polynucleotide is integrated into the genomic DNA.

[0015] Item 7. The cell according to any one of Items 1 to 6, wherein the insect of the family Erebidae is an insect of the genus Arctia.

[0016] Item 8. The cell according to Item 7, wherein the insect of the genus Arctia is Arctia caja.

[0017] Item 9. A cell having refrigeration tolerance, comprising an exogenous polynucleotide comprising a sensor protein coding sequence.

[0018] Item 10. The cell according to any one of Items 1 to 9, which is determined to have refrigeration tolerance when the following refrigeration tolerance test is performed: <Refrigeration Tolerance Test> Step (1): Cells are seeded on a plate with compartments at 1×10^5 cells / 200 μL / compartment, and allowed to stand at 27°C for 6 hours. Step (2): The plate is shielded from light with aluminum foil, allowed to stand at 4°C for 72 hours, and then the fluorescence intensity is measured. Step (3): After adding a substance that the sensor protein responds to to each compartment, the fluorescence intensity is measured. Step (4): When the fluorescence intensity measured in Step (3) is greater than the fluorescence intensity measured in Step (2), the cell is determined to have refrigeration tolerance.

[0019] Item 11. The cell according to any one of Items 1 to 10, which has a survival rate of 50% or more after storage at 4°C for 15 days.

[0020] Item 12. A cell chip comprising a compartment containing the cell according to any one of Items 1 to 11.

[0021] Item 13. The cell chip according to Item 12, which is for chemical substance detection. [Effects of the Invention]

[0022] According to this disclosure, it is possible to provide cells with excellent refrigeration resistance that can be used in chemical substance sensors, and cell chips using said cells. [Brief explanation of the drawing]

[0023] [Figure 1] This shows the cell viability measured in Test Example 1. The vertical axis represents cell viability (100% on day 0), and the horizontal axis represents the storage period at 4°C. The cells used are shown in the legend. [Figure 2] This figure shows the chemical response activity of ORA cells measured in Test Example 2. The vertical axis shows the difference in fluorescence intensity before and after the addition of the chemical substance (compound a), and the horizontal axis shows the concentration of the chemical substance. The figure on the left shows the results without refrigeration, and the figure on the right shows the results with refrigeration. [Figure 3] This graph shows the chemical response activity of SpIm cells that stably express olfactory receptors, as measured in Test Example 3. The olfactory receptors expressed by the cells are shown at the top of the graph, and the chemical substances used are shown at the bottom of the graph. The vertical axis shows the difference in fluorescence intensity before and after the addition of the chemical substance, and the horizontal axis shows the concentration of the chemical substance. [Figure 4] This graph shows the chemical response activity of SpIm cells that stably express olfactory receptors, as measured in Test Example 3. The olfactory receptors expressed by the cells are shown at the top of the graph, and the chemical substances used are shown at the bottom of the graph. The vertical axis shows the difference in fluorescence intensity before and after the addition of the chemical substance, and the horizontal axis shows the concentration of the chemical substance. [Modes for carrying out the invention]

[0024] In this specification, the terms “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consist solely of.”

[0025] This disclosure relates, in one aspect, to cells derived from Arctiidae insects (which may be referred to herein as "cells of this disclosure") containing exogenous polynucleotides including a coding sequence for a sensor protein. This is described below.

[0026] Cells derived from insects of the family Arctiidae are primary cultured cells or cell lines of the constituent cells of living organisms derived from insects of the family Arctiidae, and are not particularly limited to that extent.

[0027] Examples of the Arctiinae family include the Arctiinae, Lithosiinae, and Syntominae subfamilies, but among these, the Arctiinae subfamily is preferred. Within the Arctiinae subfamily, the genera Spilosoma, Spilarctia, and Rhagonis are preferred, with the genus Spilosoma being particularly preferred. Within the genus Spilosoma, there are no particular restrictions, but from the viewpoint of cold tolerance, Spilosoma imparilis is particularly preferred.

[0028] Cells derived from Arctiidae insects can be obtained from known biological banks, or they can be obtained by collecting and culturing them from living Arctiidae insects according to or in accordance with known methods, or by culturing them into cell lines as needed.

[0029] Examples of cells derived from the mulberry leaf miner include FFPRI-SpIm-2AM-SF cells (MAFF number: 275052) and FFPRI-SpIm-2AM-IPL411 cells (MAFF number: 275053) from the Agrobiological Resources Gene Bank.

[0030] The cells disclosed herein are derived from insects of the family Arctiidae and contain exogenous polynucleotides that include a coding sequence for a sensor protein.

[0031] Exogenous polynucleotides are polynucleotides that contain base sequences not derived from the genomic DNA (particularly chromosomal genomic DNA) of cells derived from insects of the family Arctiidae, and are not particularly limited to that extent.

[0032] In this specification, polynucleotides include not only typical polynucleotides such as DNA and RNA that are endemic to living organisms, as exemplified below, but also polynucleotides that have undergone known chemical modifications, artificial polynucleotides, etc. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residue of each nucleotide can be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate. In addition, the hydroxyl group at position 2 of the sugar (ribose) of each ribonucleotide may be replaced with -OR (where R represents, for example, CH3(2'-O-Me), CH2CH2OCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, the nucleic acid base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or cationic functional group at position 5 of the pyrimidine base, or by substituting the carbonyl group at position 2 with a thiocarbonyl group. Furthermore, examples include, but are not limited to, those in which the phosphate or hydroxyl portion is modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group. In addition, BNA (LNA), in which the conformation of the sugar portion of the nucleotide is fixed to the N-type by cross-linking the 2' oxygen and 4' carbon atoms of the sugar portion, can also be used.

[0033] The sensor protein can be selected from proteins capable of detecting the presence of a chemical substance, and can be, for example, a receptor protein that uses the chemical substance as a ligand. The sensor protein is particularly preferably an olfactory receptor protein.

[0034] Olfactory receptor proteins are membrane proteins with a seven-transmembrane structure that function as odor sensors in living organisms. From the amino terminus (sometimes referred to as the "N terminus") to the carboxyl terminus (sometimes referred to as the "C terminus") of an olfactory receptor protein, the following are linked together: the N-terminal region (NT), the first transmembrane domain (TM1), the first extracellular loop (EC1), the second transmembrane domain (TM2), the first intracellular loop (IC1), the third transmembrane domain (TM3), the second extracellular loop (EC2), the fourth transmembrane domain (TM4), the second intracellular loop (IC2), the fifth transmembrane domain (TM5), the third extracellular loop (EC3), the sixth transmembrane domain (TM6), the third intracellular loop (IC3), the seventh transmembrane domain (TM7), and the C-terminal region (CT). In this disclosure, each region is determined by structural prediction using TMpred (K. Hofmann, W. Stoffel, TMbase - a database of membrane spanning protein segments, Biol. Chem. Hoppe-Seyler, 374 (1993), p. 166, https: / / embnet.vital-it.ch / software / TMPRED_form.html) (default conditions).

[0035] From the viewpoint of suitability for detecting chemical substances, insect olfactory receptor proteins are particularly preferred. Preferred insects from which insect olfactory receptor proteins are derived include diptera insects such as Culicidae and Drosophilidae; lepidoptera insects such as Bombycidae; hymenoptera insects such as Apsidae; orthoptera insects such as Acrididae; and hemipteran insects such as Corbiculidae. More preferably, these include diptera insects such as Culicidae and Drosophilidae; orthoptera insects such as Acrididae; and hemipteran insects such as Corbiculidae. Examples of insects in the Culicidae family include Anopheles gambiae, Aedes aegypti, and Culex quinquefasciatus. Examples of insects in the Drosophilidae family include Drosophila melanogaster, Drosophila pseudoobscura, and Drosophila virillis. Examples of insects in the Bombycidae family include Bombyx mori, Bombyx mandarina, and Trilocha varians. Examples of insects in the Apidae family include Apis mellifera, Apis florea, Apis dorsata, and Bombus terrestris. Examples of insects in the Acrididae family include the migratory locust (Locusta migratoria), and examples of insects in the Pterididae family include the bed bug (Cimex lectularius).

[0036] Specifically, wild-type insect olfactory receptor proteins include, for example, AaOR1, AaOR2, AaOR4, AaOR5, AaOR6, AaOR7, AaOR8, AaOR9, AaOR10a, AaOR15, AaOR22, AaOR24, AaOR25, AaOR26, AaOR27, AaOR28, AaOR30, AaOR34, AaOR36, AaOR38, AaOR41a, AaOR41b, AaOR42, AaOR43, AaOR44, AaOR47, AaOR49, AaOR50, AaOR52, AaOR54, AaOR58, AaOR59, A aOR60, AaOR61, AaOR64, AaOR65, AaOR66, AaOR67a, AaOR69a, AaOR70, AaOR71, AaOR72a, AaOR73, AaOR74, AaOR75, AaOR77, AaOR78, AaOR79, AaOR81, AaOR 83b, AaOR84, AaOR85, AaOR86, AaOR87, AaOR91, AaOR95, AaOR97, AaOR96, AaOR99, AaOR100, AaOR102, AaOR103, AaOR104a, AaOR105, AaOR107, AaOR108, A aOR109, AaOR110, AaOR112, AaOR114, AaOR116, AaOR117, AaOR118, AaOR122, AaOR125, AaOR128, AgOR1, AgOR2, AgOR3, AgOR4, AgOR5, AgOR6, AgOR7, AgOR 8, AgOR9, AgOR10, AgOR11a, AgOR12a, AgOR12b, AgOR13, AgOR14, AgOR15, AgOR16a, AgOR17, AgOR18, AgOR20, AgOR21, AgOR23, AgOR25, AgOR26, AgOR27, A gOR28, AgOR30, AgOR34, AgOR36, AgOR37, AgOR38, AgOR39a, AgOR40, AgOR42, AgOR44, AgOR45, AgOR46, AgOR47, AgOR49, AgOR50, AgOR54, AgOR56a, AgOR5 7, AgOR60, AgOR61, AgOR62, AgOR63, AgOR64, AgOR65, AgOR69, AgOR70, AgOR71, AgOR72, AgOR74, AgOR75, AgOR76a, AmOR1, AmOR3, AmOR9, AmOR10, AmOR13,AmOR41, AmOR51, AmOR52, AmOR55, AmOR71, AmOR73, AmOR78, AmOR85, AmOR89, AmOR90, AmOR114, AmOR115, AmOR118, AmOR120, AmOR121, AmOR161, BmOR1, BmOR2, BmOR3, BmOR4, BmOR5, B mOR8, BmOR9, BmOR10, BmOR13, BmOR17, BmOR18, BmOR23, BmOR24, BmOR25, BmOR35, BmOR36, BmOR42, BmOR45, BmOR49, BmOR51, BmOR52, BmOR55, BmOR56, BmOR61, DmOR1a, DmOR9a, DmOR1 9a, DmOR22a, DmOR22b, DmOR22c, DmOR24a, DmOR30a, DmOR33a, DmOR33b, DmOR33c, DmOR35a , DmOR42b, DmOR43a, DmOR45a, DmOR45b, DmOR47a, DmOR49b, DmOR59b, DmOR65b, DmOR65c, Dm Examples include OR67b, DmOR67c, DmOR69a, DmOR71a, DmOR74a, DmOR82a, DmOR83a, DmOR83b, DmOR83c, DmOR85a, DmOR85c, DmOR85e, DmOR85f, DmOR88a, DmOR92a, DmOR94a, DmOR94b, DmOR98b, etc.

[0037] In this specification, OR indicates an olfactory receptor, Dm indicates origin from Drosophila melanogaster, Bm indicates origin from Bombyx mori, Ag indicates origin from Anopheles gambiae, and Aa indicates origin from Aedes aegypti. The amino acid sequences and coding sequences of these and other olfactory receptor proteins are publicly known or can be easily identified by sequence identity searches based on known sequences.

[0038] Sensor proteins may contain amino acid mutations from the wild-type amino acid sequence, provided that the chemical response activity is not significantly reduced. "Not significantly reduced" means, for example, that the chemical response activity of the sensor protein containing the amino acid mutation is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, even more preferably 80% or more, and even more preferably 90% or more, compared to the 100% chemical response activity of the wild-type sensor protein.

[0039] Amino acid mutations include, for example, substitutions, insertions, additions, or deletions of amino acids, preferably substitutions, and particularly preferably conservative substitutions.

[0040] In this specification, "conservative substitution" means that an amino acid residue is substituted for an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains, such as lysine, arginine, and histidine, are considered conservative substitutions. Other examples of conservative substitutions include amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains, such as glycine, asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with non-polar side chains, such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with β-branched side chains, such as threonine, valine, and isoleucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, tryptophan, and histidine.

[0041] The sensor protein may contain a wild-type amino acid sequence, or an amino acid sequence having, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 98% or more, and particularly preferably 99% or more identity with respect to the wild-type amino acid sequence.

[0042] In this specification, "identity" of amino acid sequences refers to the degree of agreement between two or more comparable amino acid sequences. Therefore, the higher the agreement between two amino acid sequences, the higher their identity or similarity. The level of amino acid sequence identity can be determined, for example, using the sequence analysis tool FASTA with default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (Karlin S, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87:2264-2268 (1990), Karlin S, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). A program called BLASTX has been developed based on this BLAST algorithm. The specific methods for these analyses are publicly known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ).

[0043] Other amino acid sequences, such as protein tags, fluorescent proteins, luminescent proteins, signal sequences, or other proteins or peptides, may be added to the sensor protein, provided that the chemical response activity is not significantly impaired. Examples of protein tags include biotin, His tags, FLAG tags, Halo tags, MBP tags, HA tags, Myc tags, V5 tags, and PA tags.

[0044] In this specification, chemical response activity refers to the property of a sensor protein to recognize a chemical substance and exhibit signal transduction activity (e.g., ion channel activity) either alone or in conjunction with other proteins. In the case of an olfactory receptor, it refers to the property of an olfactory receptor recognizing a chemical substance and activating the olfactory receptor complex formed by the olfactory receptor and its coreceptor, thereby exhibiting ion channel activity. The chemical response activity of a sensor protein can be measured using the signal transduction activity of the sensor protein in contact with the chemical substance as an indicator (e.g., by quantitatively evaluating the amount of signal molecules). In the case of an olfactory receptor, the chemical response activity of the olfactory receptor can be measured using the ion channel activity of the olfactory receptor complex formed by the olfactory receptor and its coreceptor in contact with the chemical substance as an indicator. For example, a cell expressing a protein that develops color or emits light in response to ions (such as calcium ions) flowing into the cell when (a) an olfactory receptor, (b) an olfactory receptor coreceptor, and (c) an olfactory receptor complex responds is brought into contact with a chemical substance, and the amount of light emitted by the cell is measured. The higher the measured luminescence, the higher the response activity of the chemical substance in the olfactory receptor is determined to be. Specifically, this can be measured according to the method described in Patent Document 1.

[0045] The coding sequence of a sensor protein is not particularly limited, as long as it is a nucleotide sequence that codes for a sensor protein. In one embodiment, an exogenous polynucleotide includes an expression cassette for a sensor protein. The expression cassette is not particularly limited, as long as it is a polynucleotide capable of expressing a sensor protein in a cell. A typical example of a sensor protein expression cassette is a polynucleotide containing a promoter and the coding sequence of a sensor protein placed under the control of the promoter.

[0046] The promoter is not particularly limited and can be selected as appropriate. For example, various pol II-type promoters can be used. Examples of pol II-type promoters are not particularly limited, but include the CMV promoter, EF1 promoter, SV40 promoter, MSCV promoter, and promoters of insect-derived genes.

[0047] When the sensor protein is an insect olfactory receptor, the exogenous polynucleotide preferably contains the coding sequence of an insect olfactory receptor coreceptor, from the viewpoint that the cells of this disclosure can be used directly for chemical detection applications. Although the insect olfactory receptor coreceptor is a membrane protein having a seven-transmembrane structure, similar to the olfactory receptor, it does not recognize odor molecules itself, but functions by forming a heterocomplex with the olfactory receptor. The olfactory receptor complex, which is a heterocomplex composed of the olfactory receptor and the olfactory receptor coreceptor, has ion channel activity that is activated by odor molecules, and when activated, it attracts sodium ions (Na + ), calcium ions (Ca 2+ It causes positive ions such as ) to flow into the cell.

[0048] From the viewpoint that the cells of this disclosure can be used directly for chemical substance detection, it is preferable that the exogenous polynucleotides contain a coding sequence for a protein that develops color or emits light in response to ions (such as calcium ions) that flow into the cell when a sensor protein (particularly an olfactory receptor protein) responds. Examples of such proteins include aequorin, Yellow Cameleon (YC), and GCaMP. Alternatively, it is preferable that the cells of this disclosure contain ion-dependent fluorescent dyes such as calcium ion-dependent fluorescent dyes (e.g., Fura-2, Fluo-3, Fluo-4, etc.).

[0049] From the perspective that the cells of this disclosure can be used directly for chemical substance detection applications, the cells of this disclosure contain a sensor protein, that is, the sensor protein is expressed in the cells of this disclosure. In one embodiment, the sensor protein has a seven-transmembrane structure and is therefore positioned on the cell membrane as a membrane protein.

[0050] The exogenous polynucleotide preferably contains a coding sequence for a drug resistance gene in order to enable drug selection of the cells of this disclosure. The drug resistance gene can be selected from any drug resistance gene that can be used for drug selection of insect cells, and examples include chloramphenicol resistance gene, tetracycline resistance gene, neomycin resistance gene, erythromycin resistance gene, spectinomycin resistance gene, kanamycin resistance gene, hygromycin resistance gene, and puromycin resistance gene.

[0051] The coding sequences for insect olfactory receptor coreceptors, chromogenic or bioluminescent proteins, and drug resistance genes are preferably contained in exogenous polynucleotides in the form of expression cassettes. The structure of the expression cassette is the same as that of the expression cassette for sensor proteins. The promoter of the expression cassette can be shared among multiple coding sequences.

[0052] The exogenous polynucleotide is preferably integrated into genomic DNA (particularly preferably chromosomal genomic DNA). This allows for stable expression of the sensor protein, making it suitable for chemical detection. In this case, the exogenous polynucleotide can be a single continuous region within the genomic DNA, or a combination of two or more continuous regions (for example, a configuration in which the sensor protein coding sequence is contained in continuous region A and the drug resistance gene coding sequence is contained in continuous region B, which is a different continuous region from continuous region A, or a configuration in which the sensor protein coding sequence is contained in both continuous region A and continuous region B).

[0053] In another embodiment, the exogenous polynucleotide may be in a state where it is not integrated into the genomic DNA. In this case, the exogenous polynucleotide may be, for example, in the form of a vector. In this case, the exogenous polynucleotide may be one polynucleotide molecule or two or more polynucleotide molecules (for example, a form in which the sensor protein coding sequence is contained in polynucleotide molecule A and the drug resistance gene coding sequence is contained in polynucleotide molecule B, which is a different molecule from polynucleotide molecule A, or a form in which the sensor protein coding sequence is contained in both polynucleotide molecule A and polynucleotide molecule B).

[0054] In one aspect, this disclosure relates to a refrigeration-tolerant cell (which may also be referred to as "the cell of this disclosure") containing an exogenous polynucleotide including a coding sequence for a sensor protein.

[0055] Refrigeration tolerance can be determined in one embodiment of the present invention based on the following refrigeration tolerance test 1: <Refrigeration Tolerance Test 1> Step (1) Seed cells at a rate of 1 x 10^5 / 200 μL / compartment in a plate with compartments, and leave to stand at 27°C for 6 hours. Step (2) The plate is shielded from light with aluminum foil and left to stand at 4°C for 72 hours, after which the fluorescence intensity is measured. Step (3) After adding a substance to which the sensor protein responds to each compartment, the fluorescence intensity is measured. If the fluorescence intensity measured in step (3) is greater than the fluorescence intensity measured in step (2), the cells are determined to be refrigerated.

[0056] For more detailed conditions of refrigeration tolerance test 1, follow Test Example 2 described below.

[0057] In one aspect of the present invention, the refrigeration tolerance can be defined as a survival rate that is equal to or higher than a certain level after storage at 4°C for a certain period. For example, the refrigeration tolerance can be such that the survival rate after storage at 4°C for 14 days is, for example, 50% or more, preferably 60% or more, more preferably 70% or more, and even more preferably 80% or more. The refrigeration tolerance is measured according to Test Example 1 described later.

[0058] From the viewpoint of being suitable for use in chemical sensors, the cells of the present disclosure are preferably in a form contained in a cell chip. Accordingly, in one aspect, the present disclosure relates to a cell chip (which may be referred to as "the cell chip of the present disclosure" in the present specification) comprising a compartment containing the cells of the present disclosure.

[0059] The compartment of the cell chip of the present disclosure preferably contains cells and a hydrogel. This can prevent drying of the cells.

[0060] The form of the compartment is not particularly limited as long as it is an embodiment capable of retaining cells. From the viewpoints of cell retention property, production efficiency, or chemical substance detectability, the compartment is preferably in a well shape.

[0061] The material of the compartment is not particularly limited as long as it can retain cells. The material can be, for example, a resin, a metal, or the like.

[0062] From the viewpoint of detection sensitivity, the compartment usually contains a plurality of cells. The area (cm in the compartment 2 ) per cell (number) is, for example, 1×10 3 to 1×10 9 cells / cm 2 .

[0063] From the viewpoint of detection sensitivity or the viewpoint of production efficiency, the bottom area of one compartment is preferably 0.5 to 100 mm 2 , more preferably 1 to 30 mm 2 , still more preferably 1.5 to 10 mm 2 .

[0064] The number of compartments contained in the cell chip is preferably 10 to 2000, more preferably 30 to 1000, and even more preferably 50 to 500, from the viewpoint of detection sensitivity or manufacturing efficiency.

[0065] The cells of this disclosure exhibit excellent refrigeration tolerance. In this specification, “refrigeration tolerance” means that cell viability and / or chemical response activity are maintained at a higher level after a certain period of time (e.g., 1 day or more, 1 to 30 days, 2 to 20 days, 3 to 10 days) under refrigerated storage (e.g., 0 to 10°C, 0 to 8°C, 0 to 6°C, 2 to 6°C). Cell viability and chemical response activity can be measured according to or in accordance with the methods of the examples described below.

[0066] The cells disclosed herein exhibit superior chemical response activity using sensor proteins compared to cells derived from other species. Furthermore, these cells are suitable for detecting chemical substances using a variety of sensor proteins.

[0067] The cells and cell chips of this disclosure can be used for the detection of chemical substances (in particular odorants). These chemical substances include, for example, bodily fluids (e.g., urine, blood, saliva), air (e.g., room air, air inside packaging), and water (e.g., river water, seawater, tap water, drinking water, sewage). These can be chemical substances in a sample. In this case, for example, by bringing the cells of this disclosure into contact with the sample, or by adding the sample to a compartment of the cell chip of this disclosure, the chemical substances in the sample can reach the cells and come into contact with the cell's sensor proteins. For example, the chemical substances can be detected by detecting ions flowing into the cell (for example, by detecting them with a protein that changes color or emits light in response to ions). [Examples]

[0068] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0069] Test Example 1. Refrigeration Tolerance Test 1 Spilosoma imparilis-derived cells (SpIm cells), Sf9 cells, and HEK293 cells were seeded in 96-well plates at a rate of 3 x 10^3 cells / well, n=3. The number of cells at seeding was measured by staining the cell suspension with trypan blue and using a countessII FL Automated Cell Counter (Thermo Fisher). The culture media used were as follows: SpIm cells; Sf-900III SFM medium; Sf9 cells; Sf-900III SFM medium; HEK293 cells; Dulbecco's modified Eagle medium (4.5 g / l glucose) (containing L-glutamine and HEPES, but without pyruvate).

[0070] After cell seeding, the 96-well plate was shielded from light with aluminum foil and left to stand in a refrigerator (4°C). From the start of standing, cell viability was measured using the MTT assay method (Roche kit) on days 14, 28, and 35 for SpIm cells, days 1, 3, 7, and 14 for Sf9 cells, and days 1, 2, and 5 for HEK293 cells.

[0071] The MTT assay was performed according to the instructions provided with the kit. 10 μL / well of MTT solution was added, and the samples were incubated at 27°C for 4 hours. Next, 100 μL / well of MTT eluate was added, and the samples were incubated at 37°C overnight. Subsequently, the absorbance at 560 nm was measured using a TECAN infinite M200 pro. Cell viability (%) was calculated by dividing the absorbance of the post-seeding sample by the absorbance of the sample at seeding (day 0).

[0072] The results are shown in Figure 1. SpIm cells were found to have a higher survival rate even when stored under refrigerated conditions for longer periods.

[0073] Test Example 2. Refrigeration Tolerance Test 2 <2-1. Production of stable expression cells> SpIm cells were introduced into a transboson vector in which the olfactory receptor protein (ORA) coding sequence, the olfactory receptor co-receptor coding sequence, the calcium sensor luminescence protein coding sequence, and the puromycin resistance gene coding sequence were positioned under the control of a promoter sequence, and these sequences were located between 5'ITR (reverse repeat sequence) and 3'ITR. Selection was then performed with puromycin to generate SpIm cells (hereinafter referred to as ORA cells) that stably express olfactory receptors, in which the exogenous DNA containing the above coding sequences and promoter sequence was incorporated into the chromosomal genomic DNA.

[0074] <2-2. Measurement of Sensor Function> ORA cells were seeded at 1 x 10^5 / 200 μL / well in a 96-well CORNING3909 plate and incubated at 27°C (without CO2 supply). Sf-900III SFM medium was used. Cell counts were performed by staining the cell suspension with trypan blue and then counting using a countessII FL Automated Cell Counter (Thermo Fisher). 24 hours after seeding, the culture medium was removed from the 96-well plate and replaced with 80 mL of 0.1% BSA HBSS (-) (phenol red-free). Fluorescence intensity (GCaMP) was measured using a FlexStation3 5 minutes after this exchange. Specifically, compound a (a substance to which ORA responds) was added to each well at various concentrations (0, 0.01, 0.1, 1, 10, and 100 μM), and the change in fluorescence intensity before and after addition was quantified using a microplate reader (FlexStation3, Molecular Devices).

[0075] <2-3. Measurement of sensor function after refrigeration> After seeding ORA cells in the same manner as described in 2-2 above, the cells were left to stand in a 27°C incubator (without CO2 supply) for 6 hours. Then, they were covered with aluminum foil to protect them from light and left to stand in a refrigerator (4°C). After 72 hours, the fluorescence intensity before and after the addition of compound a was measured in the same manner as described in 2-2 above.

[0076] <2-4.Results> The results are shown in Figure 2. It was found that SpIm cells can be used as chemical response sensors, and that their sensory function remains stable even after refrigeration.

[0077] Test Example 3. Chemical Response Activity Evaluation Test Using Splm cells, SpIm cells expressing olfactory receptor proteins (ORA, ORB, ORC, ORD, ORE, ORF, AaOR47) were generated in the same manner as in 2-1 above. Chemical response activity was evaluated using each of these cells in the same manner as in 2-2 above.

[0078] The results are shown in Figures 3 and 4. It was found that SpIm cells exhibit chemical response activity even when using a variety of olfactory receptors.

Claims

1. A cell derived from the mulberry tree moth, comprising an exogenous polynucleotide including a coding sequence for an insect olfactory receptor protein, a coding sequence for an insect olfactory receptor coreceptor, and a coding sequence for a protein that develops color or emits light in response to ions flowing into the cell when the insect olfactory receptor protein responds.

2. The cell according to claim 1, wherein the ion is a calcium ion.

3. The cell according to claim 1, wherein the exogenous polynucleotide includes a coding sequence for a drug resistance gene.

4. The cell according to claim 1, wherein the exogenous polynucleotide is incorporated into the genomic DNA.

5. A cell chip comprising a compartment containing cells according to any one of claims 1 to 4.

6. A cell chip according to claim 5, for the detection of chemical substances.

Images