Construction of a fluorescently traced mouse and its application in the sorting of Mcpt4-positive and negative mast cells

Abstract

This invention provides a method for constructing a fluorescently traced mouse model and its application in the sorting of Mcpt4-positive and negative mast cells. First, the Cre gene is knocked into the Mcpt4 gene sequence of C57BL / 6 mice using CRISPR / Cas9 technology. Then, through animal hybridization and Cre-loxP recombination technology, a fluorescently traced mouse model of Mcpt4-positive mast cells is obtained. The fluorescently traced mouse model possesses a dual-fluorescent reporter gene system consisting of the red fluorescent protein tdTomato and the green fluorescent protein ZsGreen. Mcpt4-positive mast cells exhibit red fluorescence, while other Mcpt4-negative cells exhibit green fluorescence. Using this fluorescently traced mouse model, Mcpt4-positive and negative mast cells can be rapidly, accurately, and effectively sorted, providing a reliable technical basis for exploring the roles and mechanisms of Mcpt4-positive and negative mast cells in immune responses.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and biomedicine, specifically relating to the construction of a fluorescently traced mouse and its application in the sorting of Mcpt4 positive and negative mast cells. Technical Background

[0002] Mast cells, as immune cells, are sentinel cells of innate immunity and core cells in the pathology of allergic diseases, playing a crucial role in tissue morphology and host defense. Mouse mast cells originate from bone marrow hematopoietic stem cells and can further differentiate into connective tissue mast cells (CTMCs) or mucosal mast cells (MMCs) in different local tissues. These two mast cell subtypes coexist in various mouse tissues inhabited by mast cells, but the proportions of the two subtypes vary in different tissues.

[0003] Mast cells contain abundant proteases, including trypsin, chymase, and carboxypeptidase A3 (CPA3), which are specifically present in mast cells. In mouse CTMCs, there are four mast cell-specific proteases (mMCP-4, -5, -6, and -8, whose encoding genes correspond to Mcpt4, Mcpt5 (Cma1), Mcpt6, and Mcpt8, respectively). Mcpt4 and Mcpt5 are homologs of the human chymase gene and are also markers of CTMCs. However, according to previous studies, not both can be used for CTMC sorting because: (1) Based on tissue distribution, substrate specificity, and proteoglycan affinity, it has been reported that mouse mMCP-4 is more similar to human chymase in terms of functional characteristics than mMCP-5. (2) Although Mcpt5-cre mice have been reported to be constructed, Mcpt5 is also expressed during the growth and development of MMCs and is not a specific marker of CTMCs. Therefore, there is an urgent need to construct a mouse model based on the Mcpt4 gene that can be used to effectively identify and sort Mcpt4-positive mast cells (CTMC) and Mcpt4-negative mast cells (MMC) in the same tissue.

[0004] The current lack of an ideal mouse model for tracing and distinguishing between CTMC and MMC hinders detailed research into the differential roles and mechanisms of different mast cell subtypes in related immune responses. To better trace and sort different subtypes of mouse mast cells, it is urgent to construct an animal model that can differentiate between CTMC and MMC. Fluorescent proteins are important tracking tools in molecular biology research. Through gene knock-in, exogenous fluorescent protein expression elements are knocked into endogenous mouse genes, allowing for the labeling and tracking of target genes. This can be applied to studies such as protein subcellular localization, expression profiling, and cell lineage tracing. For example, the red fluorescent protein tdTomato gene was combined with the green fluorescent protein ZsGreen gene and knocked into the mouse genome sequence, with loxP sites flanking the ZsGreen gene. Without hybridization with mice carrying the Cre recombinase gene, mice with ZsGreen-loxP and tdTomato genome knock-in exhibited ZsGreen green fluorescence throughout their bodies. When this mouse is crossed with a mouse carrying the Cre recombinase gene, the Cre recombinase acts in the offspring cells that specifically express the target gene, causing the ZsGreen gene to be deleted from the genome. The affected cells then display the red fluorescence of tdTomato. Therefore, this patent utilizes the fluorescence difference between these cells to specifically sort Mcpt4-positive mast cells, which exhibit a different fluorescence color from other cells in the tissue, using techniques such as flow cytometry.

[0005] Furthermore, in the same tissue of Mcpt4-cre G / R traced mice, red fluorescent Mcpt4-positive mast cells carrying the Mcpt4-cre recombinase gene, green fluorescent Mcpt4-negative mast cells, and green fluorescent non-mast cells coexist. While flow cytometry can separate red fluorescent Mcpt4-positive mast cells using only fluorescence color difference and related techniques, it cannot separate green fluorescent Mcpt4-negative mast cells from the numerous green fluorescent cells. Therefore, this patent considers utilizing the specific binding characteristics of antigen and antibody, as well as the biocompatibility and easy separation properties of magnetic beads, to enrich all mast cells from the tissue cell population, and then using the red-green fluorescence difference of the cells to separate Mcpt4-positive and negative mast cells.

[0006] Mast cells, as tissue-inhabiting cells, vary in the ratio of CTMCs to MMCs among different tissues. Therefore, the magnetic bead-binding antibodies used for enrichment of mast cells from different tissue sources may differ. CD117 (also known as c-Kit, the receptor for stem cell factor SCF) and FcεRI (a high-affinity IgE receptor) are mast cell surface marker receptors, both of which can be used to identify mast cells from cell populations. This patent utilizes magnetic beads bound to CD117 and FcεRIA antibodies to enrich peritoneal mast cells and bone marrow mast cells, respectively. The results are shown in Table 1.

[0007] Table 1

[0008]

[0009] As shown in Table 1, magnetic beads incubated with FcεRⅠA antibody achieved an enrichment rate of 86.9% for peritoneal PCMCs, while those incubated with CD117 antibody only achieved 70.3%. Conversely, magnetic beads incubated with CD117 antibody achieved an enrichment rate of 96.6% for bone marrow-derived BMMCs, while those incubated with FcεRⅠA antibody only achieved 87.3%. This indicates a significant difference in antibody selection when using surface-bound antibody-rich magnetic beads to enrich peritoneal and bone marrow-derived mast cells.

[0010] In summary, to more effectively explore the functional characteristics of Mcpt4-positive CTMC and Mcpt4-negative MMC subtypes, selectively constructing Mcpt4-cre mice and selectively utilizing FcεRIA as a magnetic bead-binding antibody are substantial advantages. Within the same tissue, the target cells to be isolated include not only Mcpt4-positive CTMCs with completely different fluorescence colors from other cells, but also Mcpt4-negative MMCs with the same fluorescence colors as other cells. Therefore, this patent not only requires the use of flow cytometry to separate different fluorescent cells, but also the combination of magnetic bead enrichment technology that specifically distinguishes cells with different surface antigens; this combination represents a significant advancement of this patent. Summary of the Invention

[0011] The purpose of this invention is to provide a fluorescently traced mouse model and its application in the sorting of Mcpt4-positive and Mcpt4-negative mast cells. This invention first utilizes CRISPR / Cas9 technology to knock the Cre gene into the Mcpt4 gene sequence of C57BL / 6 mice. Then, through animal hybridization and Cre-loxP recombination technology, a fluorescently traced mouse model of Mcpt4-positive mast cells is obtained. The fluorescently traced mouse model possesses a dual-fluorescent reporter gene system consisting of the red fluorescent protein tdTomato and the green fluorescent protein ZsGreen. Mcpt4-positive cells exhibit red fluorescence, while other Mcpt4-negative cells exhibit green fluorescence. The fluorescently traced mouse model constructed in this invention, when applied to the sorting of Mcpt4-positive and Mcpt4-negative mast cells, can rapidly, accurately, and effectively sort Mcpt4-positive and Mcpt4-negative mast cells, providing a reliable technical basis for exploring the role and mechanism of Mcpt4-positive and Mcpt4-negative mast cells in immune responses.

[0012] The technical objective of this invention is achieved through the following technical solutions.

[0013] 1. A method for constructing a fluorescently traced mouse, comprising the following steps:

[0014] (1) Using bacterial artificial chromosome clones RP24-167M24 and RP24-175A23 from the C57BL / 6 library as templates, homologous arms were generated by PCR, and “2A-Cre” was used as a homologous recombination repair template.

[0015] (2) Design gRNA targeting the Mcpt4 gene sequence on chromosome 14 of C57BL / 6 mice;

[0016] (3) Homologous recombination repair template, gRNA, Cas9 and target vector were injected together into fertilized eggs and transplanted into the uterus of pseudopregnant mice. The mice bred were identified by gene identification and mice with the genotype Mcpt4-cre were obtained.

[0017] (4) Mcpt4-cre mice were crossed with homologous mice carrying the G / R-loxP recombinant gene to obtain fluorescent tracer mice with the genotype Mcpt4-cre;G / R;

[0018] (5) In mice with the genotype Mcpt4-cre;G / R, Mcpt4-positive cells genetically express tdTomato red fluorescent protein, and Mcpt4-negative cells genetically express ZsGreen green fluorescent protein. Using the dual fluorescent reporter system of red fluorescent protein tdTomato and green fluorescent protein ZsGreen, cells that specifically express Mcpt4 can be traced by red fluorescence.

[0019] 2. A method for using fluorescently traced mice in the sorting of Mcpt4-positive and negative mast cells, comprising the following steps:

[0020] (1) Wash the peritoneal cavity of Mcpt4-cre;G / R mice with PBS buffer to obtain peritoneal cell suspension;

[0021] (2) Peritoneal cell suspension was co-incubated with magnetic beads with FcεRⅠA antibody on the surface. FcεRⅠA antibody can specifically bind to FcεRⅠ receptor on the surface of mast cells, thereby enriching mast cells.

[0022] (3) The enriched mast cells were sorted by flow cytometer to obtain Mcpt4 positive connective tissue mast cells and Mcpt4 negative mucosal mast cells.

[0023] The beneficial technical effects of this invention are reflected in the following aspects:

[0024] (1) This invention utilizes CRISPR / Cas9 technology to knock the Cre gene into the Mcpt4 gene sequence of C57BL / 6 mice, obtaining mice with the genotype Mcpt4-cre. Mcpt4-cre mice can be flexibly combined with different loxp mice, and Mcpt4-positive cells in the mice can be specifically modified, thereby objectively and systematically exploring the role and mechanism of Mcpt4-positive mast cells in immune inflammation;

[0025] (2) This invention obtains Mcpt4-cre and G / R fluorescent tracer mice by crossing Mcpt4-cre mice with G / R-loxp mice. The introduction of Mcpt4-cre mice limits the application of the red fluorescent protein tdTomato and green fluorescent protein ZsGreen dual fluorescent reporter gene system to mouse Mcpt4-positive mast cells, thereby achieving rapid, accurate and effective identification of mouse Mcpt4-positive cells;

[0026] (3) The present invention applies Mcpt4-cre;G / R fluorescent tracer mice to the sorting of Mcpt4 positive and negative mast cells, which can quickly, accurately and effectively sort Mcpt4 positive and negative mast cells, providing a reliable technical basis for exploring the role and mechanism of Mcpt4 positive and negative mast cells in immune response;

[0027] (4) This invention uses magnetic beads incubated with FcεRⅠA antibody to enrich mast cells. The magnetic beads have a large specific surface area, which can bind more FcεRⅠA antibody, thereby improving the enrichment efficiency of mast cells. The FcεRⅠ antigen on the cell surface specifically binds to the FcεRⅠA antibody on the magnetic beads. Under the action of magnetic force, the mast cells are separated from other cells. This method simplifies the enrichment operation and has the characteristics of high sensitivity, fast enrichment speed, high specificity, and good reproducibility. Attached Figure Description

[0028] Appendix Figure 1 A schematic diagram of the Mcpt4-cre mouse model constructed using CRISPR / Cas9 technology and a diagram of gene identification results;

[0029] Appendix Figure 2 Schematic diagram of Mcpt4-cre G / R mouse model construction and mouse fluorescence image;

[0030] Appendix Figure 3 Image of Mcpt4-cre; G / R mouse genotype identification results and peritoneal cell fluorescence image;

[0031] Appendix Figure 4 Schematic diagram of magnetic beads enriching mast cells;

[0032] Appendix Figure 5 Flowchart of flow sorting;

[0033] Appendix Figure 6 Image showing the results of Mcpt4 positive and negative mast cell identification. Detailed Implementation

[0034] The present invention will be further described below with reference to specific embodiments. The features and advantages of the present invention will become clearer as the description unfolds, but the embodiments are merely illustrative and do not constitute any limitation on the scope of the invention.

[0035] All raw materials and equipment used in this invention are known products, obtained by purchasing commercially available products. All animal experiments in this invention were authorized by the Experimental Animal Management Committee of Hefei University of Technology. All experimental animals were housed in the animal facility of Hefei University of Technology in a clean environment with a temperature of (23±1)℃, humidity of (35±2)%, 12 hours of day and night lighting, and free access to food and water.

[0036] Example 1

[0037] 1. Construction of Mcpt4-cre;G / R genotype fluorescent tracer mice

[0038] (1) Homologous arms were generated by PCR using bacterial artificial chromosome clones RP24-167M24 and RP24-175A23 from the C57BL / 6 library as templates.

[0039] (2) “2A-Cre” serves as a template for homologous recombination repair;

[0040] (3) Design gRNA targeting the Mcpt4 gene sequence on chromosome 14 of C57BL / 6 mice, with the sequence 5'-CTGGATTAACAGAGTTATAAAGG-3'. At the same time, introduce two synonymous mutations I243 (ATA to ATT) and K244 (AAG to AAA) to prevent the gRNA from binding to the sequence and re-cutting after homology-directed repair.

[0041] (4) The gRNA and Cas9 were injected together with the targeting vector into the fertilized eggs and transplanted into the uterus of pseudopregnant mice to breed offspring mice.

[0042] (5) The offspring mice will undergo genotyping using Southern blot to screen for Mcpt4-cre mice. A schematic diagram of the mouse model construction is attached to the instruction manual. Figure 1 A.

[0043] 2. Mcpt4-cre gene identification

[0044] Southern blot analysis of the DNA samples confirmed the successful construction of Mcpt4-cre mice. The Southern blot analysis strategy is as follows:

[0045] The probe that binds to the 5' end of the Mcpt4 gene was labeled with ScaI, and the probe that binds to the 3' end of the Mcpt4 gene was labeled with AflII.

[0046] 5' probe primer sequence:

[0047] Forward primer:5'-CAGAAATGAAGGGAGCAAGGGAAAC-3'

[0048] Reverse primer:5'-GCATCTCCGCGTCCATAAGATACAA-3'

[0049] 3' probe primer sequence:

[0050] Forward primer:5'-AAGGGTGTGGCCCTCTAACTACTGTAC-3'

[0051] Reverse primer:5'-TTTCTTAGTGCTCCCTCCCTCCTGA-3'

[0052] The DNA binding membrane was treated with the 5' and 3' probes of the Mcpt4 gene, respectively, and analyzed by radiographic imaging. Results are shown in the appendix to the instruction manual. Figure 1 B. The presence of a 6.37 kb band binding to the 5' probe of the Mcpt4 gene and a 4.81 kb band binding to the 3' probe of the Mcpt4 gene in the Mcpt4-cre mouse DNA indicates that the Mcpt4-cre mouse was successfully constructed.

[0053] Example 2

[0054] 1. Construction of Mcpt4-cre;G / R genotype fluorescent tracer mice

[0055] (1) The Mcpt4-cre mice obtained in Example 1 were crossed with red-green fluorescent mice (the green fluorescent protein ZsGreen gene was inserted between two loxP sites, and the red fluorescent protein tdTomato gene was inserted downstream of it, while the mouse H11 gene site was knocked in simultaneously. Genotype: G / R-loxp. The red-green fluorescent mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) to obtain mice with the genotype Mcpt4-cre;G / R. See the attached instruction manual. Figure 2 A and 2B.

[0056] (2) Mcpt4-cre;G / R mice appear pale green under incandescent light and exhibit green fluorescence under ultraviolet light. See the instruction manual appendix. Figure 2 C.

[0057] 2. DNA was extracted from mouse tail tissue, Mcpt4-cre; G / R mouse genotyping was performed.

[0058] (1) Tissue digestion solution: an aqueous solution containing 25 mM sodium hydroxide and 0.2 mM disodium ethylenediaminetetraacetate;

[0059] (2) Digestion termination solution: an aqueous solution containing 40 mM Tris, with the pH adjusted to 8.0 by hydrochloric acid;

[0060] (3) Take the rat tail tissue and add 25 μL of tissue digestion solution. Digest at 98°C for 45 min. After digestion, add 25 μL of digestion termination solution to stop digestion and obtain mouse DNA suspension.

[0061] (4) Constructing a DNA amplification system:

[0062]

[0063]

[0064] (5) Primer sequences for Mcpt4-cre gene identification:

[0065] Forward primer 1: 5'-ATCTTATGGACGCGGAGATGC-3'

[0066] Reverse primer 1: 5'-ACCATTTCCGGTTATTCAACTTGC-3'

[0067] Reverse primer 2: 5'-CAGGGAACAGTCCATCATCACAG-3'

[0068] Mcpt4-cre DNA amplification program: 94℃ for 3 min; 94℃ for 30 s, 60℃ for 30 s, 65℃ for 75 s, cycle number 33; 65℃ for 10 min.

[0069] G / R-loxP gene identification primer sequences:

[0070] Forward primer 1: 5'-CCTCCTCTCCTGACTACTCCCAGTC-3'

[0071] Reverse primer 1: 5'-TCACAGAAACCATATGGCGCTCC-3'

[0072] G / R-loxP DNA amplification program: 95℃ for 5 min; 98℃ for 30 s, 65℃ (-0.5℃ / cycle) for 30 s, 72℃ for 45 s, cycle number 20; 98℃ for 30 s, 55℃ for 30 s, 72℃ for 45 s, cycle number 20; 72℃ for 5 min.

[0073] The DNA amplification products were identified by 1.5% agarose gel electrophoresis. See the attached instructions for the results. Figure 3 A. Mcpt4-cre identification results showed that Mcpt4-cre mice had one 275bp band and one 368bp band, while wild-type (WT) mice had only one 275bp band. G / R-loxP identification results showed that G / R-loxP mice had a single 1229bp band, while WT mice had no band.

[0074] Example 3

[0075] 1. Obtaining peritoneal mast cells (PCMCs)

[0076] (1) Eight-week-old Mcpt4-cre;G / R mice were intraperitoneally injected with 1 mL of sterile 3% mercaptoacetate solution to induce PCMC increase for 3 days;

[0077] (2) The mice were euthanized by being placed in CO2. The limbs of the mice were fixed and the abdominal skin was peeled off with scissors to expose the abdominal cavity.

[0078] (3) Inject 5 mL of PBS solution containing 10% FBS into the peritoneal cavity, rinse the peritoneal cavity repeatedly with a pipette and collect the irrigation fluid, repeat 3 times;

[0079] (4) Transfer the recovered irrigation fluid into a sterile 50ml centrifuge tube, centrifuge at 400g for 5min, discard the supernatant, add RPMI 1640 cell culture medium, and add 30ng / mL IL-3 and 30ng / mL SCF for in vitro induction.

[0080] RPMI 1640 cell culture medium composition: RPMI 1640, 0.2% sodium bicarbonate, 10% FBS, 60 nM β-mercaptoethanol, 100 mM non-essential amino acids, 1% penicillin-streptomycin solution;

[0081] Take 1 mL of PCMC cell suspension and place it in a centrifuge (purchased from Sigma, USA). Centrifuge at 400g for 5 min at 4°C, discard the supernatant, and fix with 300 μL of paraformaldehyde for 1 h. Transfer the cells to a glass slide using a cell smear centrifuge (purchased from Hunan Kecheng Instrument Equipment Co., Ltd.). Under a fluorescence microscope (purchased from Nikon, France), both red and green fluorescent cells were observed in the peritoneal cells (see attached instruction manual). Figure 3 B.

[0082] 2. Enrichment of Mcpt4-cre; G / R mouse PCMCs

[0083] (1) Take 50 μL of magnetic beads (purchased from Invitrogen, USA), wash twice with 1 mL of PBS buffer, then resuspend in PBS buffer to a final volume of 300 μL, and add 4 μL of FcεRⅠA antibody (purchased from Abcam, UK). Incubate overnight at 4°C. Wash three times with PBS buffer to obtain FcεRⅠA antibody-coated magnetic beads. Use a magnetic rack to separate the magnetic beads during the procedure.

[0084] (2) Mix PCMCs with FcεRⅠA antibody-coated magnetic beads and incubate at 4°C for 45 min. After incubation, place the mixture on a magnetic rack and let it stand for 1 min. Use the magnetic rack to enrich mast cells bound to the magnetic beads. See the instruction manual for a diagram illustrating the magnetic bead enrichment of mast cells. Figure 4 ;

[0085] (3) Wash three times with 1 mL PBS buffer. After each wash, fix the cells with a magnetic rack and remove the supernatant. Finally, resuspend the cells in 100 μL PBS buffer.

[0086] 3. Flow cytometry was used to separate red fluorescent PCMCs and green fluorescent PCMCs.

[0087] After enrichment with magnetic beads, PCMCs with red fluorescence and those with green fluorescence are separated by flow cytometry using the FITC and PE channels. See the attached instruction manual for the flow cytometry separation flowchart. Figure 5 .

[0088] 4. RT-PCR and staining analysis were used to determine the mast cell subtypes of red fluorescent PCMCs and green fluorescent PCMCs.

[0089] Cellular mRNA was extracted using the RNeasy Micro Handbook small-sample mRNA extraction kit (purchased from QIAGEN, Germany), and cDNA was obtained by reverse transcription using the HiScript III RT SuperMix for RT-PCR mRNA reverse transcription kit (purchased from Novizan Biosciences Co., Ltd., Nanjing). The cDNA amplification system was constructed as follows:

[0090]

[0091] Mcpt4 RT-PCR primer sequences

[0092] Forward primer: 5'-CTTCTGACTTTATCAAGCCGGG-3'

[0093] Reverse primer: 5'-CAGTCCAGTTCGCCCCC-3'

[0094] The expression level of Mcpt4 gene in PCMCs was detected by RT-PCR (see attached instruction manual). Figure 6 A. The results showed that red fluorescent PCMCs highly expressed the connective tissue mast cell marker gene Mcpt4.

[0095] 5. Toluidine blue staining was used to analyze the morphology of red fluorescent PCMCs and green fluorescent PCMCs.

[0096] (1) The red and green fluorescent PCMCs separated by flow cytometry were transferred to a 1.5 mL EP tube and fixed with 300 μL of formaldehyde for 1 h.

[0097] (2) After fixation, centrifuge at 400g, 4℃ for 5min, discard the supernatant, and resuspend in single-distilled water. Drop the cell suspension onto a glass slide, protect from light, and air dry.

[0098] (3) Add 20 μL of single-distilled water to a glass slide, cover it with a coverslip, observe the red and green fluorescence of the cells using an oil immersion microscope and take a picture;

[0099] (4) Remove the coverslip and place the slide in a 0.5% toluidine blue aqueous solution for 1 min at room temperature. After staining, rinse the slide in clean water to remove excess stain.

[0100] (5) Add 20 μL of single-distilled water to a glass slide, cover with a coverslip, locate the cells in the fluorescence photograph using a microscope, and observe the cell morphology using an oil immersion microscope. The staining results show that red fluorescent PCMCs appear blue-purple, and green fluorescent PCMCs appear pale blue. (See the instruction manual appendix.) Figure 6 B.

[0101] 6. Alcian blue-saffron analysis of red-green fluorescent mast cell morphology

[0102] (1) The red and green fluorescent PCMCs separated by flow cytometry were transferred to a 1.5 mL EP tube and fixed with 300 μL of formaldehyde for 1 h.

[0103] (2) After fixation, centrifuge at 400g, 4℃ for 5min, discard the supernatant, and resuspend in single-distilled water. Drop the cell suspension onto a glass slide, protect from light, and air dry.

[0104] (3) Add 20 μL of single-distilled water to a glass slide, cover it with a coverslip, observe the red and green fluorescence of the cells using an oil immersion microscope and take a picture;

[0105] (4) Remove the coverslip and place the slide in a 1% Alcian blue staining solution prepared with 0.7 mol / L hydrochloric acid aqueous solution. Stain at room temperature for 30 min. After staining, rinse the slide in 0.7 mol / L hydrochloric acid aqueous solution to remove excess staining solution.

[0106] (5) After Alcian blue staining, place the slide in 0.5% safranin staining solution and stain for 5 minutes. After staining, rinse with water to remove excess staining solution, then add 20 μL of single-distilled water and cover with a coverslip;

[0107] (6) The cells photographed under a microscope were located, and their morphology was observed under an oil immersion microscope. The staining results showed that the nuclei of red fluorescent PCMCs were red, and the cytoplasm was dark green; green fluorescent PCMCs appeared red. (See the instruction manual appendix.) Figure 6 C.

[0108] The above description is only a preferred embodiment of the present invention. For those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing a fluorescent tracer mouse, characterized by The method comprises the following steps: (1) using bacterial artificial chromosome clones RP24-167M24 and RP24-175A23 from C57BL / 6 library as templates, generating homologous arms by PCR, and taking "2A-Cre" as a homologous recombination repair template; (2) designing gRNA targeting the sequence of the Mcpt4 gene on chromosome 14 of the C57BL / 6 mouse; (3) co-injecting the homologous recombination repair template, gRNA, Cas9 and the targeting vector into a fertilized egg, implanting it into the uterus of a pseudopregnant mouse, and breeding the mouse to obtain a Mcpt4-cre mouse with the genotype Mcpt4-cre; (4) crossing the Mcpt4-cre mouse with a homologous mouse carrying a G / R-loxP recombinant gene to obtain a fluorescent tracing mouse with the genotype Mcpt4-cre; G / R; (5) in the fluorescent tracing mouse with the genotype Mcpt4-cre; G / R, the Mcpt4-positive cells genetically express tdTomato red fluorescent protein, and the Mcpt4-negative cells genetically express ZsGreen green fluorescent protein, and the red fluorescent protein tdTomato and the green fluorescent protein ZsGreen double fluorescent reporter system can be used to trace the Mcpt4-specific cells.

2. The application of the fluorescent tracing mouse constructed by the method of claim 1 in sorting Mcpt4-positive and negative mast cells.

3. A method of using the fluorescent tracer mouse constructed in claim 1 for sorting Mcpt4 positive and negative mast cells, characterized in that The method comprises the following steps: (1) washing the peritoneal cavity of the Mcpt4-cre; G / R mouse with PBS buffer to obtain a peritoneal cell suspension; (2) co-incubating the peritoneal cell suspension with magnetic beads having FcεRⅠA antibodies bound on the surface, so that the FcεRⅠA antibodies can specifically bind to the FCεRⅠ receptor on the surface of the mast cells to achieve enrichment of the mast cells; (3) sorting the enriched mast cells by using a flow cytometer to obtain Mcpt4-positive connective tissue mast cells and Mcpt4-negative mucosal mast cells.

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