Construction method and application of a conditional knockout mouse model of the Cpt1a gene

Abstract

The present invention discloses a method for constructing a conditional knockout mouse model of the Cpt1a gene and its application. The construction method is to construct a lupus erythematosus mouse model by conditionally knocking out the Cpt1a gene in macrophages. The construction method of the lupus erythematosus mouse model provided by the present invention realizes the knockout of the Cpt1a gene in the bone marrow cell lineage, while the gene is normally expressed in other tissues or cells. It is a simple, efficient and rapid construction method. The obtained lupus erythematosus mouse model can be better applied to the mechanism research of lupus erythematosus, and the preparation and / or screening of drugs for treating lupus erythematosus.

Description

Technical Field

[0001] The present invention belongs to the technical field of mouse models, and particularly relates to a method for constructing a conditional knockout mouse model of the Cpt1a gene and its application. Background Art

[0002] Systemic lupus erythematosus (SLE), as a prototype of autoimmune diseases, can affect multiple organs and systems throughout the body. SLE has a certain degree of heterogeneity. Mild patients may present with joint pain, while severe patients may experience damage to target organs such as the heart, brain, and kidneys, and in severe cases, it can even endanger life. It has been reported that the incidence of SLE is 40 - 50 per 100,000 people. Moreover, 90% of SLE patients are women of childbearing age, which not only affects personal health but also imposes a heavy burden on families and society.

[0003] Currently, there are two mouse models for studying the pathogenesis of SLE and new treatment methods, including New Zealand Black × White F1 mice (NZB / W), Murphy Roths Large / Lpr mice (MRL / LPR), and BXSB / Yaa mice.

[0004] The NZB / NZW F1 mouse is the first-generation mouse after mating NZB mice and NZW mice, which spontaneously exhibits lupus phenotypes, and neither of its parental mice shows lesions. It was discovered by Helyer in 1963. The symptoms of this model mouse start to appear at 4 - 5 months, obvious symptoms of glomerulonephritis appear at 5 - 6 months, and it progresses to severe lupus at 10 - 12 months and dies due to renal failure. The disease symptoms of such mice are similar to those of humans, and sex hormones have a significant impact on these mice. Female mice develop the disease earlier and more severely than male mice. Its characteristics are high titers of anti-dsDNA and anti-ssDNA antibodies, and hypergammaglobulinemia.

[0005] The MRL / lpr mouse simulates the functional abnormalities caused by T cell apoptosis defects, characterized by lymphadenopathy and splenomegaly. These two organs contain a large number of lymphocytes. Therefore, this model mainly simulates the abnormalities of the adaptive immune system. While the innate immune system is normal, for example, IFN-alpha does not increase. Although the MRL / lpr mouse shows elevated anti-dsDNA antibodies, proteinuria, and hypocomplementemia, it does not fully conform to the characteristics of human diseases. For example, lupus mainly affects women of childbearing age, with a male-to-female ratio of 1:9, while the MRL / lpr model does not show gender differences. The reason for the lupus phenotype in NZB / NZW F1 mice is unknown, and its disease onset period is long (6 months), it is susceptible to environmental factors, and the experimental process is not easy to control, which brings difficulties to the study of the disease pathogenesis. Summary of the Invention

[0006] The present invention provides a method for constructing a conditional knockout mouse model of the Cpt1a gene. The constructed lupus erythematosus mice are helpful for studying the mechanism of lupus erythematosus and for preparing and / or screening drugs for treating lupus erythematosus.

[0007] The first invention of the present invention provides a method for constructing a conditional knockout mouse model of the Cpt1a gene. A lupus erythematosus mouse model is constructed by conditionally knocking out the Cpt1a gene in macrophages.

[0008] In one embodiment of the present invention, a lupus erythematosus mouse model is constructed by conditionally knocking out the Cpt1a gene in macrophages, including the following steps:

[0009] S1. Design gRNAs targeting the Cpt1a gene. The gRNAs include gRNA-A1 and gRNA-A2. The sequence of gRNA-A1 is as shown in SEQ ID No:1, and the sequence of gRNA-A2 is as shown in SEQ ID No:2;

[0010] S2. Co-inject Cas9 protein, gRNA, and Cpt1a into the nucleus of mouse fertilized eggs, and transplant the fertilized eggs into recipient female mice for pregnancy to obtain F0 generation mice;

[0011] S3. Cross the F0 generation mice with normal wild-type mice to obtain F1 generation mice;

[0012] S4. Mate the F1 generation mice with Lyz2-Cre tool mice to obtain a lupus erythematosus mouse model with successful conditional knockout of the Cpt1a gene in macrophages.

[0013] The second invention of the present invention provides a lupus erythematosus mouse model constructed by the construction method described in any one of the above.

[0014] The second invention of the present invention provides the application of the lupus erythematosus mouse model obtained by the above-mentioned method for constructing a conditional knockout mouse model of the Cpt1a gene in the mechanism research of lupus erythematosus.

[0015] The second invention of the present invention provides the application of the lupus erythematosus mouse model obtained by the above-mentioned method for constructing a conditional knockout mouse model of the Cpt1a gene in the preparation and / or screening of drugs for treating lupus erythematosus.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The method for constructing a conditional knockout mouse model of the Cpt1a gene provided by the embodiments of the present invention. The constructed lupus erythematosus mice are helpful for studying the mechanism of lupus erythematosus and for preparing and / or screening drugs for treating lupus erythematosus.

[0017] The construction method of the Cpt1a gene conditional knockout mouse model provided by the embodiments of the present invention realizes the knockout of the Cpt1a gene in the bone marrow cell lineage, while the gene is normally expressed in other tissues or cells. The obtained lupus erythematosus mouse model has a clear cause for showing lupus phenotypes, is not easily affected by environmental factors, and the experimental process is controllable. It is a simple, efficient, and rapid construction method that can be better applied to the mechanism research of lupus erythematosus and the preparation and / or screening of drugs for treating lupus erythematosus.

[0018] In the construction method of the Cpt1a gene conditional knockout mouse model provided by the embodiments of the present invention, the constructed lupus erythematosus mouse model has a simple construction method and develops the disease spontaneously. The mouse shows elevated anti-nuclear antibodies, elevated anti-dsDNA, elevated urine protein, and shows manifestations of lupus nephritis, enriching the selection of lupus models.

[0019] Description of the drawings

[0020] Figure 1 It is a comparison chart of the anti-nuclear antibody concentrations of CPT1a-cKO mice and control mice;

[0021] Figure 2 It is a comparison chart of the anti-dsDNA antibody concentrations of CPT1a-cKO mice and control mice;

[0022] Figure 3 It is a comparison chart of the urine albumin-to-creatinine ratio of CPT1a-cKO mice and control mice;

[0023] Figure 4 It is a comparison chart of the phagocytic function of peritoneal macrophages of CPT1a-cKO mice and control mice;

[0024] Figure 5 It is a comparison chart of kidney immunohistochemistry of CPT1a-cKO mice and control mice;

[0025] Figure 6 It is a comparison chart of kidney immunofluorescence of CPT1a-cKO mice and control mice. Detailed implementation manners

[0026] In this article, a range represented by "from one value to another value" is a summary representation method to avoid listing all the values in the range in the specification one by one. Therefore, the description of a specific numerical range covers any value within that numerical range and the smaller numerical ranges defined by any value within that numerical range, as if the arbitrary value and the smaller numerical range were clearly written in the specification.

[0027] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the protection scope of the present invention. In actual applications, the improvements and adjustments made by those skilled in the art based on the present invention still fall within the protection scope of the present invention.

[0028] Example 1

[0029] This example provides a method for constructing a conditional knockout mouse model of the Cpt1a gene, which specifically includes the following steps:

[0030] 1. gRNA design

[0031] Design gRNA according to http: / / crispor.tefor.net / , and select high-score gRNAs; the gRNAs include gRNA-A1 and gRNA-A2;

[0032] The sequence of gRNA-A1 is as follows: TTCAAGACTGCTCAGGTCGC-TGG (SEQ ID No:1);

[0033] The sequence of gRNA-A2 is as follows: GAGGATGACGGATCACTCCT-GGG (SEQ ID No:2);

[0034] 2. Construction of the Donor vector:

[0035] Fragment amplification (5’arm, cKO, 3’arm): Use Novoprotein P515 high-fidelity enzyme to prepare a 50ul system for PCR amplification, with 30 cycles;

[0036] Recover the gel: Electrophorese the PCR product, and use the Qiagen gel extraction kit (product number: 28706) to recover the gel at the position of the target product;

[0037] Ligation (backbone + fragment) / transformation: Use Novoprotein C115 ligase to ligate the recovered fragments, and use Takara's Stellar competent cells to transform the ligated DNA fragments into Escherichia coli, and culture overnight at 37°C;

[0038] Bacterial inspection: Pick 16 well-shaped colonies, use Novoprotein P222 Taq enzyme to prepare a 25ul system for PCR amplification; pick the colonies with the correct band and culture them in a small shaker with 4mL broth medium;

[0039] Extract the plasmid of the positive clone: Extract the plasmid using the alkaline lysis method (self-prepared reagent);

[0040] Restriction enzyme digestion identification and sequencing: Select an appropriate restriction enzyme from NEB, and prepare a 20 μL restriction enzyme digestion system with 600 ng of plasmid for digestion. Sequence the plasmid with correct digestion results.

[0041] Preparation of plasmid for injection: For the clone with correct sequencing results, inoculate 22.5 mL of broth medium and culture overnight. Use the QIAGEN plasmid purification kit (Cat. No. 27106) to purify the plasmid for injection.

[0042] 3. gRNA synthesis

[0043] Synthesize the CrRNA (CRISPR RNA) sequence (IDT) and tracrRNA (trans-activating crRNA) sequence (GenScript Biotech Corporation) artificially. The CrRNA will bind to the tracrRNA to form the gRNA sequence.

[0044] The sequence information is as follows:

[0045] The sequence of CrRNA-A1 (SEQ ID No: 3) is as follows:

[0046] 5'-UUCAAGACUGCUCAGGUCGCGUUUUAGAGCUAUGCUGUUUUG-3'

[0047] The sequence of CrRNA-A2 (SEQ ID No: 4) is as follows:

[0048] 5'-GAGGAUGACGGAUCACUCCUGUUUUAGAGCUAUGCUGUUUUG-3';

[0049] The sequence of tracrRNA (SEQ ID No: 5) is as follows:

[0050] 5'-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3'.

[0051] 4. Preparation of RNP complex

[0052] Tube 1 solution: Add 0.8 μL of 100 pmol / μL CrRNA to 5.2 μL of RNase-free water, then add 0.6 μL of 100 pmol / μL TracRNA and mix well. Incubate for 5 min, and then add 0.2 μL of Cas9 protein (NEB, Cat. No.: M0646M) and mix well. Incubate for 10 min to obtain the tube 1 solution;

[0053] Tube 2 solution: Donor plasmid with a final concentration of 15 ng / uL;

[0054] Mix the solution in tube 1 and the solution in tube 2 to obtain the RNP injection complex.

[0055] 5. Preparation of Fertilized Eggs

[0056] Preparation of fertilized eggs: 3-4 week old C57BL / 6 female mice were selected and injected with pregnant mare serum (PMSG) and human chorionic gonadotropin (hCG) respectively, with an interval of 46-48 hours between the two.

[0057] After the HCG injection, the female mouse is mated with an adult fertile male mouse to fertilize the female mouse;

[0058] The next day, the female mice were euthanized, and the fertilized eggs were collected from the oviduct and placed in a 37°C incubator with 5% CO2 for later use.

[0059] 6. Pronuclear Microinjection

[0060] Prepare microinjection needles and fixation needles;

[0061] The prepared RNP complex and plasmid DNA are diluted and mixed to form an injection solution, which is then loaded into a microinjection needle;

[0062] The fertilized eggs with normal morphology were screened and placed in an injection dish. Under an inverted microscope with a magnification of 200-400 times, the exogenous gene injection solution was injected into the nucleus of the fertilized egg by microinjection.

[0063] Transfer the injected fertilized eggs into M16 culture medium and place them in a 37°C 5% CO2 incubator for 0.5-1 hour before transplantation; or culture them until they reach 2 cells and transplant them the next day.

[0064] 7. Preparation of Surrogate Mice and Embryo Transfer

[0065] Prepare pseudo-pregnant female mice: Select fertile female mice of appropriate age and mate them with male mice that have been sterilized after vasectomy. Stimulate the female mice to undergo a series of pregnancy changes to obtain pseudo-pregnant female mice, which will serve as surrogate mice for the transgenic fertilized eggs.

[0066] The fertilized eggs injected with foreign genes were transplanted into the oviduct of the surrogate mother mouse on the day of thrombosis;

[0067] After transplantation, place the surrogate mother mouse in a clean cage and keep it warm until it wakes up and then return it to the cage for breeding;

[0068] After successful fallopian tube transplantation, the female mouse will generally give birth 19-20 days after the operation;

[0069] One week after the mice are born, their claws can be clipped for numbering, and PCR identification can be carried out at the same time; three weeks after the mice are born, they can be separately caged and raised independently.

[0070] 8. Identification of F0 newborn mice

[0071] Collect tissues of young mice at 1-2 weeks of age (tail or toe tissues);

[0072] Lyse and extract the genome from the tissues;

[0073] Perform PCR amplification and electrophoresis detection with specific primers for the target gene to screen for offspring with integrated foreign genes; specifically, use two pairs of primers to perform PCR and sequencing verification on the two loxps of F0 mice (F1: 5’-GTGTGTTGTTCAAGACTGCTCA-3’ (SEQ ID No: 6), R1: 5’-TCAGCATGGAAGGTGGTAATGG-3 (SEQ ID No: 7); F2: 5’-TAAGCTTAGCTCTCTGGAGTAGGG-3’ (SEQ ID No: 8), R2: 5’-GCATTTGAAAGGCAATGTTACCAG-3’ (SEQ ID No: 9));

[0074] Mice with integration are called founder mice, which can be passaged and established as a strain, and protein expression level identification can be carried out if necessary;

[0075] 9. Passage and strain establishment of transgenic mice

[0076] Passage F0 mice with wild-type mice to obtain F1 generation floxed mice with normal germline transmission.

[0077] Before the floxed mice are crossed with mice expressing Cre recombinase, the gene is normally expressed; after crossing the floxed mice with Lyz2-Cre mice, the Cpt1a gene can be knocked out in the bone marrow cell lineage, while the gene is normally expressed in other tissues or cells, obtaining Cpt1a-cKO mice.

[0078] Example 2

[0079] In this example, it was verified that the Cpt1a-cKO mice constructed in Example 1 showed lupus-like manifestations, and the Cpt1a-cKO mice were a successful lupus erythematosus mouse model with Cpt1a gene knockout.

[0080] The specific steps are as follows:

[0081] 1. The Cpt1a-cKO mice constructed in Example 1 were raised in a SPF-class animal house until they reached 32 weeks of age, and then the mice were sacrificed by cervical dislocation.

[0082] 2. Blood was collected by enucleating the eyeballs, and then serum was obtained by centrifuging at 10,000 rpm for 10 minutes. A mouse anti-nuclear antibody kit and an anti-double-stranded antibody kit (purchased from Shanghai Yuchun Biotechnology) were used to perform tests according to the instructions, and the OD450 value was read. The results are as Figure 1 、 Figure 2 shown. Compared with WT mice, the anti-nuclear antibody level in Cpt1a-cKO mice was increased (p < 0.05). Compared with WT mice, the anti-double-stranded antibody level in Cpt1a-cKO mice was increased (p < 0.05).

[0083] 3. A 1.5-ml centrifuge tube was used to collect mouse urine, and urinary albumin and creatinine (purchased from Nanjing Jiancheng Bioengineering Institute) were detected. Tests were performed according to the instructions, and values were read on a spectrophotometer.

[0084] The results are as Figure 3 shown. Compared with WT mice, the ratio of urinary albumin to creatinine in Cpt1a-cKO mice was increased (p < 0.05).

[0085] 4. Detection of the phagocytic function of macrophages derived from the mouse peritoneal cavity:

[0086] The mice were sacrificed by cervical dislocation, soaked in 75% alcohol for 1 - 2 minutes, transferred to a laminar flow hood, placed on an anatomical table, fixed at the four limbs with needles, and the skin was torn open with forceps held in both hands and pulled to both sides to expose the peritoneum, but the peritoneal wall was not damaged. After wiping the peritoneal wall with 70% alcohol, 5 ml of sterile PBS solution was injected into the peritoneal cavity with a syringe. At the same time, the peritoneal wall was kneaded from both sides with fingers to make the liquid flow fully in the peritoneal cavity. The abdominal wall was gently lifted with a needle to make the animal body tilt slightly to one side, so that the liquid in the peritoneal cavity was collected under the needle and aspirated into the syringe. The needle was carefully removed, and the liquid was injected into a centrifuge tube and centrifuged at 1,000 rpm for 5 minutes. The supernatant was discarded, and the cells were resuspended in complete culture medium. After adherent culture for 4 hours, non-adherent cells were discarded to obtain peritoneal macrophages. Then, the human T cell line Jurkat cells were induced to become apoptotic cells with staurosporine (purchased from Sangon Biotech), stained with green fluorescence with PHrodo (purchased from Life Technology), and after phagocytosis by macrophages, the phagocytic function was detected by flow cytometry.

[0087] The results are as Figure 4 shown. Compared with WT mice, the phagocytic function of peritoneal macrophages derived from Cpt1a-cKO mice was decreased (p < 0.05).

[0088] 5. The bilateral kidneys of the mice were collected and stored in 4% paraformaldehyde for HE and PAS staining.

[0089] The steps of HE are as follows:

[0090] Deparaffinize the paraffin sections to water: sequentially place the sections into environment-friendly deparaffinizing solution I for 20 min - environment-friendly deparaffinizing solution II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min, and wash with tap water;

[0091] Restore the temperature and fix the frozen sections: take the frozen sections out of the -20°C refrigerator and restore them to room temperature, fix them with tissue fixing solution for 15 min, and then rinse with running water;

[0092] Pretreatment: place the sections into the high-definition constant staining pretreatment solution for 1 min;

[0093] Hematoxylin staining: place the sections into hematoxylin staining solution for 3 - 5 min, wash with tap water, differentiate with differentiating solution, wash with tap water, blue with blueing solution, and rinse with running water;

[0094] Eosin staining: dehydrate the sections in 95% alcohol for 1 min, and stain them in eosin staining solution for 15 s;

[0095] Dehydrate and mount: sequentially place the sections into absolute ethanol I for 2 min - absolute ethanol II for 2 min - absolute ethanol III for 2 min - n-butanol I for 2 min - n-butanol II for 2 min - xylene I for 2 min - xylene II for 2 min for clearing, and mount with neutral gum;

[0096] Examine under a microscope and collect and analyze images.

[0097] The steps of PAS:

[0098] (1) Deparaffinize the paraffin sections to water: sequentially place the sections into environment-friendly deparaffinizing solution I for 20 min - environment-friendly deparaffinizing solution II for 20 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 75% alcohol for 5 min, and wash with tap water;

[0099] Restore the temperature and fix the frozen sections: take the frozen sections out of the -20°C refrigerator and restore them to room temperature, fix them with tissue fixing solution for 15 min, and then rinse with running water

[0100] (2) Place the sections into PAS staining solution B and stain for 10 - 15 min, wash with tap water, and wash twice with distilled water;

[0101] (3) Immerse the sections in PAS staining solution A for 25 - 30 min, avoid light, and rinse with running water for 5 min;

[0102] (4) Place the sections into PAS staining solution C and stain for 30 s, wash with tap water, differentiate with hydrochloric acid aqueous solution, wash with tap water, blue with ammonia water, and rinse with running water.

[0103] (5) Dehydration and sealing: The sections were sequentially placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene for 5 min, and xylene II for 5 min until transparent, and then sealed with neutral gum.

[0104] (6) Microscopic examination, image acquisition and analysis.

[0105] The results are as follows Figure 5 As shown, compared with WT mice, HE and PAS staining of the kidneys of Cpt1a-cKO mice showed that the renal pathology of RCP-cKO mice showed increased glomerular mesangial matrix, accompanied by mesangial cell proliferation, and increased cells in the capillary lumen of some glomerular segments.

[0106] 6. Perform immunofluorescence staining of IgG and C3 on mouse kidneys.

[0107] The immunofluorescence steps are as follows:

[0108] (1) Dewaxing of paraffin sections: Place the sections in environmentally friendly dewaxing solution I for 10 min, environmentally friendly dewaxing solution II for 10 min, environmentally friendly dewaxing solution III for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, and then wash with distilled water.

[0109] (2) Antigen retrieval: See the table above for retrieval conditions. During the retrieval process, prevent excessive evaporation of the buffer and do not allow the slides to dry out. After retrieval, allow the slides to cool naturally. Wash the slides three times in PBS (pH 7.4) on a decolorizing shaker, shaking for 5 minutes each time.

[0110] (3) Circle serum blocking: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue, add BSA (if the primary antibody is from goat, use 10% donkey serum to block; if the primary antibody is from other sources, use 3% BSA to block) and block for 30 minutes.

[0111] (4) Add primary antibody: Add the prepared primary antibody dropwise and place the slices flat in a humidified chamber and incubate at 4°C overnight.

[0112] (5) Adding secondary antibody: Place the slides in PBS (pH 7.4) and wash on a decolorizing shaker three times for 5 minutes each. Add the corresponding secondary antibody and incubate at room temperature in the dark for 50 minutes.

[0113] (6) DAPI counterstaining of cell nuclei: Place the slide in PBS (pH 7.4) and wash on a decolorizing shaker three times for 5 minutes each time. Add DAPI staining solution and incubate at room temperature for 10 minutes in the dark.

[0114] (7) Quench the autofluorescence of the tissue: Place the slide in PBS (pH 7.4) and wash it 3 times on a shaker for 5 minutes each time. Add the B solution of the autofluorescence quenching agent for 5 minutes and rinse with running water for 10 minutes. (If the customer needs to observe the autofluorescence of the tissue, this step can be removed.)

[0115] (8) Mounting: Mount with an anti-fluorescence quenching mounting medium.

[0116] (9) Image acquisition: For DAPI, the excitation wavelength is 330 - 380 nm and the emission wavelength is 420 nm; for 488, the excitation wavelength is 465 - 495 nm and the emission wavelength is 515 - 555 nm; for CY3, the excitation wavelength is 510 - 560 nm and the emission wavelength is 590 nm; for CY5, the excitation wavelength is 608 - 648 nm and the emission wavelength is 672 - 712 nm.

[0117] The results are as Figure 6 shown. Compared with WT mice, immunofluorescence in the kidneys of Cpt1a - cKO mice showed increased fluorescence intensity of IgG and C3 in the mesangial region.

[0118] The above - disclosed are only the preferred embodiments of the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to the specific embodiments described. Obviously, many modifications and variations can be made according to the content of this specification. These embodiments are selected and specifically described in this specification to better explain the principles and practical applications of the present invention, so that those skilled in the art can make good use of the present invention. The present invention is only limited by the claims and their full scope and equivalents.

[0119] Under the teachings of the present invention and the above - mentioned embodiments, those skilled in the art can easily foresee that the various raw materials or their equivalent substitutes, and the various processing methods or their equivalent substitutes listed or exemplified in the present invention can all implement the present invention, and the upper and lower limits and interval values of the parameters of the various raw materials and processing methods can all implement the present invention. Examples are not listed one by one here.

Claims

1. A method for constructing a conditional knockout mouse model of the Cpt1a gene, characterized in that, Construct a lupus mouse model by conditional knockout of the Cpt1a gene in macrophages, including the following steps: S1. Design gRNAs targeting the Cpt1a gene. The gRNAs include gRNA-A1 and gRNA-A2. The sequence of gRNA-A1 is as shown in SEQ ID No:1, and the sequence of gRNA-A2 is as shown in SEQ ID No:2; S2. Co-inject Cas9 protein, gRNA, and Cpt1a into the nucleus of mouse fertilized eggs, and transplant the fertilized eggs into recipient female mice for pregnancy to obtain F0 generation mice; S3. Cross the F0 generation mice with normal wild-type mice to obtain F1 generation mice; S4. Mate the F1 generation mice with Lyz2-Cre tool mice to obtain a lupus mouse model with successful conditional knockout of the Cpt1a gene in macrophages.

2. Use of the lupus mouse model obtained by the method for constructing a conditional knockout mouse model of the Cpt1a gene according to claim 1 in screening drugs for treating lupus.

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