A mouse model of renal resident macrophage depletion and construction method and application thereof

Abstract

The application belongs to the technical field of genetic engineering and experimental animal models, and provides a kidney resident macrophage deletion mouse model and a construction method and application thereof. flox / flox The Csf1r-P2A-CreERT2 transgenic mouse is crossed with the DTA flox / flox The reporter mouse is crossed to obtain a double-positive mouse; after induction by tamoxifen, the CreERT2 enters the nucleus to excise the stop element between LoxP in the macrophage, to start the expression of DTA, to inhibit the peptide chain elongation factor-2 and to induce the apoptosis of target cells, to realize the conditional inducible knockout of the kidney resident macrophage. The model has high induction efficiency and strong targeting specificity; when not induced, the mouse develops normally and has a survival period of more than half a year, is suitable for long-term animal experiments, and avoids the non-specific side effects of traditional whole-body knockout and chemical clearance. The model provides a precise and controllable animal tool for the research of kidney immune mechanisms and the screening of drug targets for kidney diseases.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering and experimental animal model technology, specifically relating to a mouse model lacking intrinsic kidney macrophages, its construction method, and its application. Background Technology

[0002] Macrophages are an important component of the body's innate immune system, widely distributed throughout tissues and organs. Kidney-resident macrophages are one of the most important immune cell populations in the kidney, playing crucial roles in maintaining renal homeostasis, clearing apoptotic cells, regulating inflammatory responses, and participating in tissue repair. Recent studies have shown that kidney-resident macrophages play a significant role in various kidney diseases, including acute kidney injury, chronic kidney disease, diabetic nephropathy, autoimmune nephritis, renal fibrosis, and transplant rejection. However, due to the widespread distribution and functional heterogeneity of macrophages in the body, their specific mechanisms of action in particular kidney diseases remain not fully elucidated.

[0003] Traditional methods for studying macrophage function mainly include pharmacological clearance (such as using clophosphonate liposomes) and systemic gene knockout. However, clophosphonate liposome-mediated macrophage clearance lacks cell type specificity, can simultaneously clear macrophages from multiple tissues, and the clearance effect varies significantly depending on the route of administration and dosage. Systemic gene knockout strategies often lead to embryonic lethality or severe developmental defects, making them unsuitable for functional studies in adult animals. Furthermore, none of the above methods can achieve precise manipulation of macrophages in specific tissues or at specific developmental stages, severely limiting in-depth research on the function of intrinsic kidney macrophages.

[0004] The Cre-LoxP recombination system is one of the most widely used conditional gene manipulation techniques. Combined with the tamoxifen-inducible CreERT2 system, it can achieve time-specific and cell-type-specific gene modification. Diphtheria toxin A subunit (DTA) is a highly efficient cell-killing molecule; after entering the cell, it catalyzes the inactivation of peptide chain elongation factor-2, leading to protein synthesis termination and target cell apoptosis. Combining the above techniques with macrophage-specific promoters holds promise for achieving inducible, conditional clearance of intrinsic renal macrophages, providing an ideal animal model for studying their role in kidney diseases. Summary of the Invention

[0005] The purpose of this invention is to provide a mouse model of renal intrinsic macrophage deficiency, its construction method and application. By combining the Csf1r-mediated tamoxifen-induced Cre-LoxP system with the DTA toxin killing strategy, the invention achieves spatiotemporally specific, efficient and controllable conditional clearance of renal intrinsic macrophages. This solves the problems of existing macrophage clearance methods, such as strong nonspecificity, easy embryonic lethality, inability to target renal intrinsic macrophages alone, and difficulty in conducting long-term animal experiments on kidney diseases.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: A method for constructing a mouse model lacking intrinsic renal macrophages, comprising the following steps:

[0007] (1) Breeding Csf1r-P2A-CreERT2 mice: Csf1r-P2A-CreERT2 mice were crossbred with C57BL / 6JGpt wild-type mice, and the genomic DNA of the offspring tails was extracted. The genotypes of the offspring were identified by PCR using 3' arm primer pair T006204-F1 / T006204-R1 and wild-type primer pair T006204-F2 / T006204-R2. Csf1r-P2A-CreERT2 mice were obtained by screening.

[0008] (2) Breeding DTA flox / flox Mice: Rosa26-LoxP-ZsGreen-STOP-LoxP-DTA mice were bred with C57BL / 6JGpt mice. Genomic DNA was extracted from the tails of the offspring. PCR genotyping was performed using 5' ARM primer pair T009408-F1 / T009408-R1 and wild-type primer pair T009408-F2 / T009408-R2 to screen for DTA mice. flox / flox Mice;

[0009] (3) Preparation of F1 generation double-positive mice: The Csf1r-P2A-CreERT2 mice obtained in step (1) and the DTA mice obtained in step (2) were used to prepare the F1 generation double-positive mice. flox / flox Mice were mated, and the offspring were subjected to Csf1r-P2A-CreERT2 and DTA treatments. flox / flox Dual-gene PCR identification was used to screen F1 generation double-positive mice that simultaneously carry two gene modifications.

[0010] (4) Breeding F2 generation double-positive mice: F1 generation double-positive mice were bred with DTAf lox / flox Mouse backcrosses were performed, and the offspring were subjected to dual-gene PCR identification to screen for Csf1r-P2A-CreERT2-DTA. flox / flox Double-positive mice;

[0011] (5) Tamoxifen was administered to the double-positive mice obtained in step (4) to induce CreERT2 to enter the nucleus and specifically remove the termination element between two LoxP sites in the intrinsic renal macrophages to enable DTA expression and induce apoptosis of the intrinsic renal macrophages, thus obtaining the intrinsic renal macrophage-deficient mouse model.

[0012] Preferably, the identification primer sequence for Csf1r-P2A-CreERT2 is as follows:

[0013] T006204-F1: 5'-ACAGCATGAAGTGCAAAGAACGTGGT-3',

[0014] T006204-R1: 5'-GTGGTTCCACCCACTCTCACCT-3';

[0015] T006204-F2: 5'-GGAAGAGGAGCCAGTGCAAACA-3',

[0016] T006204-R2: 5'-GTGGTTCCACCCACTCTCACCT-3'.

[0017] Preferably, the DTA flox / flox The identification primer sequences are as follows:

[0018] T009408-F1: 5'-TCAAATGGCTCTCCCTCAAGCGT-3',

[0019] T009408-R1: 5'-TGGTACATGACGTTTCTCCCACC-3';

[0020] T009408-F2: 5'-CCAAAAGTCGCTCTGAGTTGTTA-3',

[0021] T009408-R2: 5'-TCGGGTGAGCATGTCTTTTAATCT-3'.

[0022] Preferably, the administration of tamoxifen in step (5) is by intraperitoneal injection, with an injection dose of 100 mg / kg based on the mouse's body weight. The injection is given once every other day for a total of 4 injections, with an injection cycle of 7 days.

[0023] Preferably, the induction time in step (5) is from day 42 to day 49 after the mice are born.

[0024] Preferably, the macrophages mentioned in step (5) are intrinsic renal macrophages, including F4 / 80. hi CD11blo Macrophage subsets.

[0025] The present invention further provides a mouse model of kidney-specific macrophage deficiency obtained by the above construction method.

[0026] Preferably, in the mouse model, macrophages develop normally and DTA is not expressed when tamoxifen is not induced; after tamoxifen induction, intrinsic renal macrophages are absent, and the absence efficiency is detected by flow cytometry.

[0027] The present invention further provides the application of the above-mentioned mouse model with specific deficiency of renal intrinsic macrophages in the preparation of animal models for studying the involvement of renal intrinsic macrophages in acute kidney injury, chronic kidney disease, autoimmune nephritis, renal fibrosis or transplant rejection.

[0028] The present invention further provides the application of the above-mentioned mouse model of specific deficiency of renal intrinsic macrophages in screening or evaluating drugs or treatment strategies that regulate the function of renal intrinsic macrophages.

[0029] The beneficial effects of this invention are:

[0030] This invention provides a mouse model of renal intrinsic macrophage deficiency. It utilizes a cross between Csf1r-P2A-CreERT2 transgenic mice and Rosa26-LoxP-ZsGreen-STOP-LoxP-DTA reporter mice, achieving conditional and inducible clearance of renal intrinsic macrophages through tamoxifen induction. Csf1r promoter-driven CreERT2 expression ensures high macrophage specificity, avoiding the non-specific side effects of traditional systemic knockout or chemical clearance methods. The specific induction strategy of this invention results in a mouse model that develops normally and exhibits good viability without induction, with a long survival period exceeding six months, effectively ensuring the smooth progress of long-term experiments. The renal intrinsic macrophage deficiency mouse model obtained through this invention shows great promise for applications in renal immunology, renal disease mechanism research, and drug target screening. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the primer design strategy for Csf1r-P2A-CreERT2 genotyping in this invention (wild type: no amplification band in PCR reaction ①; wild type band in PCR reaction ②; heterozygous: site-directed mutation band in PCR reaction ①; wild type band in PCR reaction ②; homozygous mutant: site-directed mutation band in PCR reaction ①; no amplification band in PCR reaction ②).

[0032] Figure 2 DTA in the embodiments of the present inventionflox / flox Schematic diagram of primer design strategy for genotyping (Wild type: No amplification band in PCR reaction ①; Wild type band appears in PCR reaction ②; Heterozygote: Site-directed mutation band appears in PCR reaction ①; Wild type band appears in PCR reaction ②; Homozygous mutant: Site-directed mutation band appears in PCR reaction ①; No amplification band appears in PCR reaction ②).

[0033] Figure 3 The electrophoresis results of 3' arm PCR identification and WT PCR identification of Csf1r-P2A-CreERT2 transgenic mice in this embodiment of the invention are shown.

[0034] Figure 4 DTA in the embodiments of the present invention flox / flox Results of PCR identification electrophoresis in mice and PCR identification electrophoresis in WT mice;

[0035] Figure 5 Csf1r-P2A-CreERT2-DTA in this embodiment of the invention flox / flox Genotyping results of double-positive mice (①: DTA reporter gene allele-specific amplification band; ②: Csf1r modified allele-specific amplification band; HO: homozygous; HE: heterozygous).

[0036] Figure 6 This is the result of flow cytometry analysis of renal intrinsic macrophages (KRMs) after tamoxifen induction in this embodiment of the invention (phenotype F4 / 80). hi CD11b lo Infiltrative macrophages (MDRMs), phenotype F4 / 80 lo CD11b hi WT group: wild-type mice with intact KRMs (as control group); DTA group: mice carrying diphtheria toxin A chain (DTA) transgene, which can specifically induce KRM ablation (as experimental group). Detailed Implementation

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] Example 1: Construction of a mouse model lacking intrinsic renal macrophages

[0041] 1. Materials

[0042] Both Csf1r-P2A-CreERT2 mice (strain number T006204) and Rosa26-LoxP-ZsGreen-STOP-LoxP-DTA mice (strain number T009408) are commercially available.

[0043] 2. Methods

[0044] 2.1 Mouse gene extraction method

[0045] (1) Method 1: Use the TaKaRa MiniBEST Universal Genomic DNA Extraction Kit to obtain high-purity genomic DNA. The specific steps are as follows:

[0046] ① Cut off approximately 3 mm of the mouse's tail;

[0047] ② Add 180 μL buffer GL, 20 μL proteinase K, 10 μL RNase A (provided by TaKaRaMiniBEST Universal Genomic DNA Extraction Kit) and the mouse tail from step ① to a microcentrifuge tube, and incubate overnight at 56 ℃;

[0048] ③ Place the microcentrifuge tube from step ② into a microcentrifuge and centrifuge at 12000 rpm for 2 min. Take the supernatant and discard the precipitate.

[0049] ④ Add 200 μL Buffer GB (provided by TaKaRa MiniBEST Universal Genomic DNA Extraction Kit) and 200 μL anhydrous ethanol to the supernatant from step ③, mix thoroughly, and then transfer to a centrifuge column (provided by TaKaRa MiniBEST Universal Genomic DNA Extraction Kit).

[0050] ⑤ Place the centrifuge column in the collection tube and centrifuge at 12,000 rpm for 2 minutes, then discard the liquid in the collection tube;

[0051] ⑥ Add 500 μL of Buffer WA (provided by TaKaRa MiniBEST Universal Genomic DNA Extraction Kit) to the centrifuge column, centrifuge at 12000 rpm for 1 min, and discard the liquid in the collection tube;

[0052] ⑦ Add 700 μL of Buffer WB (provided by TaKaRa MiniBEST Universal Genomic DNA Extraction Kit) to the centrifuge column, centrifuge at 12000 rpm for 1 min, and discard the liquid in the collection tube (Note: Ensure that Buffer WB has been premixed with anhydrous ethanol. Add Buffer WB along the tube wall to wash away any residual salts on the collection tube wall).

[0053] ⑧ Repeat step ⑦;

[0054] ⑨ Place the centrifuge column in the collection tube and centrifuge at 12,000 rpm for 2 minutes, then discard the liquid in the collection tube;

[0055] ⑩ Transfer the centrifuge column to a new 1.5 mL centrifuge tube, add 50-200 μL of sterile water or elution buffer to the center of the column membrane, let stand for 5 min, then centrifuge at 12000 rpm for 2 min. The elution buffer at the bottom of the 1.5 mL centrifuge tube is the eluted genomic DNA (Note: Preheating the sterile water or elution buffer to 65 ℃ before use can improve the elution efficiency of genomic DNA).

[0056] Add the elution buffer from step 10 and / or 50-200 μL of sterile water or elution buffer to the center of the column membrane of the centrifuge column. After standing for 5 min, centrifuge at 12000 rpm for 2 min to increase the DNA yield. The liquid at the bottom of the 1.5 mL centrifuge tube after centrifugation is the mouse genomic DNA.

[0057] Genomic DNA quantification: The genomic DNA obtained by elution can be quantified by electrophoresis.

[0058] (2) Method 2: A low-cost, crude method for extracting genomic DNA, the specific steps of which are as follows:

[0059] ① Cut off approximately 3 mm of the mouse's tail;

[0060] ② Add 100 μL of mouse tail digestion buffer from step ① to a microcentrifuge tube and incubate overnight at 56 ℃;

[0061] ③ Transfer the microcentrifuge tube from step ② to 98 °C and incubate for 13 min to denature proteinase K;

[0062] ④ Centrifuge the microcentrifuge tube at 15,000 rpm for 15 min, and collect the supernatant, which is the crude extracted mouse genomic DNA. The mouse tail digestion buffer contains 50 mM KCl, 10 mM Tris-HCl (pH 9.0), 0.1% Triton X-100, and 0.4 mg / mL proteinase K.

[0063] 2.2 Model Construction Method

[0064] (1) Breeding and Genotyping of Csf1r-P2A-CreERT2 Transgenic Mice: Csf1r-P2A-CreERT2 mice were bred by mating with C57BL / 6JGpt wild-type mice. Mouse genomes were extracted using a mouse genome extraction method, and genotyping was performed by PCR. Primers used for PCR identification included the 3' arm identification primer pair T006204-F1 / T006204-R1 and the wild-type identification primer pair T006204-F2 / T006204-R2 (design strategy as follows). Figure 1 (As shown in Table 1). The 3' arm primer pair was used to amplify the 3' homologous arm sequence of the Csf1r gene, producing a positive band of 469 bp, confirming correct integration of the targeting vector. The wild-type primer pair was used to amplify the wild-type allele, producing a wild-type band of 334 bp and a targeting allele band of 2368 bp. Primer sequences are shown in Table 1.

[0065] Table 1. Amplification primer sequences (SEQ ID NIO.1~3)

[0066] T006204-F1 SEQ ID NO.1 5'-ACAGCATGAAGTGCAAAGAACGTGGT-3' T006204-R1 SEQ ID NO.2 5'-GTGGTTCCACCCACTCTCACCT-3' T006204-F2 SEQ ID NO.3 5'-GGAAGAGGAGCCAGTGCAAACA-3' T006204-R2 Same as SEQ ID NO.2 5'-GTGGTTCCACCCACTCTCACCT-3'

[0067] The PCR reaction system for the primer pairs is shown in Table 2. The PCR amplification program was as follows: 95 ℃ pre-denaturation for 5 min; 98 ℃ denaturation for 30 s, 65 ℃ annealing for 30 s (decreasing by 0.5 ℃ per cycle), 72 ℃ extension for 45 s, for a total of 20 cycles; 98 ℃ denaturation for 30 s, 55 ℃ annealing for 30 s, 72 ℃ extension for 45 s, for a total of 20 cycles; and finally, 72 ℃ final extension for 5 min, and storage at 4 ℃.

[0068] The results of the identification show that ( Figure 3 ), 3' arm PCR of Csf1r-P2A-CreERT2 heterozygous mice showed a 469 bp positive band, while wild-type mice showed no band; WT PCR showed that heterozygous mice had both a 334 bp wild-type band and a 2368 bp target band.

[0069] Table 2 PCR reaction system

[0070] Components Volume (μL) Rat tail genomic DNA 3 Forward primer (10 μM) 0.5 Reverse primer (10 μM) 0.5 Premixed Taq polymerase 13 Double distilled water 8 total 25

[0071] (2) DTA flox / floxBreeding and Genotyping of Report Mice: Rosa26-LoxP-ZsGreen-STOP-LoxP-DTA mice were bred by mating with C57BL / 6JGpt wild-type mice. Mouse genomes were extracted using a mouse genome extraction method, and genotyping was performed by PCR. Primers used for PCR identification included the 5' arm identification primer pair T009408-F1 / T009408-R1 and the wild-type identification primer pair T009408-F2 / T009408-R2 (design strategy as follows). Figure 2 (As shown in Table 3). The 5' arm primer pair was used to amplify the 5' homologous arm sequence, producing a positive band of 597 bp, confirming correct integration of the targeting vector. The wild-type primer pair was used to amplify the wild-type allele, producing a wild-type band of 479 bp and a targeting allele band of 8217 bp. Primer sequences are shown in Table 3.

[0072] Table 3. Amplification primer sequences (SEQ ID NIO.4~7)

[0073] name serial number sequence T009408-F1 SEQ ID NO.4 5'-TCAAATGGCTCTCCCTCAAGCGT-3' T009408-R1 SEQ ID NO.5 5'-TGGTACATGACGTTCTCTCCACC-3' T009408-F2 SEQ ID NO.6 5'-CCAAAAGTCGCTCTGAGTTGTTA-3' T009408-R2 SEQ ID NO.7 5'-TCGGGTGAGCATGTCTTTTAATCT-3'

[0074] The PCR reaction system for the primer pairs is shown in Table 2. The PCR amplification procedure was the same as above. The identification results showed ( Figure 4 DTA flox / flox Heterozygous mice showed a 597 bp positive band in 5' arm PCR, while wild-type mice showed no band; WT PCR showed that heterozygous mice had both a 479 bp wild-type band and an 8217 bp target band.

[0075] (3) Construction and screening of F1 generation double-positive mice: The Csf1r-P2A-CreERT2 heterozygous mice identified as positive above were compared with DTA mice. flox / flox Csf1r-P2A-CreERT2-DTA was obtained by mating homozygous mice. flox / flox F1 generation double-positive mice were obtained. Genomes of F1 generation mice were extracted using a mouse genome extraction method, and genotyping was performed by PCR. The aforementioned Csf1r-P2A-CreERT2 and DTA were also used. flox / flox The primer pairs were used for dual-gene identification to screen out double-positive mice that simultaneously carry two gene modifications.

[0076] (4) Construction and screening of F2 generation double-positive homozygous mice: F1 generation double-positive heterozygous mice were crossed with DTA. flox / flox Backcrossing homozygous mice yielded Csf1r-P2A-CreERT2-DTA. flox / floxF2 generation double-positive homozygous mice. Genomes were extracted using mouse genome extraction methods, and genotyping was performed by PCR. The identification results are as follows: Figure 5 As shown, it was successfully screened that individuals simultaneously carry Csf1r-P2A-CreERT2 (3'arm positive) and DTA. flox / flox (5'arm positive, WT negative) double-positive homozygous mice.

[0077] Example 2: Detection of intrinsic renal macrophages in tamoxifen-induced conditional knockout mice

[0078] Specific experimental methods:

[0079] (1) Preparation of tamoxifen reagent: Dissolve tamoxifen in corn oil at a concentration of 50 mg / mL, shake overnight at 37 °C, and sonicate at 37 °C for 30 min until completely dissolved.

[0080] (2) Intraperitoneal injection: Take 8-10 week old Csf1r-P2A-CreERT2-DTAf lox / flox Double-positive mice were administered the drug at a dose of 100 mg / kg (based on a tamoxifen concentration of 50 mg / mL), via intraperitoneal injection every other day for a total of four injections over a seven-day period. A control group without tamoxifen, Csf1r-P2A-CreERT2-DTA, was also included. flox / flox Double-positive mice served as the negative control group, and wild-type mice injected with an equal amount of corn oil served as the solvent control group.

[0081] (3) Flow cytometry analysis of intrinsic renal macrophages: Mice were sacrificed on day 7 after the last injection, and kidney tissue was collected. After perfusion and washing with sterile PBS, the tissue was minced and digested with collagenase IV and DNase I at 37 °C for 30 min. Single-cell suspension was obtained after filtration through a 70 μm cell sieve. APC-labeled F4 / 80 antibody and PE-labeled CD11b antibody were added sequentially, and the mixture was gently vortexed and incubated at room temperature in the dark for 30 min. After washing with 2 mL of PBS, the cells were resuspended and analyzed by flow cytometry. CD45 + F4 / 80 lo CD11b hi The cell population was defined as intrinsic renal macrophages, and their proportion in each group of mice was analyzed.

[0082] Flow cytometry results showed ( Figure 6 ), Csf1r-P2A-CreERT2-DTA induced by tamoxifen flox / flox The F4 / 80 ratio in the kidney tissue of mice was higher than that in the uninduced control group. hi CD11blo The proportion of macrophages decreased significantly, and the macrophage loss efficiency reached more than 80%, indicating that the mouse model of kidney intrinsic macrophage loss was successfully constructed.

[0083] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for constructing a mouse model lacking intrinsic renal macrophages, characterized in that, Includes the following steps: (1) Breeding Csf1r-P2A-CreERT2 mice: Csf1r-P2A-CreERT2 mice were crossbred with C57BL / 6JGpt wild-type mice, and the genomic DNA of the offspring tails was extracted. The genotypes of the offspring were identified by PCR using 3' arm primer pair T006204-F1 / T006204-R1 and wild-type primer pair T006204-F2 / T006204-R2. Csf1r-P2A-CreERT2 mice were obtained by screening. (2) Breeding DTA flox / flox Mice: Rosa26-LoxP-ZsGreen-STOP-LoxP-DTA mice were bred with C57BL / 6JGpt mice. Genomic DNA was extracted from the tails of the offspring. PCR genotyping was performed using 5' ARM primer pair T009408-F1 / T009408-R1 and wild-type primer pair T009408-F2 / T009408-R2 to screen for DTA mice. flox / flox Mice; (3) Preparation of F1 generation double-positive mice: The Csf1r-P2A-CreERT2 mice obtained in step (1) and the DTA mice obtained in step (2) were used to prepare the F1 generation double-positive mice. flox / flox Mice were mated, and the offspring were subjected to Csf1r-P2A-CreERT2 and DTA treatments. flox / flox Dual-gene PCR identification was used to screen F1 generation double-positive mice that simultaneously carry two gene modifications. (4) Breeding F2 generation double-positive mice: F1 generation double-positive mice were bred with DTAf lox / flox Mouse backcrosses were performed, and the offspring were subjected to dual-gene PCR identification to screen for Csf1r-P2A-CreERT2-DTA. flox / flox Double-positive mice; (5) Tamoxifen was administered to the double-positive mice obtained in step (4) to induce CreERT2 to enter the nucleus and specifically remove the termination element between two LoxP sites in the intrinsic renal macrophages to enable DTA expression and induce apoptosis of the intrinsic renal macrophages, thus obtaining the intrinsic renal macrophage-deficient mouse model.

2. The construction method according to claim 1, characterized in that, The identification primer sequences for Csf1r-P2A-CreERT2 are as follows: T006204-F1: 5'-ACAGCATGAAGTGCAAAGAACGTGGT-3', T006204-R1: 5'-GTGGTTCCACCCACTCTCACCT-3'; T006204-F2: 5'-GGAAGAGGAGCCAGTGCAAACA-3', T006204-R2: 5'-GTGGTTCCACCCACTCTCACCT-3'.

3. The construction method according to claim 1, characterized in that, The DTA flox / flox The identification primer sequences are as follows: T009408-F1: 5'-TCAAATGGCTCTCCCTCAAGCGT-3', T009408-R1: 5'-TGGTACATGACGTTCTCTCCACC-3'; T009408-F2: 5'-CCAAAAGTCGCTCTGAGTTGTTA-3', T009408-R2: 5'-TCGGGTGAGCATGTCTTTTAATCT-3'.

4. The construction method according to claim 1, characterized in that, The administration of tamoxifen in step (5) is by intraperitoneal injection. The injection dose is 100 mg / kg based on the mouse's body weight. The injection is given once every other day for a total of 4 injections, with an injection cycle of 7 days.

5. The construction method according to claim 1, characterized in that, The induction time mentioned in step (5) is from day 42 to day 49 after the mice are born.

6. The construction method according to claim 1, characterized in that, The macrophages mentioned in step (5) are intrinsic renal macrophages, including F4 / 80. hi CD11b lo Macrophage subsets.

7. A mouse model specifically lacking intrinsic kidney macrophages obtained using the construction method described in any one of claims 1 to 6, characterized in that, In the mouse model, macrophages developed normally and DTA was not expressed when tamoxifen was not induced; after tamoxifen induction, intrinsic renal macrophages were absent, and the absence efficiency was detected by flow cytometry.

8. The use of the mouse model of specific deficiency of renal intrinsic macrophages as described in claim 7 in the preparation of animal models for studying the involvement of renal intrinsic macrophages in acute kidney injury, chronic kidney disease, autoimmune nephritis, renal fibrosis or transplant rejection.

9. The use of the mouse model of renal intrinsic macrophage deficiency as described in claim 7 in screening or evaluating drugs or therapeutic strategies that regulate renal intrinsic macrophage function.

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