Construction of fluorescent tracing mouse and application of fluorescent tracing mouse in sorting Mcpt4 positive and negative mast cells

Abstract

The invention provides a construction method of a fluorescent tracing mouse and application of the fluorescent tracing mouse in sorting Mcpt4 positive and negative mast cells. According to the invention, a Cre gene is knocked into an Mcpt4 gene sequence of a C57BL / 6 mouse by using a CRISPR / Cas9 technology, and then a fluorescence tracing mouse model of Mcpt4 positive mast cells is obtained through animal hybridization and a Cre-loxP recombination technology. The fluorescent tracing mouse model has a red fluorescent protein tdTomato and green fluorescent protein ZsGreen double-fluorescence reporter gene system, Mcpt4 positive mast cells show red fluorescence, and other Mcpt4 negative cells show green fluorescence. By utilizing the fluorescence tracing mouse model, the Mcpt4 positive and negative mast cells can be quickly, accurately and effectively sorted, and a reliable technical basis is provided for exploring the action and mechanism of the Mcpt4 positive and negative mast cells in immunoreaction.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering and biomedicine, and specifically relates to the construction of a fluorescent tracer mouse and the application of the fluorescent tracer mouse 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 of allergic disease pathology, playing a key role in tissue morphogenesis and host defense. Mouse mast cells are derived from bone marrow hematopoietic stem cells and can further differentiate into connective tissue mast cells (CTMC) or mucosal mast cells (MMC) in different local tissues. The chymotrypsin encoding gene Mcpt4 is specifically expressed in connective tissue mast cells, while Mcpt4 is not expressed in mucosal mast cells.

[0003] Due to the lack of an ideal mouse model for tracing and distinguishing CTMC from MMC, it is impossible to study in detail the differential roles and mechanisms of different mast cell subtypes in related immune responses. In order to better trace and sort different subtypes of mouse mast cells, it is urgent to construct an animal model that can be used to distinguish CTMC from MMC. Fluorescent protein is an important tracing tool in molecular biology research. By knocking in exogenous fluorescent protein expression elements into endogenous genes in mice, the target gene can be marked and traced, and applied to studies such as subcellular localization, expression profile, and cell lineage tracing of proteins. For example, the red fluorescent protein tdTomato gene is combined with the green fluorescent protein ZsGreen gene, knocked into the mouse genome sequence, and loxP sites are buried on both sides of the ZsGreen gene. When not hybridized with mice carrying the Cre recombinase gene, mice with ZsGreen-loxP and tdTomato knocked in the genome show green fluorescence of ZsGreen throughout the body. When this mouse is crossed with a mouse carrying the Cre recombinase gene, in the cells of their offspring that specifically express the target gene, the Cre recombinase acts to cause the ZsGreen gene to be deleted in the genome, and the relevant cells instead display the red fluorescence of tdTomato, thereby specifically fluorescently tracing the cells expressing the target gene. Summary of the invention

[0004] The object of the present invention is to provide a construction of a fluorescent tracer mouse and its application in the sorting of Mcpt4 positive and negative mast cells. The present invention first uses CRISPR / Cas9 technology to knock the Cre gene into the Mcpt4 gene sequence of C57BL / 6 mice, and then obtains a fluorescent tracer mouse model of Mcpt4 positive mast cells through animal hybridization and Cre-loxP recombination technology. The fluorescent tracer mouse model has a red fluorescent protein tdTomato and a green fluorescent protein ZsGreen dual fluorescent reporter gene system, Mcpt4 positive cells show red fluorescence, and other Mcpt4 negative cells show green fluorescence. The fluorescent tracer mouse constructed by the present invention is applied to the sorting of Mcpt4 positive and negative mast cells, and 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.

[0005] The technical purpose of the present invention is achieved through the following technical solutions.

[0006] 1. A method for constructing a fluorescent tracer mouse, comprising the following steps:

[0007] (1) Using bacterial artificial chromosome clones RP24-167M24 and RP24-175A23 from the C57BL / 6 library as templates, homology arms were generated by PCR, and “2A-Cre” was used as a homologous recombination repair template;

[0008] (2) Designing a gRNA targeting the Mcpt4 gene sequence on chromosome 14 of C57BL / 6 mice;

[0009] (3) injecting the homologous recombination repair template, gRNA, Cas9 and targeting vector into the fertilized egg, transplanting it into the uterus of pseudo-pregnant mice, and obtaining mice with the genotype of Mcpt4-cre through genetic identification;

[0010] (4) Mcpt4-cre mice were crossed with isogenic mice carrying the G / R-loxP recombinant gene to obtain fluorescent tracer mice with the genotype of Mcpt4-cre; G / R;

[0011] (5) In the fluorescent tracing mice with the genotype of Mcpt4-cre;G / R, Mcpt4-positive cells genetically express the tdTomato red fluorescent protein, and Mcpt4-negative cells genetically express the ZsGreen green fluorescent protein. The red fluorescent protein tdTomato and green fluorescent protein ZsGreen dual fluorescent reporter system can be used to perform red fluorescent tracing of cells that specifically express Mcpt4.

[0012] 2. A method for using fluorescent tracer mice in Mcpt4 positive and negative mast cell sorting, comprising the following steps:

[0013] (1) Wash the peritoneal cavity of Mcpt4-cre;G / R mice with PBS buffer to obtain peritoneal cell suspension;

[0014] (2) The peritoneal cell suspension is co-incubated with magnetic beads with FcεRⅠA antibodies on the surface. The FcεRⅠA antibodies can specifically bind to the FcεRⅠ receptors on the surface of mast cells, thereby enriching mast cells.

[0015] (3) The enriched mast cells were sorted by flow cytometry to obtain Mcpt4-positive connective tissue mast cells and Mcpt4-negative mucosal mast cells.

[0016] The beneficial technical effects of the present invention are embodied in the following aspects:

[0017] (1) The present invention uses CRISPR / Cas9 technology to knock the Cre gene into the Mcpt4 gene sequence of C57BL / 6 mice to obtain mice with the genotype of Mcpt4-cre. Mcpt4-cre mice can be flexibly used with different loxp mice, and Mcpt4-positive cells in mice can be specifically modified, thereby objectively and systematically exploring the role and mechanism of Mcpt4-positive mast cells in immune inflammation;

[0018] (2) The present invention obtains Mcpt4-cre; G / R fluorescent tracer mice by hybridizing 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;

[0019] (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;

[0020] (4) The present invention uses magnetic beads incubated with FcεRⅠA antibodies to enrich mast cells. The magnetic beads have a large specific surface area and can bind more FcεRⅠA antibodies, thereby improving the efficiency of mast cell enrichment. The cell surface FCεRⅠ antigen specifically binds to the FcεRⅠA antibody on the magnetic beads, and under the action of magnetic force, mast cells are separated from other cells. While simplifying the enrichment operation, it has the characteristics of high sensitivity, fast enrichment speed, high specificity, and good repeatability. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Attached Figure 1 Schematic diagram of the Mcpt4-cre mouse model constructed using CRISPR / Cas9 technology and the results of gene identification;

[0022] Attached Figure 2 Schematic diagram of Mcpt4-cre;G / R mouse model construction and mouse fluorescence images;

[0023] Attached Figure 3 Mcpt4-cre; G / R mouse genotype identification results and peritoneal cell fluorescence images;

[0024] Attached Figure 4 Schematic diagram of mast cell enrichment using magnetic beads;

[0025] Attached Figure 5 Flow sorting flow chart;

[0026] Attached Figure 6 Figure 1. Mcpt4-positive and -negative mast cell identification results. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with specific examples. The features and advantages of the present invention will become clearer with the description, but the examples are only used to illustrate the present invention and do not constitute any limitation to the scope of the present invention.

[0028] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products. All animal experiments in the present invention are approved by the Experimental Animal Management Committee of Hefei University of Technology. All experimental animals are raised in the animal room of Hefei University of Technology in a clean environment, with a temperature of (23±1)°C, a humidity of (35±2)%, 12h day and night lighting, and free access to food and water.

[0029] Example 1

[0030] 1. Construction of Mcpt4-cre; G / R genotype fluorescent tracer mice

[0031] (1) Using bacterial artificial chromosome clones RP24-167M24 and RP24-175A23 from the C57BL / 6 library as templates, homology arms were generated by PCR;

[0032] (2) “2A-Cre” as homologous recombination repair template;

[0033] (3) Design a gRNA targeting the Mcpt4 gene sequence on chromosome 14 of C57BL / 6 mice, whose sequence is 5'-CTGGATTAACAGAGTTATAAAGG-3', and introduce two synonymous mutations I243 (ATA to ATT) and K244 (AAG to AAA) to prevent gRNA binding and re-cutting of the sequence after homology-directed repair;

[0034] (4) The gRNA and Cas9 are co-injected into the fertilized egg together with the targeting vector, and then transplanted into the uterus of pseudo-pregnant mice to breed offspring mice;

[0035] (5) The offspring mice will be tested for genotype by Southern blot and Mcpt4-cre mice will be selected. Figure 1 A.

[0036] 2. Mcpt4-cre gene identification

[0037] Southern blot analysis of DNA samples confirmed that Mcpt4-cre mice were successfully constructed. The strategy for Southern blot analysis is as follows:

[0038] The probe binding to the 5' end of the Mcpt4 gene was labeled with ScaI, and the probe binding to the 3' end of the Mcpt4 gene was labeled with AflII;

[0039] 5' probe primer sequence:

[0040] Forward primer:5'-CAGAAATGAAGGGAGCAAGGGAAAC-3'

[0041] Reverse primer:5'-GCATCTCCGCGTCCATAAGATACAA-3'

[0042] 3' probe primer sequence:

[0043] Forward primer:5'-AAGGGTGTGGCCCTCTAACTACTGTAC-3'

[0044] Reverse primer:5'-TTTCTTAGTGCTCCCTCCCTCCTGA-3'

[0045] The DNA binding membrane was treated with the 5' end probe and 3' end probe of Mcpt4 gene respectively, and analyzed by radiography. The results are shown in the appendix of the instruction manual. Figure 1 B. The presence of a 6.37 kb band that binds to the 5' end probe of the Mcpt4 gene and a 4.81 kb band that binds to the 3' end probe of the Mcpt4 gene in the DNA of the Mcpt4-cre mouse indicated that the Mcpt4-cre mouse was successfully constructed.

[0046] Example 2

[0047] 1. Construction of Mcpt4-cre; G / R genotype fluorescent tracer mice

[0048] (1) The Mcpt4-cre mice obtained in Example 1 were hybridized with red-green fluorescent mice (the green fluorescent protein ZsGreen gene was inserted between two loxP sites, the red fluorescent protein tdTomato gene was inserted downstream, and the mouse H11 gene site was knocked in at the same time. Genotype: G / R-loxp. The red-green fluorescent mice were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) to obtain mice with the genotype of Mcpt4-cre; G / R. See the attached manual Figure 2 A and 2B.

[0049] (2) Mcpt4-cre; G / R mice appear light green under incandescent light and green fluorescence under ultraviolet light. Figure 2 C.

[0050] 2. Extract DNA from mouse tail tissue and identify the genotype of Mcpt4-cre; G / R mice

[0051] (1) Tissue digestion solution: an aqueous solution containing 25 mM sodium hydroxide and 0.2 mM disodium EDTA;

[0052] (2) Digestion stop solution: an aqueous solution containing 40 mM Tris, with pH adjusted to 8.0 with hydrochloric acid;

[0053] (3) Take the mouse tail tissue and add 25 μL of tissue digestion solution, digest at 98°C for 45 min, and add 25 μL of digestion stop solution to terminate the digestion to obtain a mouse DNA suspension;

[0054] (4) Construction of DNA amplification system:

[0055]

[0056]

[0057] (5) Mcpt4-cre gene identification primer sequence:

[0058] Forward primer 1: 5'-ATCTTATGGACGCGGAGATGC-3'

[0059] Reverse primer 1: 5'-ACCATTTCCGGTTATTCAACTTGC-3'

[0060] Reverse primer 2: 5'-CAGGGAACAGTCCATCATCACAG-3'

[0061] Mcpt4-cre DNA amplification program: 94°C for 3 min; 94°C for 30 s, 60°C for 30 s, 65°C for 75 s, the number of cycles is 33; 65°C for 10 min.

[0062] G / R-loxP gene identification primer sequence:

[0063] Forward primer 1: 5'-CCTCCTCTCCTGACTACTCCCAGTC-3'

[0064] Reverse primer 1: 5'-TCACAGAAACCATATGGCGCTCC-3'

[0065] G / R-loxP DNA amplification program: 95°C for 5 min; 98°C for 30 s, 65°C (-0.5°C / cycle) for 30 s, 72°C for 45 s, the number of cycles is 20; 98°C for 30 s, 55°C for 30 s, 72°C for 45 s, the number of cycles is 20; 72°C for 5 min.

[0066] The DNA amplification product was subjected to 1.5% agarose gel electrophoresis, and the identification results are shown in the appendix of the instruction manual. Figure 3 A. Mcpt4-cre identification results showed that Mcpt4-cre mice had a 275bp band and a 368bp band, while wild type (WT) mice had only a 275bp band. G / R-loxP identification results showed that G / R-loxP mice had a single 1229bp band, while WT mice had no band.

[0067] Example 3

[0068] 1. Obtaining peritoneal mast cells (PCMC)

[0069] (1) 8-week-old Mcpt4-cre;G / R mice were intraperitoneally injected with 1 mL of sterilized 3% thioglycolate solution to induce PCMC increase for 3 days;

[0070] (2) The mice were placed in CO2 and asphyxiated to death. The limbs of the mice were fixed and the abdominal skin was peeled open with scissors to expose the abdominal cavity.

[0071] (3) Inject 5 mL of PBS solution containing 10% FBS into the peritoneal cavity, repeatedly flush the peritoneal cavity with a pipette and recover the lavage fluid, repeating this process three times;

[0072] (4) The recovered lavage fluid was transferred into a sterile 50 ml centrifuge tube, centrifuged at 400 g for 5 min, the supernatant was discarded, RPMI 1640 cell culture medium was added, and 30 ng / mL IL-3 and 30 ng / mL SCF were added for in vitro induction;

[0073] 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 double antibody solution;

[0074] 1 mL of PCMC cell suspension was placed in a centrifuge (purchased from Sigma, USA), centrifuged at 400 g for 5 min at 4°C, the supernatant was discarded, and fixed with 300 μL of paraformaldehyde for 1 h. The cells were transferred to a glass slide using a cell smear centrifuge (purchased from Hunan Kecheng Instrument Equipment Co., Ltd.), and the presence of red fluorescent cells and green fluorescent cells in the peritoneal cells was observed under a fluorescence microscope (purchased from Nikon, France), see the attached manual Figure 3 B.

[0075] 2. PCMC enrichment in Mcpt4-cre;G / R mice

[0076] (1) Take 50 μL of magnetic beads (purchased from Invitrogen, USA), wash them twice with 1 mL of PBS buffer, resuspend them to 300 μL with PBS buffer, add 4 μL of FcεRⅠA antibody (purchased from Abcam, UK), and incubate them at 4°C overnight. Wash them three times with PBS buffer to obtain magnetic beads coated with FcεRⅠA antibody. Use a magnetic stand to separate the magnetic beads during the operation;

[0077] (2) Mix PCMC and magnetic beads coated with FcεRⅠA antibody and incubate at 4°C for 45 minutes. After incubation, place on a magnetic rack for 1 minute and use the magnetic rack to enrich mast cells bound to magnetic beads. See the attached manual for a schematic diagram of mast cell enrichment with magnetic beads. Figure 4 ;

[0078] (3) Wash three times with 1 mL of PBS buffer. After each wash, fix the cells with a magnetic rack, remove the supernatant, and finally resuspend them in 100 μL of PBS buffer.

[0079] 3. Flow cytometry sorting of red fluorescent PCMC and green fluorescent PCMC

[0080] After PCMC is enriched by magnetic beads, red fluorescent PCMC and green fluorescent PCMC are separated through the FITC channel and PE channel of flow cytometry. See the attached manual for the flow chart of flow cytometry separation. Figure 5 .

[0081] 4. RT-PCR and staining analysis to determine the mast cell subtypes of red fluorescent PCMC and green fluorescent PCMC

[0082] Cellular mRNA was extracted using the small sample mRNA extraction kit RNeasy Micro Handbook (purchased from QIAGEN, Germany), and reverse transcribed using the mRNA reverse transcription kit HiScript III RT SuperMix for RT-PCR (purchased from Novozyme Biotech Co., Ltd., Nanjing) to obtain cDNA. Construction of cDNA amplification system:

[0083]

[0084] Mcpt4 RT-PCR primer sequences

[0085] Forward primer: 5'-CTTCTGACTTTATCAAGCCGGG-3'

[0086] Reverse primer: 5'-CAGTCCAGTTCGCCCCC-3'

[0087] The expression level of Mcpt4 gene in PCMC was detected by RT-PCR. Figure 6 A, The results showed that red fluorescent PCMCs highly expressed the connective tissue mast cell marker gene Mcpt4.

[0088] 5. Toluidine blue staining to analyze the morphology of red fluorescent PCMC and green fluorescent PCMC

[0089] (1) The red fluorescent and green fluorescent PCMC separated by flow cytometry were transferred to a 1.5 mL EP tube and fixed with 300 μL formaldehyde for 1 h;

[0090] (2) After fixation, centrifuge at 400g, 4°C for 5 min, discard the supernatant, and resuspend in distilled water. Drop the cell suspension on a glass slide, protect from light, and air dry.

[0091] (3) Add 20 μL of distilled water on the slide and cover it with a coverslip. Use the oil objective of a fluorescence microscope to observe the red and green fluorescence of the cells and take pictures.

[0092] (4) Remove the coverslip and place the slide in a 0.5% toluidine blue aqueous solution for staining at room temperature for 1 min. After staining, wash off the excess stain in clean water;

[0093] (5) Add 20 μL of distilled water to the slide and cover it with a coverslip. Use a microscope to locate the cells for fluorescence photography and use an oil immersion lens to observe the cell morphology. The staining results show that red fluorescent PCMC is blue-purple and green fluorescent PCMC is light blue. See the attached manual for details. Figure 6 B.

[0094] 6. Analysis of red and green fluorescent mast cell morphology using Alcian blue-safranin

[0095] (1) The red fluorescent and green fluorescent PCMC separated by flow cytometry were transferred to a 1.5 mL EP tube and fixed with 300 μL formaldehyde for 1 h;

[0096] (2) After fixation, centrifuge at 400g, 4°C for 5 min, discard the supernatant, and resuspend in distilled water. Drop the cell suspension on a glass slide, protect from light, and air dry.

[0097] (3) Add 20 μL of distilled water on the slide and cover it with a coverslip. Use the oil objective of a fluorescence microscope to observe the red and green fluorescence of the cells and take pictures.

[0098] (4) Remove the cover glass and place the slide in 1% Alcian blue dye solution prepared in 0.7 mol / L hydrochloric acid aqueous solution and dye at room temperature for 30 min. After dyeing, place the slide in 0.7 mol / L hydrochloric acid aqueous solution to wash away excess dye solution;

[0099] (5) After the Alcian blue staining is completed, place the slide in 0.5% safranin staining solution and stain for 5 minutes. After the staining is completed, wash off the excess stain in clean water, then add 20 μL of single distilled water and cover with a coverslip;

[0100] (6) Use a microscope to locate the cells for fluorescence photography, and use an oil immersion lens to observe the morphology of the cells. The staining results show that the nucleus of red fluorescent PCMC is red and the cytoplasm is dark green; green fluorescent PCMC is red. See the instructions for Figure 6 C.

[0101] The above is only a preferred embodiment of the present invention. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the technical principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for constructing a fluorescent tracer mouse, characterized in that The following steps are involved: (1) Using bacterial artificial chromosome clones RP24-167M24 and RP24-175A23 from the C57BL / 6 library as templates, homology arms were generated by PCR, and "2A-Cre" was used as a homologous recombination repair template; (2) Designing a gRNA targeting the Mcpt4 gene sequence on chromosome 14 of C57BL / 6 mice; (3) The homologous recombination repair template, gRNA, Cas9 and targeting vector were co-injected into fertilized eggs, which were then transplanted into the uterus of pseudo-pregnant mice. The bred mice were genetically identified and the genotype was Mcpt4-cre mice; (4) Mcpt4-cre mice were crossed with isogenic mice carrying the G / R-loxP recombinant gene to obtain fluorescent tracer mice with the genotype of Mcpt4-cre; G / R; (5) In the fluorescent tracing mice with the genotype of Mcpt4-cre;G / R, Mcpt4-positive cells genetically express the tdTomato red fluorescent protein, and Mcpt4-negative cells genetically express the ZsGreen green fluorescent protein. The red fluorescent protein tdTomato and green fluorescent protein ZsGreen dual fluorescent reporter system can be used to perform red fluorescent tracing of cells that specifically express Mcpt4.

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

3. A method for using the fluorescent tracer mouse constructed according to claim 1 in the separation of Mcpt4 positive and negative mast cells, characterized in that The following steps are involved: (1) Wash the peritoneal cavity of Mcpt4-cre;G / R mice with PBS buffer to obtain peritoneal cell suspension; (2) The peritoneal cell suspension is co-incubated with magnetic beads with FcεRⅠA antibodies on the surface. The FcεRⅠA antibodies can specifically bind to the FCεRⅠ receptors on the surface of mast cells, thereby enriching mast cells. (3) The enriched mast cells were sorted by flow cytometry to obtain Mcpt4-positive connective tissue mast cells and Mcpt4-negative mucosal mast cells.

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