Human lung cell system introduced with fluorescent reporter for evaluating inhalation toxicity

Abstract

The present invention relates to a human lung cell system introduced with a fluorescent reporter for evaluating inhalation toxicity. Using the CRISPR-Cas9 system, a human lung cell line was established in which a fluorescent marker is permanently expressed endogenously at the 3'-terminal portion of the G3BP1 gene, which is one of the representative proteins constituting phase separation phenomena, and after treating the human lung cells with test substances for toxicity evaluation, the formation of phase separation phenomena, specifically stress granules, could be confirmed in real time, thereby providing an effect of simply confirming pulmonary toxicity in real time according to exposure to medicines, chemicals, environmental harmful factors, and biological harmful factors, and thus the present invention can be effectively used for inhalation toxicity evaluation.

Description

Human lung cell system with fluorescent reporter for inhalation toxicity assessment

[0001] The present invention relates to a human lung cell system incorporating a fluorescent reporter for inhalation toxicity assessment that detects phase separation phenomena in real time.

[0002] This research was supported by the 'Development of a real-time lung toxicity assessment method for mixtures containing household chemical products using human lung organoids with fluorescent labels' project (Project No.: 1485019341, RS-2022-KE002021), a project of the Korea Environmental Industry & Technology Institute under (or affiliated with) the Ministry of Environment's household chemical product safety management technology development project.

[0003] Alternative animal testing (AAT) is a technology that replaces laboratory animals to evaluate the efficacy and toxicity of chemicals. It is considered a promising approach to overcome the inconsistencies between animal testing and clinical trials due to species differences. Furthermore, the technology holds significant future potential, with potential applications in regenerative therapeutics, personalized medicine, and disease modeling.

[0004] As interest in laboratory animal welfare grows, particularly in advanced economies, a growing movement is underway within related industries to discourage the use of laboratory animals. Beginning with the UK, at least 23 countries around the world have enacted laboratory animal protection laws. Europe has banned the sale of cosmetics tested on animals within the EU since 2013. In the US, state governments like New York and California are regulating the sale of cosmetics tested on animals.

[0005] As ethical issues surrounding laboratory animals have emerged, the basic principles of animal testing, considered international ethical standards, have been established. The "3Rs" principle for animal testing refers to 1) replacing animal testing with non-animal testing as much as possible (Replacement), 2) reducing the number of animals used in animal testing (Reduction), and 3) minimizing animal suffering as much as possible during animal testing (Refinement). Among these, the need for test methods that can replace laboratory animals (Replacement) is growing, and the development and revision of related test method guidelines is on the rise.

[0006] Meanwhile, with the increase in patients with respiratory infections such as the coronavirus, the use of disinfectants has increased, leading to more frequent exposure to chemicals in daily life. This has highlighted the importance of assessing human lung toxicity following exposure to various chemicals in daily life. However, while existing in vitro toxicity assessment methods include measuring alveolar viability and ROS measurement following chemical exposure, no method has been reported for real-time monitoring of lung cell toxicity following short-term exposure. Therefore, it is necessary to discover new acute toxicity markers following chemical exposure and develop new technologies to monitor them.

[0007] Meanwhile, Korean Patent No. 1455244 discloses a quantitative cytotoxicity evaluation system and method using an AFM and a probe-type sensor, and Korean Patent No. 2466092 discloses a human liver chimeric non-human animal having a defective P450 oxidoreductase and a method for using the same. However, there is no disclosure at all regarding a human lung cell system with a fluorescent reporter for inhalation toxicity evaluation that detects the phase separation phenomenon of the present invention in real time.

[0008] The present invention was derived from the above-mentioned needs, and provides a human lung cell system in which a fluorescent marker is introduced at the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system for inhalation toxicity evaluation, and completes the present invention by confirming the real-time phase separation phenomenon after treating the human lung cells with a test substance for toxicity evaluation (a test substance causing toxicity).

[0009] To solve the above problem, the present invention provides a human lung cell line into which a fluorescent reporter for inhalation toxicity evaluation is introduced, which detects the phase separation phenomenon in real time.

[0010] In addition, the present invention provides a composition for evaluating inhalation toxicity comprising the human lung cell line.

[0011] In addition, the present invention provides a composition for screening for inhalation toxicity-inducing substances comprising the human lung cell line.

[0012] In addition, the present invention provides a method for producing a human lung cell line having a fluorescent reporter for inhalation toxicity evaluation, comprising the step of introducing a fluorescent label to the 3'-end of a G3BP1 gene using a CRISPR-Cas9 system.

[0013] In addition, the present invention

[0014] 1) A step of treating the human lung cell line with a test substance for toxicity evaluation; and

[0015] 2) A method for screening inhalation toxic substances is provided, including a step of confirming a real-time phase separation phenomenon after the above step 1).

[0016] The present invention relates to a human lung cell system into which a fluorescent reporter for inhalation toxicity evaluation has been introduced, and a human lung cell line that permanently and endogenously expresses a fluorescent marker at the 3'-terminal portion of the G3BP1 gene, which is one of the representative proteins constituting the phase separation phenomenon, is established using the CRISPR-Cas9 system, and after treating the human lung cells with a test substance for toxicity evaluation, the phase separation phenomenon, specifically, the formation of stress granules, is confirmed in real time, thereby having the effect of easily confirming in real time lung toxicity due to exposure to pharmaceuticals, chemicals, environmental toxic factors, and biological toxic factors.

[0017] Figure 1 is a schematic diagram of a CRISPR / Cas9-based gene insertion (knock-in) system for introducing a fluorescent marker into a phase separation marker gene (G3BP1).

[0018] Figure 2 shows the results of confirming the expression of fluorescent protein (EGFP) and G3BP1 protein in a human lung cell line (KI#1) into which a specific fluorescent marker for the phase separation marker gene (G3BP1) of the present invention has been introduced, and confirming the signal transduction pathway related to stress granule formation. Oxidative stress was induced by treatment with sodium arsenite (NaAsO2, Sodium arsenite, Ars.). WT is the A549 wild-type cell group.

[0019] Figure 3 shows the results of confirming the expression of the phase separation marker (G3BP1) and the expression of the fluorescent marker according to sodium arsenite (Ars.) treatment in a human lung cell line (KI#1) into which the phase separation marker gene (G3BP1) specific fluorescent marker of the present invention has been introduced. WT is the A549 wild-type cell group.

[0020] Figure 4 shows the results of confirming the expression of fluorescent protein (GFP) and G3BP1 protein according to treatment with bactericidal disinfectant components (BAC, BIT, DCB) in a human lung cell line (KI#1) into which a specific fluorescent marker for the phase separation marker gene (G3BP1) of the present invention has been introduced, and confirming the signal transduction pathway related to stress granule formation. BAC is benzalkonium chloride, BIT is benzisothiazolinone, and DCB is 1,4-dichlorobenzene. WT is the A549 wild-type cell group.

[0021] Figure 5 shows the results of confirming the expression of the endogenous phase separation marker (G3BP1) and the expression of the fluorescent marker according to the treatment with bactericidal disinfectant components (BIT, DCB, BAC) in a human lung cell line (KI#1) into which a specific fluorescent marker of the phase separation marker gene (G3BP1) of the present invention has been introduced, through fluorescence imaging analysis. Cont. is a control group in which the lung cell line established in the present invention was not treated with the bactericidal disinfectant components. BIT is benzisothiazolinone, DCB is 1,4-dichlorobenzene, and BAC is benzalkonium chloride. WT is the A549 wild-type cell group.

[0022] Figure 6 is a live cell image photograph confirming the phase separation phenomenon (stress granule formation) of a fluorescent marker according to treatment with a bactericidal disinfectant component (BIT) in a human lung cell line (KI#1) into which a specific fluorescent marker of the phase separation marker gene (G3BP1) of the present invention has been introduced (A), and the result confirming the degree of formation of stress granules (SGs) according to BIT treatment over time (B). BIT is benzisothiazolinone.

[0023] In order to achieve the purpose of the present invention, the present invention provides a human lung cell line into which a fluorescent reporter for inhalation toxicity evaluation is introduced, which detects a phase separation phenomenon in real time.

[0024] The term 'phase separation phenomenon' of the present invention refers to a process in which macromolecules within a cell are separated into a dense phase and a relatively dilute phase by stimulation, and the phase separation phenomenon of the present invention is preferably characterized by the formation of stress granules.

[0025] The fluorescent reporter for the above inhalation toxicity evaluation may be any protein that expresses fluorescence, and is preferably one selected from the group consisting of firefly luciferase, renilla luciferase, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), and DsRed, but is not limited thereto.

[0026] In one embodiment of the present invention, the human lung cell line into which the fluorescent reporter for inhalation toxicity evaluation is introduced may be one in which a fluorescent label is introduced into the G3BP1 gene using the CRISPR-Cas9 system, preferably one in which a fluorescent label is introduced immediately before the stop codon at the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system, and more preferably one in which a fluorescent label is introduced into the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system, including firefly luciferase, renilla luciferase, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), Any one fluorescent marker selected from the group consisting of yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), and DsRed is introduced, but is not limited thereto.

[0027] In one embodiment of the present invention, the human lung cell line into which the fluorescent reporter for inhalation toxicity evaluation has been introduced is the A549 cell line, but any human lung cell line that can be used to evaluate inhalation toxicity can be used without limitation.

[0028] The 'inhalation toxicity evaluation' of the present invention is to investigate the harm that a test substance (a pesticide, a pharmaceutical, a household chemical product, an industrial chemical, other environmental pollutants, or a biological hazard (bacteria and virus)) causes to the lungs, and to evaluate the degree of harm to a human lung cell line into which a fluorescent reporter for inhalation toxicity evaluation, which detects the phase separation phenomenon of the present invention in real time, has been introduced.

[0029] In addition, the present invention provides a composition for evaluating inhalation toxicity comprising the human lung cell line.

[0030] The above inhalation toxicity may be, but is not limited to, inhalation toxicity caused by any one of pesticides; household chemical products or industrial chemicals; and biological hazards including bacteria or viruses.

[0031] The above-mentioned household chemical product or industrial chemical is preferably, but not limited to, sodium arsenite or a disinfectant. The above-mentioned disinfectant is preferably, but not limited to, any one of benzalkonium chloride, benzisothiazolinone, and 1,4-dichlorobenzene.

[0032] The composition for evaluating the above inhalation toxicity is characterized by measuring the degree of stress granule formation after treatment with a test substance using a fluorescent reporter, and the degree of stress granule formation can be confirmed using a microscope.

[0033] In addition, the present invention provides a composition for screening for inhalation toxicity-inducing substances comprising the human lung cell line.

[0034] The above composition can be used to screen for substances that cause inhalation toxicity by treating a test substance to determine whether it causes inhalation toxicity and then checking the degree of stress granule formation.

[0035] The above inhalation toxicity causing substance is preferably, but is not limited to, one of pesticides; household chemical products or industrial chemicals; and biological hazards including bacteria or viruses.

[0036] In addition, the present invention provides a method for producing a human lung cell line having a fluorescent reporter for inhalation toxicity evaluation, comprising the step of introducing a fluorescent label to the 3'-end of a G3BP1 gene using a CRISPR-Cas9 system.

[0037] The above manufacturing method preferably includes a step of transfecting lung cells with a CRISPR-Cas9-sgRNA vector including sgRNA; and a gene insertion (Knock-in) vector for introducing a fluorescent label into the G3BP1 gene; and introducing a fluorescent label into the 3'-end of the G3BP1 gene; and more preferably, it includes a step of introducing a fluorescent label into the 3'-end of the G3BP1 gene using a CRISPR-Cas9 system including sgRNA of SEQ ID NO: 1; but is not limited thereto.

[0038] In one embodiment of the present invention, a human lung cell line was established by transfecting lung cells with a CRISPR-Cas9-sgRNA vector for CRISPR-Cas9-based gene editing; and a gene insertion (Knock-in) vector for introducing a fluorescent marker into the phase separation marker (G3BP1) gene, thereby permanently endogenously expressing a fluorescent marker immediately before the 3'-terminal stop codon of the G3BP1 gene through CRISPR-Cas9-based gene insertion (Knock-in).

[0039] There is no particular limitation on the type of Cas9 vector as long as it is a vector that expresses the Cas9 protein, and the pX330 plasmid is preferred.

[0040] In order to produce a gene insertion (knock-in) vector for introducing a fluorescent label into the phase separation marker (G3BP1) gene, it is preferable to design a 5' homology arm (5'HA) of 500 to 600 bp and a 3' homology arm (3'HA) of 700 to 900 bp based on the stop codon sequence located in exon 12 of the G3BP1 gene, and to insert the eGFP gene therebetween, and more preferably, a 5' homology arm (5'HA) of 550 bp and a 3' homology arm (3'HA) of 800 bp based on the stop codon sequence located in exon 12 of the G3BP1 gene, and to insert the eGFP gene therebetween, but the present invention is not limited thereto.

[0041] In one embodiment of the present invention, a pDSRed vector may be used to produce a gene insertion (knock-in) vector for introducing a fluorescent marker into a phase separation marker (G3BP1) gene, but is not limited thereto.

[0042] In addition, the present invention

[0043] 1) A step of treating the human lung cell line with a test substance for toxicity evaluation; and

[0044] 2) A method for screening inhalation toxic substances is provided, including a step of confirming a real-time phase separation phenomenon after the above step 1).

[0045] The test substance for toxicity evaluation in step 1) above refers to any substance that is expected to induce phase separation, specifically stress granule formation, when treated on cells.

[0046] The step of confirming the phase separation phenomenon in the above step 2) is a step of confirming the phase separation phenomenon, specifically the formation of stress granules, by comparing it with a control group that was not treated with the test substance, and the method of confirming the phase separation phenomenon is not particularly limited as long as it is a method that can confirm the formation of stress granules with the naked eye.

[0047]

[0048] Hereinafter, the present invention will be described in more detail using examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0049]

[0050] Materials and Methods

[0051] 1. Cell culture

[0052] Human lung epithelial cell line A549 was purchased from ATCC. A549 cells were cultured in RPMI 1640 (Roswell Park Memorial Institute 1640; Corning, NY, USA, Cat# 10-040-CV) medium containing 10% (v / v) fetal bovine serum (Gibco, Waltham, MA, USA, Cat# 12483-020) and 1% (v / v) penicillin / streptomycin (WELGENE, Cat# LS 202-02) in a humidified 37°C, 5% CO2 incubator.

[0053]

[0054] 2. Cell transfection

[0055] A549 cells are Amaxa Cell Line Nucleofector 　 1×10 according to the manufacturer's protocol using Kit T (Lonza, Basel, Switzerland) 6Cells were transfected with 2 μg of the knock-in construct (pX330-sgRNA+homologous arm vector) per cell using Nucleofector program X-01. 　 Transfection was performed using a 2b device, and after transfection, cells were transferred to 100 mm dishes and cultured in a humidified 37°C, 5% CO2 incubator until analysis.

[0056]

[0057] 3. pX330-sgRNA design construction

[0058] The sgRNA targeting G3BP1 was designed using CRISPOR, the CRISPR sgRNA design tool (crispor.tefor.net). Forward and reverse oligos were annealed in annealing buffer (10 mM Tris pH 7.5, 50 mM NaCl, 1 mM EDTA) and ligated to 50 ng of the BbsI-linearized pX330 vector. The sgRNA sequence is shown in SEQ ID NO: 1 in Table 1 below.

[0059] Sequence number sgRNA sequence 15'-CGACGAGATAATCGCCTTCG-3'

[0060]

[0061] 4. Construction of homologous arm vector

[0062] A homologous arm containing the genomic gene of G3BP1 was designed, and a homologous arm vector was constructed with the EGFP gene inserted between them. To construct a gene insertion (knock-in) vector for introducing a fluorescent marker into the G3BP1 gene, a 550-bp 5' homology arm (5'HA) and an 800-bp 3' homology arm (3'HA) were designed based on the stop codon sequence located in exon 12 of the G3BP1 gene, and the eGFP gene was inserted between them. The promoter-less pDsRed-Express 2-1 plasmid was used as the donor vector, and the DsRed-Express 2 region was removed by digestion with BglII and NotI (Fig. 1). The primer sequences used for constructing the homologous arm vector are as disclosed in SEQ ID NOs: 2 to 7 of Table 2 below.

[0063] SEQ ID NO: primer sequence 2BglII-G3BP1 5' HA forward5'-ATATAGATCTCAATGGCGTGATCTTGGCT-3'3G3BP1 5' HA reverse5'-CTGTCGTGGCGCAAGC-3'4G3BP1 5' HA-GFP forward5'-AAGGGGGCTTGCGCCACGACAGATGGTGAGCAAGGGCGA-3'5GFP-G3BP1 3' HA reverse5'-GCATGAAGATCCATGAAGATTTACTTGTACAGCTCGTCCATG-3'6G3BP1 3' HA forward5'-ATCTTCATGGATCTTCATGCAG-3'7G3BP1 3' HA-NotI reverse5'-TAGCGGCCGCAGGCACAACAGTTTTGCTC-3'

[0064]

[0065] 5. Immunoblot analysis

[0066] Protein lysates were prepared using M-PER™ mammalian protein extraction reagent (Thermo Scientific, USA, Cat# 78501) containing protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland).

[0067] Protein lysates were denatured and reduced with SDS and β-mercaptoethanol, respectively, and separated by protein size through SDS-polyacrylamide gel electrophoresis, and then transferred to nitrocellulose membranes (Pall Life Science, Port Washington, NY, USA). 5% (w / v) skim milk (Rockland Immunochemicals, West Grove, PA, USA) was prepared using 0.05% (v / v) Tween-20 dissolved in PBS, and the membranes to which the proteins were transferred were treated with primary antibodies and reacted overnight at 4°C. The membranes were then washed and incubated with horseradish peroxidase-conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA, USA) at 25°C for 1 h. Proteins were detected using a WSE-6200H LuminoGraph II (ATTO, Tokyo, Japan) with a SuperSignal system (Thermo Fisher Scientific, Waltham, MA, USA).

[0068] The primary antibodies used in the present invention are anti-G3BP1 (1:500, Santa Cruz Biotechnology, TX, USA, Cat# sc-365338), anti-GFP (1:500, Santa Cruz Biotechnology, Cat# sc-9996), anti-phospho-PERK (1:1000, Affinity Biosciences, Cat# DF7576), anti-PERK (1:500, Santa Cruz Biotechnology, Cat# sc-377400), anti-phospho-eIF2α (1:1000, Cell Signaling, Cat# 3597), anti-eIF2α (1:500, Santa Cruz Biotechnology, Cat# sc-133132), and anti-GAPDH (1:4000, Meridian Life Science, Memphis, TN, USA).

[0069]

[0070] 6. Immunofluorescence

[0071] To detect stress granule formation, A549 wild-type (A549 WT) and G3BP1-GFP transgenic cell lines (G3BP1-GFP KI) were treated with chemicals for 1 h, fixed in 4% (v / v) paraformaldehyde for 10 min, and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) in phosphate-buffered saline (PBS) for 15 min. Cells were then blocked with 5% (v / v) goat serum and 0.1% (v / v) bovine serum albumin for 1 h and incubated with anti-G3BP1 (1:500, Santa Cruz Biotechnology, Cat# sc-365338) and anti-GFP (1:500, MBL International Corporation, Woburn, MA, USA, Cat# 598) antibodies. After washing the slides, Alexa Fluor 488 and 594-conjugated goat antibodies (1:2000, Thermo Fisher Scientific, Cat# A11008, A11005) corresponding to rabbit and mouse IgG (Thermo Fisher Scientific, Cat# A28175, A-11012) were used as secondary antibodies. Nuclei were co-stained with DAPI (1:1000, Thermo Fisher Scientific, Cat# D1306).

[0072] Images were acquired using a Zeiss LSM 880 confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).

[0073]

[0074] 7. Live cell imaging

[0075] For real-time analysis of stress granule formation, G3BP1-GFP transgenic cell lines (G3BP1-GFP KI) were treated with BIT for 70 min. Live-cell imaging was performed at 37°C in a humidified 5% CO2 atmosphere. Fluorescence was excited with a 488-laser diode and detected with a Zeiss LSM 880 confocal laser scanning microscope.

[0076]

[0077] Example 1. Analysis of Fluorescent Protein and G3BP1 Protein Expression in Human Lung Cell Lines Transduced with a Phase Separation Marker (G3BP1) Gene-Specific Fluorescent Label; and Identification of Signaling Pathways Associated with Stress Granule Formation - Western Blotting

[0078] The expression of fluorescent protein and G3BP1 protein in the human lung cell line constructed in the present invention was compared with that of A549 wild-type cells.

[0079] As a result, it was confirmed that the GFP-labeled G3BP1 protein was expressed as disclosed in Fig. 2, and when oxidative stress was induced by treating with sodium arsenite (NaAsO2, Sodium arsenite, Ars.) to confirm the signaling pathway related to stress granule formation, it was confirmed that there was no change in G3BP1 protein expression, but phosphorylation of PERK and elF2α was similar to that of the wild type, thereby confirming that the fluorescently labeled G3BP1 expressed in the human lung cell line constructed in the present invention does not affect the function of G3BP1 and is expressed in a fluorescently labeled form.

[0080]

[0081] Example 2. Confirmation of the expression of phase separation marker (G3BP1) and fluorescent marker expression according to sodium arsenite (Ars.) treatment.

[0082] To verify whether the human lung cell line constructed in the present invention can be used for actual toxicity evaluation, phase separation phenomenon, specifically stress granule formation, was confirmed after treatment with sodium arsenite.

[0083] As a result, it was confirmed that GFP was normally expressed by gene insertion as disclosed in Fig. 3, and it was confirmed that phase separation, specifically stress granules, were formed at the same location as the G3BP1 protein (stained in red) when treated with sodium arsenite.

[0084]

[0085] Example 3. Analysis of fluorescent protein and G3BP1 protein expression following treatment with sterilizing disinfectant ingredients (BAC, BIT, DCB); and confirmation of signaling pathways related to stress granule formation - Western blotting.

[0086] After treating the human lung cell line constructed in the present invention with BAC, BIT or DCB, which are known as bactericidal disinfectant components, the expression of fluorescently labeled G3BP1 protein and the signal transduction pathway related to stress granule formation were confirmed. As a result, as shown in Fig. 4, it was confirmed that phosphorylation of PERK and elF2α occurred similarly compared to the wild type, thereby confirming that the fluorescently labeled G3BP1 expressed in the human lung cell line constructed in the present invention does not affect the expression amount and function of G3BP1, is expressed in a fluorescently labeled form, and can be used for inhalation toxicity evaluation.

[0087]

[0088] Example 4. Confirmation of the expression of an endogenous phase separation marker (G3BP1) and a fluorescent marker in an established lung cell line following treatment with sterilizing disinfectant ingredients (BIT, DCB, BAC).

[0089] After treating the human lung cell line constructed in the present invention with a sterilizing disinfectant component, the phase separation phenomenon, specifically the formation of stress granules, was confirmed.

[0090] As a result, as disclosed in Fig. 5, it was confirmed that phase separation, specifically stress granule formation, occurred after treatment with a sterilizing disinfectant component, and through this, it was confirmed that the human lung cell line constructed in the present invention can be used for inhalation toxicity evaluation.

[0091]

[0092] Example 5. Live cell imaging analysis of established lung cell lines following treatment with a bactericidal disinfectant component (BIT).

[0093] After treating the human lung cell line constructed in the present invention with a sterilizing disinfectant component, the phase separation phenomenon, specifically the formation of stress granules, was confirmed in real time in live cells.

[0094] As a result, as disclosed in Fig. 6, it was confirmed that phase separation, specifically stress granule formation, occurred after treatment with the sterilizing disinfectant component, and it was confirmed in real time that the number of stress granules formed per cell increased as the treatment time of the sterilizing disinfectant component passed. Through this, it was confirmed that the human lung cell line constructed in the present invention can be used for real-time inhalation toxicity evaluation.

Claims

1. A human lung cell line with a fluorescent reporter for inhalation toxicity assessment that detects phase separation phenomena in real time.

2. A human lung cell line having a fluorescent reporter for inhalation toxicity evaluation, characterized in that the phase separation phenomenon in paragraph 1 is stress granule formation.

3. In the first paragraph, the human lung cell line into which the fluorescent reporter for inhalation toxicity evaluation has been introduced is a human lung cell line into which a fluorescent reporter for inhalation toxicity evaluation has been introduced, characterized in that a fluorescent labeling factor has been introduced to the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system.

4. A human lung cell line having a fluorescent reporter for inhalation toxicity evaluation introduced therein, wherein the fluorescent reporter for inhalation toxicity evaluation in the first paragraph is any one selected from the group consisting of firefly luciferase, renilla luciferase, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), and DsRed.

5. In the third paragraph, the human lung cell line into which the fluorescent reporter for inhalation toxicity evaluation is introduced is prepared by using the CRISPR-Cas9 system to introduce firefly luciferase, renilla luciferase, green fluorescent protein (GFP), modified green fluorescent protein, enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP), enhanced red fluorescent protein (ERFP), blue fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), enhanced yellow fluorescent protein (EYFP), cyan fluorescent protein (CFP), enhanced cyan fluorescent protein (ECFP), and A human lung cell line introduced with a fluorescent reporter for inhalation toxicity assessment, characterized in that any one fluorescent marker selected from the group consisting of DsRed is introduced.

6. A composition for evaluating inhalation toxicity comprising a human lung cell line according to any one of claims 1 to 5.

7. A composition for evaluating inhalation toxicity, characterized in that the inhalation toxicity in paragraph 6 is inhalation toxicity caused by any one of pesticides; household chemical products or industrial chemicals; and biological hazards including bacteria or viruses.

8. A composition for evaluating inhalation toxicity in accordance with claim 7, wherein the household chemical product or industrial chemical substance is sodium arsenate or a sterilizing disinfectant component.

9. A composition for evaluating inhalation toxicity, characterized in that in paragraph 8, the sterilizing disinfectant component is any one of benzalkonium chloride, benzisothiazolinone, and 1,4-Dichlorobenzene.

10. A composition for screening for inhalation toxicity-inducing substances comprising a human lung cell line according to any one of claims 1 to 5.

11. A composition for screening inhalation toxicity-causing substances, characterized in that in the 10th paragraph, the inhalation toxicity-causing substance is any one of pesticides; household chemical products or industrial chemicals; and biological hazards including bacteria or viruses.

12. A method for producing a human lung cell line having a fluorescent reporter for inhalation toxicity evaluation, comprising the step of introducing a fluorescent label to the 3'-end of the G3BP1 gene using the CRISPR-Cas9 system.

13. In claim 12, the manufacturing method comprises a step of transfecting lung cells with a CRISPR-Cas9-sgRNA vector including sgRNA; and a gene insertion (Knock-in) vector for introducing a fluorescent marker into the G3BP1 gene; thereby introducing a fluorescent marker into the 3'-end of the G3BP1 gene. A method for manufacturing a human lung cell line into which a fluorescent reporter for inhalation toxicity evaluation has been introduced.

14. A method for producing a human lung cell line having a fluorescent reporter for inhalation toxicity evaluation, characterized in that the sgRNA in claim 13 consists of a base sequence of sequence number 1. 15.(1) A step of treating a test substance for toxicity evaluation to a human lung cell line according to any one of clauses 1 to 5; and (2) A method for screening inhalation toxic substances, comprising: a step of confirming a real-time phase separation phenomenon after the above step (1).

16. A method for screening inhalation toxic substances, characterized in that in the step of confirming the real-time phase separation phenomenon in clause 15, the degree of stress granule formation is confirmed compared to a control group that was not treated with the test substance.

Images