Mouse model for specifically inducing apoptosis, necrosis and pyroptosis of intestinal epithelial cells and construction method thereof

Abstract

The invention relates to a mouse model for specifically inducing apoptosis, necrosis and pyroptosis of intestinal epithelial cells and a construction method thereof, the method comprises the following steps: designing sgRNA targeting a first exon and a second exon of a Rosa26 gene, the sequence of the sgRNA being as shown in SEQ ID NO: 1 and SEQ ID NO: 2; the method comprises the following steps: constructing an expression cassette carrying fusion genes of Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2, and connecting the fusion genes through a Linker sequence as shown in SEQ ID NO: 12; integrating the expression cassette between a first exon and a second exon of a Rosa26 site of a mouse by adopting a CRISPR / Cas9 technology through a homologous recombination mode to obtain a gene editing mouse; a gene editing mouse with positive PCR amplification and sequencing identification is mated with a Vilin-cre < + / -> mouse, and the mouse model of which the intestinal epithelial cells specifically express the fusion gene is obtained. The mouse model can specifically induce single-type intestinal epithelial cell death, and avoids non-specific signal activation and complex environment disturbance accompanied by a traditional method to the greatest extent.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a mouse model for specifically inducing apoptosis, necrosis and pyroptosis of intestinal epithelial cells and a construction method thereof. BACKGROUND

[0002] Apoptosis, necrosis and pyroptosis, as the main forms of programmed cell death, are widely involved in development, homeostasis maintenance and injury and aging processes, and trigger distinct body effect responses. At present, the methods for inducing specific cell death types (apoptosis, necrosis or pyroptosis) in mice mainly rely on gene manipulation (such as specific gene knockout or overexpression) or exogenous stimulation (such as specific drugs, pathogens). However, these methods usually indirectly induce death by activating upstream signaling pathways, and this process is often accompanied by complex microenvironment changes and is prone to cause mixed occurrence of non-targeted death types, making it difficult to accurately analyze the in vivo effects triggered by a single death type and its pathological significance. SUMMARY

[0003] The present application aims to at least partially solve one of the technical problems in the related art. To this end, one object of the present application is to provide a mouse model for specifically inducing apoptosis, necrosis and pyroptosis of intestinal epithelial cells and a construction method thereof, which can specifically induce a single type of intestinal epithelial cell death and maximize the avoidance of non-specific signal activation and complex environmental disturbance accompanying traditional methods, thereby providing a more accurate and reliable research tool for in-depth elucidation of the inherent differences between different cell death types and their unique mechanisms of action in specific pathological processes (such as inflammation, tissue repair, tumor occurrence, etc.).

[0004] To this end, in one aspect of the present application, the present application proposes a construction method of a mouse model for specifically inducing apoptosis, necrosis and pyroptosis of intestinal epithelial cells, which comprises the following steps:

[0005] designing sgRNAs targeting the first and second exons of the Rosa26 gene, the sequences of the sgRNAs being shown in SEQ ID NO: 1 and SEQ ID NO: 2;

[0006] constructing an expression frame carrying Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2 fusion genes, the fusion genes being connected by a Linker sequence shown in SEQ ID NO: 12;

[0007] using CRISPR / Cas9 technology, the expression frame is integrated between the first and second exons of the Rosa26 site of the mouse by homologous recombination, to obtain a gene edited mouse;

[0008] The gene editing mouse is mated with a Villin-cre+ / - mouse to obtain a mouse model in which the fusion gene is specifically expressed in intestinal epithelial cells.

[0009] According to the method for constructing a mouse model for specifically inducing apoptosis, necrosis and pyroptosis of intestinal epithelial cells, the method first inserts an expression frame carrying a Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2 fusion gene between two exons at a Rosa26 site by using a CRISPR / Cas9 gene editing technology, and then crosses the gene editing mouse with a Villin-cre+ / - mouse to obtain a mouse model in which Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2 are specifically expressed in intestinal epithelial cells; when Tamoxifen is intraperitoneally injected into the mouse model, 4-OHT can be metabolized in the mouse model, can be combined with HBD / ERT2, and can induce oligomerization and activation of Gsdmd, Caspase8 and Mlkl connected thereto, so as to specifically induce death of different types of intestinal epithelial cells. The mouse model thus constructed can specifically induce death of a single type of intestinal epithelial cell and can avoid non-specific signal activation and complex environmental disturbance that are accompanied by traditional methods to the greatest extent, thereby providing a more accurate and reliable research tool for in-depth elucidation of the internal differences between different cell death types and the unique mechanism of the different cell death types in a specific pathological process (such as inflammation, tissue repair, tumor occurrence, etc.).

[0010] In addition, the method for constructing a mouse model for specifically inducing apoptosis, necrosis and pyroptosis of intestinal epithelial cells according to the above embodiments of the present application can have the following additional technical features:

[0011] Optionally, the sequence of the Gsdmd gene is as shown in SEQ ID NO: 7, the sequence of the Caspase8 gene is as shown in SEQ ID NO: 8, the sequence of the Mlkl gene is as shown in SEQ ID NO: 9, the sequence of the HBD gene is as shown in SEQ ID NO: 10, and the sequence of the ERT2 gene is as shown in SEQ ID NO: 11.

[0012] Optionally, the method further comprises the following steps:

[0013] After the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 2 are combined together, the sequences are inserted into a gRNA-Cas9-P2A-mcherry vector to obtain an sgRNA plasmid;

[0014] The Rosa26 homologous left arm, the Rosa26 homologous right arm, the Gsdmd / Caspase8 / Mlkl, the HBD / ERT2 gene fragment are connected into the pBKs-CKO vector by LIC to obtain the plasmid of the Gsdmd-HBD genotype mouse, the plasmid of the Caspase8-ERT2 genotype mouse and the plasmid of the Mlkl-ERT2 genotype mouse;

[0015] The sgRNA plasmid, the plasmid of the Gsdmd-HBD genotype mouse, the plasmid of the Caspase8-ERT2 genotype mouse and the plasmid of the Mlkl-ERT2 genotype mouse are injected into the fertilized eggs of C57BL / 6 mice by cytoplasm injection or using haploid embryonic stem cells to obtain the gene editing mouse.

[0016] In the second aspect of the present application, the application provides the above-mentioned method for constructing a mouse model for specifically inducing apoptosis, necrosis and pyroptosis of intestinal epithelial cells, and the mouse model constructed by the method.

[0017] Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2.

[0018] According to the mouse model of the present application, after intraperitoneal injection of Tamoxifen to the mouse model, 4-OHT can be metabolized in the mouse model, can be combined with HBD / ERT2, and can induce oligomerization and activation of Gsdmd, Caspase8 and Mlkl connected thereto, thereby specifically inducing different types of intestinal epithelial cell death. The mouse model thus constructed can specifically induce death of a single type of intestinal epithelial cell and can avoid non-specific signal activation and complex environmental disturbance accompanying traditional methods to the greatest extent, thereby providing a more accurate and reliable research tool for in-depth elucidation of the intrinsic differences between different types of cell death and the unique mechanism of action of the different types of cell death in specific pathological processes (such as inflammation, tissue repair and tumor occurrence).

[0019] In the third aspect of the present application, the application provides the use of the above-mentioned mouse model in the preparation of a research tool for inflammation, tissue repair or tumor occurrence.

[0020] Additional aspects and advantages of the application will be in part apparent and in part pointed out hereinafter. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 A flow chart for constructing the mouse model according to the embodiments of the present application is shown in the figure;

[0022] Figure 2A gRNA-Cas9-P2A-mcherry vector plasmid map according to an embodiment of the present application;

[0023] Figure 3 A pBKs-CKO vector plasmid map according to an embodiment of the present application;

[0024] Figure 4 A Gsdmd-HBD mouse plasmid map according to an embodiment of the present application;

[0025] Figure 5 A Caspase8-ERT2 mouse plasmid map according to an embodiment of the present application;

[0026] Figure 6 A Mlkl-ERT2 mouse plasmid map according to an embodiment of the present application;

[0027] Figure 7 A genotype identification result map according to an embodiment of the present application;

[0028] Figure 8 A western blot verification map according to an embodiment of the present application, wherein A is the HBD expression of each intestinal segment of the Gsdmd-HBD mouse, B is the HBD expression of each intestinal segment of the Caspase8-ERT2 mouse, and C is the HBD expression of each intestinal segment of the Mlkl-ERT2 mouse;

[0029] Figure 9 A mouse in vivo induction experiment result according to an embodiment of the present application, wherein A is the survival of three mice after injection of different concentrations of tamoxifen, B is the colon HE staining result of the Gsdmd-HBD mouse after injection of tamoxifen, C is the colon HE staining result of the Caspase8-ERT2 mouse after injection of tamoxifen, and D is the HE staining result of each intestinal segment of the Mlkl-ERT2 mouse after injection of tamoxifen;

[0030] Figure 10 A cell in vitro induction experiment result according to an embodiment of the present application. DETAILED DESCRIPTION

[0031] The technical solutions of the present application are described below through specific examples. It should be understood that the one or more method steps mentioned in the present application do not exclude the presence of other method steps before and after the described combination steps or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these examples are only used to illustrate the present application and not to limit the scope of the present application. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool to identify each method step, and not a limitation on the arrangement order of each method step or a limitation on the scope of the present application that can be implemented, the change or adjustment of the relative relationship thereof is also considered as the scope of the present application that can be implemented without substantial change of the technical content.

[0032] In order to better understand the above technical solutions, the exemplary embodiments of the present application are described in more detail below. Although exemplary embodiments of the present application are shown, it should be understood that the present application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present application and to fully convey the scope of the present application to those skilled in the art.

[0033] The test materials used in the present application are all ordinary commercially available products and can be purchased in the market; the experiments involved are all conventional experimental methods unless otherwise specified.

[0034] The mice used are wild-type C57BL / 6 mice.

[0035] The Villin-cre+ / -mice are derived from The Jackson Laboratory (JAX).

[0036] DMEM (Dulbecco's Modified Eagle Medium, dry powder purchased from Gibco, item number 12800-082);

[0037] 10% fetal bovine serum (FBS, purchased from Youshi Biology, item number A1025);

[0038] Dual antibody (penicillin mixture, purchased from Gibco, item number 15140122);

[0039] ATP kit (CellTiter-Glo Luminescent Cell Viability Assay, company: Promega, item number: G7571);

[0040] LDH kit (Cytotoxicity LDH Assay kit-WST, company: Dongren Chemical, Shanghai, item number: CK12-2000 wells).

[0041] The present application will be described below with reference to specific embodiments, which are merely illustrative and not in any way limit the present application.

[0042] Example 1 Construction of mouse model

[0043] The mechanism of constructing the mouse model is shown in Figure 1 , and the specific operation steps are as follows:

[0044] (1) Construction of sgRNA plasmid: two sgRNAs were designed between the first and second exons of Rosa26, and the sgRNA sequences are shown in Table 1. After annealing to combine the complementary strands together, they were connected to the gRNA-Cas9-P2A-mcherry vector (vector plasmid map as shown in Figure 2 ) digested by Esp3I enzyme. The annealing system is shown in Table 2, and the annealing program is 98℃ for 10 min and 14℃ for 1 min. The T4 ligation system is shown in Table 3, and the ligation conditions are 22℃ for 30 min or room temperature overnight; to obtain the sgRNA plasmid.

[0045] Table 1 sgRNA sequence

[0046]

[0047] Table 2 gRNA annealing system (20 μL system as an example)

[0048]

[0049] Table 3 T4 ligation system

[0050]

[0051] (2) Construction of insertion gene plasmid:

[0052] The Rosa26 homologous left arm, Rosa26 homologous right arm, Gsdmd / Caspase8 / Mlkl, and HBD / ERT2 gene fragments were respectively connected to the pBKs-CKO vector (with Loxp and Stop genes, vector plasmid map as shown in Figure 3 ) by LIC (Ligation-independent cloning), and a linker sequence was required between Gsdmd / Caspase8 / Mlkl and HBD / ERT2. The relevant gene sequences are shown in Table 4. The plasmid map of Gsdmd-HBD genotype mouse is shown in Figure 4 , the plasmid map of Caspase8-ERT2 genotype mouse is shown in Figure 5 , and the plasmid map of Mlkl-ERT2 genotype mouse is shown in Figure 6 .

[0053] Table 4 Insertion gene sequence

[0054]

[0055]

[0056]

[0057] (3) Construction of mice: the constructed sgRNA plasmid with Cas9 and the above insertion gene plasmid are transferred into the zygote of C57BL / 6 mice by cytoplasmic injection or using haploid embryonic stem cells by the transgenic platform of Xiamen University Experimental Animal Center, to obtain gene editing mice.

[0058] (4) The gene editing mice are mated with Villin-cre+ / - mice, and the mating is performed by natural mating or IVF. The gene editing mice used for mating are female or male mice, and 8-week-old mice can be used for subsequent modeling experiments to obtain a mouse model specifically expressing the fusion gene in intestinal epithelial cells. Figure 1 As can be seen from the left side of the figure, Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2 are preceded by a stop gene with Loxp, and only after the Loxp is cut off by Cre enzyme to remove the stop gene, Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2 can be normally expressed. Therefore, by crossing with Villin-cre+ / - mice, since Villin is mainly expressed in intestinal epithelial cells, the intestinal epithelial cells contain Cre, which removes the stop gene, and thus the mice specifically expressing Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2 in intestinal epithelial cells are obtained.

[0059] (5) Genotype identification: the mouse tail is digested with EB, and then genotype identification and sequencing verification are performed. The identification of related sequences, systems and procedures are shown in the following table.

[0060] Table 5 Genotype identification PCR primer sequence

[0061]

[0062] Table 6 PCR system (for example, 20 μL system)

[0063]

[0064] Table 7 PCR program

[0065]

[0066]

[0067] The results of the identification are as follows Figure 7 As shown, the mouse genotypes were distinguished based on the different band sizes of the PCR product electrophoresis gel images. WT mice should have a band of approximately 750 bp in size; homozygous KI mice (homozygous knock-in mice with the target gene inserted in Rosa26 of both DNAs) should have a band of approximately 340 bp in size; heterozygous HT mice should have two bands, one of approximately 340 bp and one of approximately 750 bp.

[0068] Example 2 Induction of cell death

[0069] Expression was verified by western blot: Tubulin / GAPDH was used as the internal reference protein, the primary antibody was Anti-β-Tubulin (C66) Mouse mAb, Abmart, Catalog No. M20005M, the WB band size was approximately 55 kDa, the primary antibody was GAPDH antibody, Wuhan Tri-Tac Co., Ltd., Catalog No. 60004-1-Ig, the secondary antibodies for the internal reference protein were goat anti-mouse IgG, Thermo Fisher Scientific, Catalog No. 31430; the primary antibody was anti-ERα antibody (F-10), Santa Cruz Biotechnology, Catalog No. sc-8002, the WB band size was approximately 70 kDa, the secondary antibody was goat anti-mouse IgG, Thermo Fisher Scientific, Catalog No. 31430).

[0070] After the mice were sacrificed by cervical dislocation, the whole intestine was taken out by laparotomy, soaked in pre-cooled 1xPBS, and the small intestine was washed with ice-cold PBS; the small intestine was turned over and cut into 1 cm pieces, cut as much as possible, immersed in ice-cold 5mM EDTA, and mixed at 4°C for 45 min or more to separate the crypts, and the suspension containing the intestinal contents was aspirated and filtered through a 70μm filter. 500g, 4°C, centrifugal 5min, to collect the crypts, discard the supernatant, add 2mL PBS resuspend; again 500g, 4°C, centrifugal 5min; discard the supernatant, add 1mL PBS, blow and resuspend, and transfer to 1.5mL EP tube 500g, 4°C, centrifugal 5min; discard the supernatant, add a certain amount of 2xSDS according to the amount of cells. After ultrasonic crushing, continue to be placed on ice; metal bath 100℃, boil 10min, stored at -20℃ for standby. The collected intestinal epithelial samples were added to 10% protein separation gel, and an appropriate amount of electrophoresis buffer was added to the tank, and electrophoresis was carried out at a voltage of 100 volts. After the protein migrated to the desired site in the gel, the protein in the gel was transferred to a PVDF membrane, the transfer buffer was added to the tank, and the transfer was carried out at a voltage of 100 volts for 1 hour. The PVDF membrane was placed in 5% BSA and incubated at room temperature for 1 hour. The membrane was placed in a diluted primary antibody solution and incubated at 4°C overnight. The next day, wash 3 times with 1xTBST on a fast shaker for 5 minutes each time. The membrane was placed in a diluted secondary antibody solution and incubated at room temperature for 1 hour, washed 3 times with 1xTBST on a fast shaker for 5 minutes each time. The membrane was mixed with the chemiluminescent substrate ECLA+B (Xin Saimei Biotechnology, P10300), incubated at room temperature for 1 minute, exposed with Image Quant LAS 4000mini, and the image was saved.

[0071] Formulation:

[0072] 10x Lower buffer: 1% SDS, 3.5M Tris-HCl, pH 8.8.

[0073] 4x Stacking buffer: 0.4% SDS, 0.5M Tris-HCl, pH 6.8.

[0074] 5x SDS sample buffer (50mL): 5mL 3M Tris-HCl (pH6.8), 5g SDS, 25mL glycerol, 12.5mL β-mercaptoethanol, 25mg bromophenol blue, 7.3mL ultrapure water. When collecting the sample, dilute it to 1.2x sample buffer.

[0075] 10% separation gel (500 mL): 167 mL 30% Ac-Bi, 39 mL 65% sucrose, 50 mL 10× Lower buffer, 244 mL ultrapure water.

[0076] 4% stacking gel (500 mL): 66.7 mL 30% Ac-Bi, 125 mL 4× stacking buffer, 308.3 mL ultrapure water;

[0077] 10× electrophoresis buffer (10 L): 100 g SDS, 303 g Tris, 1440 g glycine, add 8 L ultrapure water, stir for at least 2 hours, and dilute to 10 L. Store at room temperature and dilute to 1× working concentration before use.

[0078] 10× Transfer Buffer (10 L): 303 g Tris, 1440 g Glycine, add 8.5 L ultrapure water and stir until completely dissolved, dilute to 10 L, store at room temperature, and dilute to 1× working concentration before use, and add 10% methanol.

[0079] 20× TBST buffer (10 L): 484 g Tris, 1600 g NaCl, 200 mL Tween-20, 270 mL HCl, add 8 L ultrapure water, adjust the pH to 7.6, mix well, and make up to 10 L. Dilute to 1× TBST with ultrapure water before use.

[0080] 5% BSA blocking solution: Dissolve 25 g BSA in TBST and adjust the volume to 500 mL.

[0081] The results are as follows Figure 8 As shown, A is the expression of HBD in each intestinal segment of Gsdmd-HBD mice, B is the expression of HBD in each intestinal segment of Caspase8-ERT2 mice, and C is the expression of HBD in each intestinal segment of Mlkl-ERT2 mice; all three mouse strains were expressed only in Villin-Cre + / - The HBD bands can be seen in each intestinal segment of mice. - / - No HBD band was detected in mice, indicating that Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2 were successfully expressed in all segments of the mouse intestine and were expressed in Villin-Cre + / - Specific to mice, i.e., intestinal epithelial cell-specific expression.

[0082] Tamoxifen (MCE, Cat# HY-13757A) was dissolved in corn oil (Sigma, Cat# C8267-500ML) and injected intraperitoneally at 10 μL / g based on the body weight of mice to the mouse model of intestinal epithelial cell-specific expression of Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2 constructed in Example 1.

[0083] Figure 9 All of B, C and D are paraffin-embedded sections, HE staining. The specific process is as follows: after being sacrificed by CO2 asphyxiation or cervical dislocation, the mouse is disinfected with 75% alcohol, the peritoneum is cut along the abdominal midline, the abdominal cavity is exposed, the duodenum to the colon is taken out, and the segment is handled (divided into five segments according to the duodenum, jejunum, ileum, cecum and colon), the excess connective tissue such as fat is removed, the segment is cut longitudinally, the intestinal contents are removed, and each segment is rolled into a "Swiss roll", placed into an embedding box, and after being fixed with 4% paraformaldehyde for 24 hours, dehydrated and embedded (dehydration procedure as follows: 50% ethanol immersion for 25 minutes; 70% ethanol immersion for 25 minutes; 80% ethanol immersion for 25 minutes; 95% ethanol immersion for 15 minutes; 95% ethanol immersion for 15 minutes; 100% ethanol immersion for 30 minutes; 100% ethanol immersion for 30 minutes; xylene and ethanol 1:1 solution immersion for 30 minutes; xylene immersion for 20 minutes; xylene immersion for 20 minutes; paraffin immersion for 90 minutes; paraffin immersion for 90 minutes). The embedded tissue is sectioned at a thickness of 5 μm, and then HE staining is performed. Before staining, the section is dried in an oven at 56°C for 1-2 hours, and then rehydrated (rehydration procedure as follows: xylene immersion for 15 minutes; xylene immersion for 15 minutes; xylene immersion for 15 minutes; 100% ethanol immersion for 5 minutes; 100% ethanol immersion for 5 minutes; 90% ethanol immersion for 5 minutes; 80% ethanol immersion for 5 minutes; 70% ethanol immersion for 5 minutes; water immersion for 5 minutes), after rehydration, immersion in hematoxylin for 2 minutes, then immersion in tap water for 3-5 times, immersion in 0.2% ammonia water for 2-5 seconds, then immersion in tap water for 3-5 times, after immersion, dehydration and transparency, the section can be mounted with neutral resin (dehydration and transparency procedure as follows: 70% ethanol immersion for 30 seconds; 80% ethanol immersion for 30 seconds; 95% ethanol immersion for 30 seconds; 100% ethanol immersion for 30 seconds; 100% ethanol immersion for 30 seconds; xylene immersion for 1 minute; xylene immersion for 1 minute), after the section is dried, it can be scanned by an automatic scanner and subsequent data analysis can be performed.

[0084] Results are as shown in Figure 9As shown in the results, mice whose intestinal epithelial cells specifically express Gsdmd-HBD, Caspase8-ERT2, and Mlkl-ERT2 showed obvious intestinal damage and different degrees of mouse death after being injected with tamoxifen, proving that tamoxifen did successfully induce cell death. Figure 9 As shown in Figure A, the survival of three types of mice at different concentrations shows that the death characteristic of Gsdmd-HBD mice is that some mice died at various time periods during the injection process, and the median lethal dose is about 90 mg / kg; the death characteristic of Caspase8-ERT2 mice is that during the injection process, mice died only in the first two days, and no mouse deaths were observed in the subsequent process; the death characteristic of Mlkl-ERT2 mice is that they died very quickly and were very sensitive to tamoxifen, with all mice dying within one day at only 2 mg / kg. This shows that intestinal epithelial cell death caused by different death types has different effects on the death of individual mice. Figure 9 Figure B shows the damage to the colon of Gsdmd-HBD mice at a half-lethal dose. It can be seen that the intestine was severely damaged from the first day, with the overall structure destroyed and accompanied by a certain degree of immune infiltration. The damage continued until the sixth day, which may be the reason for the continuous death during the injection process. Figure 9 As shown in Figure C, the damage to the colon of Caspase8-ERT2 mice is mainly concentrated on the first day. The colon structure is relatively intact and no obvious damage is observed in the subsequent days, which corresponds to the fact that no more deaths occur. Figure 9 As shown in Figure D, the damage to the intestinal segments of Mlkl-ERT2 mice shows that compared with the control group without tamoxifen injection, significant damage can be observed in all intestinal segments of mice just two hours after tamoxifen injection, including bleeding, villus shortening, structural damage, immune infiltration, etc., and the degree of damage to the small intestine is more serious than that to the colon. Such large-scale damage may also be related to the rapid death of individuals. And combined with Figure 1 As can be seen from the figure on the right, Gsdmd-HBD genotype mice induce pyroptosis type cell death, Caspase8-ERT2 genotype mice induce apoptosis type cell death, and Mlkl-ERT2 genotype mice induce necrosis type cell death.

[0085] In vitro cell induction experiments were performed using MEF cells overexpressing Gsdmd-HBD / Caspase8-ERT2 / Mlkl-HBD via lentiviral transfection. DMEM culture medium was supplemented with 10% fetal bovine serum and 100 mg / mL of dual antibody. After adding 4-OHT, cell death was detected at different concentrations and time points using ATP and LDH kits. The results are shown in the figure below. Figure 10As shown, the upper three graphs from left to right are cell survival results of Mlkl-HBD, Gsdmd-HBD and Caspase8-ERT2 overexpression MEF cells after adding culture medium containing different concentrations of 4-OHT for 3 hours, and the lower three graphs from left to right are cell survival results of Mlkl-HBD, Gsdmd-HBD and Caspase8-ERT2 overexpression MEF cells after adding culture medium containing different concentrations of 4-OHT for 3 hours. It can be seen from the above that 4-OHT can successfully induce the death of overexpression MEF cells, and the sensitivities of the three cells to 4-OHT are different. After adding the culture medium containing 4-OHT for 3 hours, the 50% death dose of Mlkl-HBD cells is about 100 ng / mL, the 100% death dose is about 400 ng / mL; the 50% death dose of Gsdmd-HBD cells is about 1 μg / mL, the 100% death dose is about 9 μg / mL; the 50% death dose of Caspase8-ERT2 cells is about 7 μg / mL, and the 100% death dose is about 10 μg / mL. Figure 10

[0086] In summary, according to the embodiment of the present application, first, the CRISPR / Cas9 gene editing technology is used to insert an expression frame carrying the Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2 fusion gene between the two exons of the Rosa26 site, and then the gene edited mouse is crossed with the Villin-cre+ / - mouse to obtain a mouse model specifically expressing Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2 in intestinal epithelial cells. When Tamoxifen is injected intraperitoneally into the mouse model, it can be metabolized to 4-OHT in the mouse model, can be combined with HBD / ERT2, and can induce the oligomerization and activation of Gsdmd, Caspase8 and Mlkl connected thereto, thereby specifically inducing the death of different types of intestinal epithelial cells. The mouse model constructed in this way can specifically induce the death of a single type of intestinal epithelial cell and maximize the avoidance of non-specific signal activation and complex environmental disturbance accompanying traditional methods, thereby providing a more precise and reliable research tool for further elucidating the inherent differences between different cell death types and their unique mechanisms of action in specific pathological processes (such as inflammation, tissue repair, tumor occurrence, etc.).

[0087] ​In the description of the specification, the description of the terms "one embodiment", "some embodiments", "an example", "a specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any suitable manner in one or more embodiments or examples. In addition, those skilled in the art can combine and integrate different embodiments or examples described in the specification.

[0088] Although the embodiments of the present application have been shown and described above, it is understood that the above-described embodiments are exemplary, and should not be construed as limiting the present application, and those skilled in the art can make changes, modifications, replacements and variations to the above-described embodiments within the scope of the present application.

Claims

1. A method for constructing a mouse model that specifically induces apoptosis, necrosis, pyroptosis of intestinal epithelial cells, characterized in that, The method comprises the following steps: designing sgRNAs targeting the first and second exons of the Rosa26 gene, the sequences of the sgRNAs being shown as SEQ ID NO: 1 and SEQ ID NO: 2; constructing an expression frame carrying Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2 fusion genes, the fusion genes being connected by a Linker sequence shown as SEQ ID NO: 12; integrating the expression frame into the first and second exons of the Rosa26 site of a mouse by homologous recombination through CRISPR / Cas9 technology to obtain a gene edited mouse; crossing the gene edited mouse with a Villin-cre+ / - mouse to obtain a mouse model specifically expressing the fusion genes in intestinal epithelial cells.

2. The construction method of claim 1, wherein, The sequence of the Gsdmd gene is shown as SEQ ID NO: 7, the sequence of the Caspase8 gene is shown as SEQ ID NO: 8, the sequence of the Mlkl gene is shown as SEQ ID NO: 9, the sequence of the HBD gene is shown as SEQ ID NO: 10, and the sequence of the ERT2 gene is shown as SEQ ID NO:

11.

3. The construction method of claim 1, wherein, The method comprises the following steps: combining the sequences shown as SEQ ID NO: 1 and SEQ ID NO: 2 together and then connecting into a gRNA-Cas9-P2A-mcherry vector to obtain an sgRNA plasmid; connecting Rosa26 homologous left arm, Rosa26 homologous right arm, Gsdmd / Caspase8 / Mlkl and HBD / ERT2 gene fragments into a pBKs-CKO vector through LIC to obtain a plasmid of a Gsdmd-HBD genotype mouse, a plasmid of a Caspase8-ERT2 genotype mouse and a plasmid of a Mlkl-ERT2 genotype mouse; injecting the sgRNA plasmid, the plasmid of the Gsdmd-HBD genotype mouse, the plasmid of the Caspase8-ERT2 genotype mouse and the plasmid of the Mlkl-ERT2 genotype mouse into zygotes of C57BL / 6 mice through cytoplasmic injection or using haploid embryonic stem cells to obtain a gene edited mouse.

4. The mouse model for specifically inducing apoptosis, necrosis, and pyroptosis of intestinal epithelial cells, which is constructed by the method for constructing a mouse model according to any one of claims 1 to 3, characterized in that, The mouse model specifically expresses Gsdmd-HBD, Caspase8-ERT2 and Mlkl-ERT2 in intestinal epithelial cells.

5. The mouse model of claim 4, wherein the mouse model is a mouse model of Alzheimer's disease. After induction by tamoxifen, a single type of death among apoptosis, necrosis and pyroptosis of intestinal epithelial cells of the mouse model can be specifically activated.

6. Use of the mouse model of claim 4 or 5 in the preparation of a research tool for inflammation, tissue repair or tumor occurrence.

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