Construction method and application of dyrk1a gene knockout mouse model specific to type i classical dendritic cells

Abstract

This invention discloses a method for constructing a type I classical dendritic cell-specific knockout Dyrk1a gene mouse model and its application. The method includes the following steps: (1) [The text abruptly ends here, likely due to an incomplete sentence or missing information.] flox / + Donor mice were crossed with XCR1-Cre positive tool mice, and the genotype Dyrk1a was obtained through screening. flox / + XCR1-Cre+ and Dyrk1a flox / + (2) The F1 generation hybrid offspring of XCR1-Cre- were crossbred; (3) The F1 generation hybrid offspring obtained in step (1) were crossbred with siblings or backcrossed to obtain the F2 generation population; (4) The F2 generation population obtained in step (2) was identified and screened for genotype Dyrk1a. flox / flox Individuals with XCR1-Cre+ are thus established. This invention overcomes the embryonic lethality limitation caused by whole-body Dyrk1a knockout, and for the first time achieves in-depth research on the long-term regulatory mechanism of Dyrk1a in the cDC1 subpopulation in adult live mice. This effect is achieved through the conditional design of the Cre-loxP system, avoiding early developmental disorders and providing a reliable genetic tool for elucidating immune system function, significantly expanding the applicability of the research.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and immunology, specifically relating to the construction method and application of a mouse model with type I classical dendritic cell-specific knockout of the Dyrk1a gene. Background Technology

[0002] Dendritic cells (DCs) are a crucial bridge connecting innate and adaptive immunity. Among DC subsets, type I classical dendritic cells (cDC1) occupy a central position in tumor immune surveillance due to their unique antigen-presenting capacity. cDC1 specifically expresses the chemokine receptor XCR1, and its most significant biological characteristic is its exceptional antigen cross-presentation ability, making it widely recognized as the "chief commander" in inducing the activation of anti-tumor effector CD8+ T cells. cDC1 can efficiently take up exogenous antigens released by tumor cells and process them, presenting them to MHCI class molecules, thereby inducing the activation, proliferation, and differentiation of naïve CD8+ T cells into cytotoxic T lymphocytes (CTLs) with cytotoxic functions. Furthermore, cDC1 recruits T cells into tumor tissues by secreting chemokines such as CXCL9 / 10. Therefore, elucidating the deep molecular mechanisms driving cDC1 maturation and activation is key to overcoming resistance to tumor immunotherapy. A deeper understanding of this mechanism will help reveal how tumor cells evade the surveillance of the immune system and provide a foundation for developing more effective immunotherapies.

[0003] Dual-specificity tyrosine-phosphorylation-regulated kinase 1A (Dyrk1a) is an evolutionarily highly conserved protein kinase located in a key region of human chromosome 21, playing a central role in regulating the cell cycle, neural development, and gene transcription. Its unique dual-specificity enzymatic activity allows it to be activated via autophosphorylation, further phosphorylating key transcription factors such as NFAT and STAT3. This activation process ensures precise regulation of downstream signaling pathways, thereby influencing various cellular physiological functions. However, recent studies have revealed that Dyrk1a is also widely expressed in hematopoietic and immune cells, suggesting its potential involvement in complex immune regulatory processes. Although studies have explored the role of Dyrk1a in B cells, whether it acts as a "switch" in antigen-presenting cells, particularly the cDC1 subset, remains an unresolved scientific question. This gap reflects the limitations of current research in elucidating the specific functions of immune cells, necessitating more precise tools to fill this gap.

[0004] Although Dyrk1a exhibits significant regulatory potential in various cellular physiological activities, research into its function within the cDC1 subset has long been hampered, primarily due to the embryonic lethality resulting from its systemic deletion. The Dyrk1a gene plays an irreplaceable role in early development. Current techniques confirm that homozygous knockout (Dyrk1a- / -) mice die between 10.5 and 13.5 days of embryonic development. This lethality precludes the possibility of studying the long-term regulatory mechanisms of Dyrk1a on the immune system, particularly the highly differentiated cDC1 subset, in adult mice. Previous studies have often used Cd11c-Cre tool mice to knock out the target gene in dendritic cells. However, Cd11c (Itgax) is expressed not only in cDC1 but also significantly in cDC2, monocytes, macrophages, activated B cells, and some NK cells. This pan-myeloid knockout leads to highly heterogeneous experimental phenotypes, making it impossible to determine whether changes in the immune response are driven by cDC1 or due to a cascading reaction of other cell subsets. This lack of resolution severely limits the scientific definition of the precise immune function of Dyrk1a. These limitations of existing technologies make it difficult for researchers to isolate the specific contributions of cDC1, thus affecting the accurate assessment of the role of Dyrk1a in tumor immunity. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a method for constructing a mouse model of type I classic dendritic cell-specific knockout of the Dyrk1a gene.

[0006] Another objective of this invention is to provide the application of the type I classical dendritic cell-specific knockout Dyrk1a gene mouse model constructed by the above-described construction method.

[0007] The technical solution of the present invention is as follows:

[0008] The method for constructing a type I classical dendritic cell-specific knockout Dyrk1a gene mouse model is characterized by the following steps:

[0009] (1) Dyrk1a flox / + Donor mice were crossed with XCR1-Cre positive tool mice, and the genotype Dyrk1a was obtained through screening. flox / + XCR1-Cre+ and Dyrk1a flox / + F1 hybrid offspring of XCR1-Cre-;

[0010] (2) The F1 generation hybrid offspring obtained in step (1) are crossbred with siblings or backcrossed to obtain the F2 generation population;

[0011] (3) Identify and screen for genotype Dyrk1a from the F2 generation population obtained in step (2). flox / flox Individuals with XCR1-Cre+ are the type I classic dendritic cell-specific knockout Dyrk1a gene mouse model.

[0012] In a preferred embodiment of the present invention, loxP sites are inserted upstream of exon 5 and downstream of exon 6 of the Dyrk1a gene, respectively.

[0013] Further preferably, the Dyrk1a flox / + The donor mice were constructed by using a PGK-Neo selection box with FRT sites on the flanks, which was then inserted downstream of exon 6 of the Dyrk1a gene in conjunction with the downstream loxP site. Another loxP site was precisely inserted upstream of exon 5.

[0014] In a preferred embodiment of the present invention, the identification and screening in step (3) is as follows: genomic DNA of mouse tissues from the F2 generation population is extracted as a template, and PCR amplification is performed using Dyrk1a gene primers and XCR1-Cre recombinase gene primers, respectively. Individuals that meet the following requirements are the type I classical dendritic cell-specific knockout Dyrk1a gene mouse model: homozygous Dyrk1a gene amplification product containing the loxP site, Cre recombinase transgene amplification product, and amplification product containing internal reference.

[0015] More preferably, the individual satisfying the following requirements is further defined as: using Dyrk1a as shown in SEQ ID NO. 01 and 02 respectively. flox / flox Upstream and downstream primers amplified only a single band, 232 bp in size; and using XCR1-Cre primers 1, 2 and 3 as shown in SEQ ID NO.03, 04 and 05 respectively, two bands of 160 bp and 287 bp were amplified.

[0016] A type I classic dendritic cell-specific knockout Dyrk1a gene mouse model was constructed using the above-described method.

[0017] In a preferred embodiment of the present invention, the strain background is selected from C57BL / 6J, C57BL / 6N or its substrains.

[0018] The above-mentioned type I classic dendritic cell-specific knockout Dyrk1a gene mouse model is used in screening or preparing drugs that regulate the immune function of cDC1 cells.

[0019] In a preferred embodiment of the present invention, it can be used as an in vivo pharmacodynamic evaluation model for DYRK1A agonists, or as a research platform for evaluating the immune escape mechanism caused by impaired cDC1 antigen cross-presentation function, or in the preparation of immune-enhancing drugs for the treatment of malignant solid tumors.

[0020] More preferably, the malignant solid tumor is selected from melanoma and colon cancer.

[0021] The beneficial effects of this invention are:

[0022] 1. This invention breaks through the embryonic lethality limitation caused by whole-body knockout of Dyrk1a and achieves for the first time an in-depth study of the long-term regulatory mechanism of Dyrk1a in the cDC1 subpopulation in adult live mice. This effect is achieved through the conditional design of the Cre-loxP system, avoiding early developmental disorders and providing a reliable genetic tool for the analysis of immune system function, significantly improving the applicability of the research.

[0023] 2. This invention provides extremely high lineage resolution. By utilizing the cDC1-specific expression of XCR1-Cre, background interference from other myeloid cells such as cDC2, monocytes, and macrophages is eliminated, ensuring the cell specificity of the observed anti-tumor immune phenotype. This improves the scientific confidence and reliability of experimental conclusions and has a significant advantage in accuracy compared to traditional pan-myeloid tools such as Cd11c-Cre.

[0024] 3. This invention precisely defines the role of Dyrk1a as a core molecular switch regulating cDC1 maturation and antigen presentation. Experiments show that its absence leads to downregulation of co-stimulatory signals and reduced MHC expression, blocking the initiation of CD8+ T cells. This discovery provides a new perspective for understanding the immune escape mechanism induced by tumor microenvironment-induced cDC1 incompetence. It is applicable to mechanistic studies in the fields of immunology and oncology and serves as an excellent research model, promoting the exploration of Dyrk1a-related functions of cDC1.

[0025] 4. This invention constructs a high-confidence reverse evaluation platform. This functional impairment model assesses the salvage ability of candidate drugs for cDC1 function with higher sensitivity. It is suitable for screening DYRK1A agonists or cDC1 enhancers, and helps to develop novel combination therapies to enhance anti-tumor immune responses. It has direct guiding significance and commercial translation potential, especially in playing a role in the paradigm shift from immunosuppression to enhancement.

[0026] 5. The method of this invention is scientific, reliable and highly reproducible, showing consistency in B16F10 melanoma and MC38 colon cancer models. It not only supports basic mechanism research, but also provides a standardized experimental carrier for vaccine adjuvant screening, adoptive cell therapy evaluation and the formulation of personalized precision immunization programs, further expanding its application value in medicine, life sciences and experimental research. Attached Figure Description

[0027] Figure 1 This is a schematic diagram of Dyrk1a gene targeting in Example 1 of the present invention.

[0028] Figure 2 This diagram illustrates the breeding strategy of mice with conditional knockout of the Dyrk1a gene in cDC1 cells in Example 1 of this invention.

[0029] Figure 3 This is an agarose gel electrophoresis image showing the PCR identification of Dyrk1a and XCR1-Cre during the reproduction process of Example 1 of the present invention.

[0030] Figure 4 For the WB analysis of Dyrk1a in Embodiment 2 of the present invention flox / flox XCR1-Cre+ (cKO) and Dyrk1a flox / flox Image showing the expression level of Dyrk1a protein in XCR1-Cre-(WT) mouse cDC1 cells.

[0031] Figure 5 Subcutaneous immunization with Dyrk1a for MC38 colon cancer cells in Example 3 of this invention. flox / flox XCR1-Cre+ (cKO) and Dyrk1a flox / flox A statistical graph showing the tumor growth rate, tumor size, and survival rate of XCR1-Cre- (WT) mice.

[0032] Figure 6 Subcutaneous immunization with Dyrk1a for MC38 colon cancer cells in Example 3 of this invention. flox / flox XCR1-Cre+ (cKO) and Dyrk1a flox / flox Flow cytometry and statistical graph of co-stimulatory molecule expression on the surface of tumor-infiltrating cDC1 cells in XCR1-Cre-(WT) mice.

[0033] Figure 7 Subcutaneous immunization with Dyrk1a for MC38 colon cancer cells in Example 3 of this invention. flox / flox XCR1-Cre+ (cKO) and Dyrk1a flox / flox Flow cytometry and statistical analysis of cytokines secreted by XCR1-Cre- (WT) mouse tumor-infiltrating T cells. Figure 7The levels of IFNγ, TNFα, and Granzyme B secreted by T cells, in descending order, are as follows.

[0034] Figure 8 Subcutaneous immunization of B16F10 melanoma cells with Dyrk1a in Example 4 of this invention flox / flox XCR1-Cre+ (cKO) and Dyrk1a flox / flox A statistical graph showing the tumor growth rate, tumor size, and survival rate of XCR1-Cre- (WT) mice.

[0035] Figure 9 Subcutaneous immunization of B16F10 melanoma cells with Dyrk1a in Example 4 of this invention flox / flox XCR1-Cre+ (cKO) and Dyrk1a flox / flox Flow cytometry and statistical graph of co-stimulatory molecule expression on the surface of tumor-infiltrating cDC1 cells in XCR1-Cre-(WT) mice.

[0036] Figure 10 Subcutaneous immunization of B16F10 melanoma cells with Dyrk1a in Example 4 of this invention flox / flox XCR1-Cre+ (cKO) and Dyrk1a flox / flox Flow cytometry and statistical graphs of cytokines secreted by tumor-infiltrating T cells in XCR1-Cre-(WT) mice, from left to right: levels of IFNγ, TNFα, and Granzyme B secreted by T cells. Detailed Implementation

[0037] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0038] Example 1: Genotyping of cDC1 cell-specific Dyrk1a gene knockout mice

[0039] laboratory animals

[0040] Dyrk1a flox / + Both the mice and the XCR1-Cre mice were obtained from the Jackson Laboratory in the United States, and both were of the C57BL / 6J strain. After introduction, both types of mice were housed in a specific pathogen-free (SPF) environment at the Experimental Animal Center of Xiamen University.

[0041] Dyrk1a flox / + The mice were constructed by using homologous recombination to perform site-specific modifications on target genes. Figure 1The specific process includes: using the PGK-Neo selection box with the FRT site placed on the flank, and co-inserting it with the downstream loxP site downstream of the 6th exon of the Dyrk1a gene, while another loxP site is precisely inserted upstream of the 5th exon, thereby achieving the modification of the target gene.

[0042] Primers and main reagents

[0043] Dyrk1a flox / flox Upstream primer: 5'-tacctggagaagagggcaag-3' (SEQ ID NO.01);

[0044] Dyrk1a flox / flox Downstream primer: 5'-ggcataacttgcatacagtgG-3' (SEQ ID NO.02);

[0045] XCR1-Cre primer 1: 5'-tcaagttccgcagacaccta-3' (SEQ ID NO.03);

[0046] XCR1-Cre primer 2: 5'-gtgcacgaagtgttgctttg-3' (SEQ ID NO.04);

[0047] XCR1-Cre primer 1: 5'-accgtcgaccccatagtcat-3' (SEQ ID NO.05).

[0048] TIANamp Genomic DNA Kit (TIANGEN, DP304-03)

[0049] 2 × Rapid Taq Plus Master Mix (Vazyme, P223-02-AA)

[0050] method

[0051] (1) Breeding of conditional cDC1 cell Dyrk1a gene knockout mice (e.g.) Figure 2 (As shown)

[0052] Mice were first mated at 8 weeks of age, with a 2:1 female-to-male ratio and long-term cohabitation. Pups were weaned at 3 weeks of age. The flowchart for constructing conditional cDC1 cell Dyrk1a gene knockout mice is shown below. Figure 1 As shown.

[0053] For Dyrk1a flox / + Crossing with XCR1-Cre mice yielded a Dyrk1a genotype. flox / + XCR1-Cre+ and Dyrk1aflox / + XCR1-Cre- generation F1 mice were obtained, and then F1 mice were mated with their siblings to obtain F2 offspring mice.

[0054] Dyrk1a was identified by PCR. flox / flox The F2 generation mice with the XCR1-Cre+ genotype are mice with the Dyrk1a gene conditionally knocked out in cDC1 cells.

[0055] (2) Identification of Dyrk1a gene and XCR1-Cre recombinase gene

[0056] The PCR identification in step (1) was performed as follows: When mice reached 2 weeks of age, 4 mm of their tails were cut off, and genomic DNA was extracted from the tails. Genotype identification was performed using PCR. Simultaneously, the genomic DNA from the tails was amplified using primers for the Dyrk1a gene and primers for the XCR1-Cre recombinase gene, respectively, for related gene identification. The amplified product of the homozygous Dyrk1a gene containing the loxP site was 232 bp in length, the amplified product of the Dyrk1a gene (wild type) without the loxP site was 132 bp in length, and the heterozygous products were 232 bp and 132 bp respectively. The length of the Cre recombinase transgene product was 160 bp, and the internal control was 287 bp.

[0057] result

[0058] (1) Genotyping of mice

[0059] Based on Mendel's laws of gene segregation and independent assortment, the F1 generation mouse Dyrk1a flox / + XCR1-Cre+ and Dyrk1a flox / + The genotype of the F2 generation mice obtained by crossing XCR1-Cre- is likely to be: Dyrk1a flox / flox XCR1-Cre+, Dyrk1a flox / flox XCR1-Cre-, Dyrk1a flox / + XCR1-Cre+, Dyrk1a flox / + XCR1-Cre-, Dyrk1a + / + XCR1-Cre+, Dyrk1a + / + XCR1-Cre-. The genotype is Dyrk1a. flox / flox XCR1-Cre+ mice are Dyrk1a gene knockout mice in conditional cDC1 cells.

[0060] PCR identification results of various genotypes in F2 generation mice are as follows: Figure 3 As shown.

[0061] (2) Identification of the XCR1-Cre gene

[0062] When the loxP site binds to Cre recombinase, gene deletion occurs. In PCR amplification using Dyrk1a primers, the genomic product showing only one band at 232 bp indicates a flux homozygote, only one band at 132 bp indicates a wild-type, and one band at both 232 bp and 132 bp indicates a heterozygote. Subsequent PCR amplification of the candidate mouse genome using Cre primers yielded a 160 bp product, indicating the presence of the XCR1-Cre gene in that mouse. If the amplified product lacks the 160 bp, the mouse does not carry the Cre gene.

[0063] Example 2: Detection of DYRK1A protein expression level in cDC1 cell-specific Dyrk1a gene knockout mice

[0064] Take Dyrk1a prepared in Example 1 flox / flox XCR1-Cre+ (Dyrk1a-cKO), Dyrk1a flox / flox cDC1 cells were enriched in the spleen tissue of XCR1-Cre-(WT) mice.

[0065] Extraction of cDC1 cell proteins: Add 200 μL of RIPA lysis buffer containing protease inhibitors, phosphatase inhibitors, and PMSF to a centrifuge tube containing cDC1 cells. Lyse on ice for 30 min. After lysis, place the sample at 4 °C and centrifuge at 12,000 rpm for 10 min to remove cell debris and other insoluble components from the lysis buffer. Carefully transfer the supernatant to a new EP tube. Dilute the supernatant with 4×SDS and heat in a 100 °C metal bath for 10 min to denature the proteins.

[0066] Add the prepared protein sample to the wells of the SDS-PAGE gel. Electrophoresis program: 80 V, 30 min; 140 V, 1.5 h. Transfer the runnated protein to a PVDF membrane using wet transfer. Electrophoresis program: 100 V, 1 h. Use 5% skim milk powder as blocking buffer and incubate at room temperature (30 rpm) for 2 h. Wash off the blocking buffer with TBST. Cut the PVDF membrane to the size of the target protein and incubate with diluted rabbit-derived DYRK1A primary antibody and rabbit-derived β-actin primary antibody overnight at 4 °C. The next day, wash the membrane three times with TBST for 10 min each time and incubate with diluted goat anti-rabbit IgG secondary antibody at room temperature (30 rpm) for 1 h. Wash the membrane three more times with TBST for 10 min each time, then develop and photograph.

[0067] The results are as follows Figure 4As shown, the expression level of DYRK1A protein in cDC1 cells of Dyrk1a-ckO mice was significantly lower than that in control WT mice.

[0068] Example 3: Phenotypic analysis of subcutaneous colonoma in cDC1 cell-specific Dyrk1a gene knockout mice

[0069] Take Dyrk1a prepared in Example 1 flox / flox XCR1-Cre+ (Dyrk1a-cKO), Dyrk1a flox / flox Six 8-week-old XCR1-Cre-(WT) mice of the same sex were subcutaneously injected with 2 × 10⁻⁶ XCR1-Cre-(WT) mice. 6 Mice were treated with MC38 colon cancer cells. Tumor size was recorded starting on day 22, and mice were treated on day 34. The treatment process was as follows:

[0070] (1) Real-time monitoring of mouse tumors

[0071] Tumor growth in mice was monitored starting on day 22 after inoculation with MC38 colon cancer cells. The length and width of the subcutaneous tumor tissue were measured at fixed time points every other day to calculate the tumor area. Monitoring continued for about 12 days. On day 34 after tumor cell inoculation, the tumor tissue was collected and weighed.

[0072] The results are as follows Figure 5 As shown, after tumor cell inoculation, Dyrk1a-cKO mice exhibited a trend of larger tumor growth compared to WT mice, accompanied by rapid tumor growth and a significantly shortened survival. These results suggest that DYRK1A deficiency may weaken the host's anti-tumor immune response.

[0073] (2) Flow cytometry staining of cell surface

[0074] Mouse tumor tissue was collected, mechanically minced, and the cell pellet was resuspended in 1640 medium (R2) containing DNase I, Collagenase IV, and 2% fetal bovine serum. The mixture was digested in a 37°C water bath for 50 min, gently mixing every 10 min to ensure complete digestion. After digestion, the tissue was filtered through a 70 mm sieve and then gently ground with a 1 mL syringe plunger until no contents remained. The filtered cell suspension was centrifuged at 1500 rpm for 5 min at 4°C, the supernatant was discarded, and the cell pellet was retained. The cell pellet was resuspended in 8 mL of 45% Percoll solution and slowly added to a 15 mL centrifuge tube containing 4 mL of 70% Percoll solution. The tube was centrifuged at 400 g at room temperature for 30 min. Both the ramp and deceleration rates were set to 0 to avoid disturbing the stratification. After centrifugation, lymphocytes were observed to accumulate between the 45% Percoll and 70% Percoll layers, forming a distinct white cell layer, which was the lymphocyte layer. Carefully aspirate the intermediate layer using a pipette and transfer it to a clean 15 mL centrifuge tube. Wash the cells once with 1640 medium containing 2% FBS, and finally resuspend the cell pellet in 1640 medium containing 10% FBS. Count the cells, using 5 million total cells per sample for flow cytometry staining. Place the cells in a 5 mL flow cytometry tube, add 800 μL of FACS Buffer to wash the cells, centrifuge at 1500 rpm for 5 min, discard the supernatant, add pre-chilled cell surface antibody mixture, and incubate at 4 °C in the dark for 30 min. After incubation, add 800 μL of FACS Buffer to stop staining, and centrifuge at 1500 rpm for 5 min at 4 °C. Discard the supernatant, leaving approximately 100 μL, and resuspend the cells in 300 μL of FACS Buffer for flow cytometry analysis.

[0075] The results are as follows Figure 6 As shown, the expression levels of MHCII, MHCII, CD80, CD86, and CD40 were significantly reduced in tumor-infiltrating cDC1s. These co-stimulatory molecules play a crucial role in the interaction between cDC1s and T cells, especially during T cell activation and proliferation. CD80 (B7-1) and CD86 (B7-2) provide necessary second signals by binding to the CD28 receptor on T cells, thereby promoting an effective immune response. CD40 also plays an important role in the activation and maintenance of cDC1 function, promoting cDC1 maturation and antigen presentation capacity through interaction with CD40L on T cells. The results indicate that DYRK1A plays a key role in promoting cDC1 maturation, antigen processing, and antigen presentation, while the absence of DYRK1A significantly weakens the efficacy of cDC1s in initiating and maintaining anti-tumor immune responses.

[0076] (3) Flow cytometry detection of intracellular factors

[0077] Approximately 3 × 10⁻⁶ immune cells infiltrated by tumor were collected. 6 Cells were seeded in 24-well suspension plates and stimulated with PMA, ionomycin, and monensin at 37 °C for approximately 4.5 h. Cells were then resuspended and collected in 5 mL flow cytometry tubes. The cells were centrifuged at 1500 rpm for 5 min at 4 °C, and the culture medium was discarded. 800 μL of FACS Buffer was added to wash the cells, and the cells were centrifuged at 1500 rpm for 5 min at 4 °C. The supernatant was carefully removed, leaving approximately 100 μL of residual liquid. The cell pellet was gently resuspended. Pre-chilled cell surface antibody mixture was added, and the cells were incubated at 4 °C in the dark for 30 min. After incubation, 800 μL of FACS Buffer was added to stop staining, and the cells were centrifuged at 1500 rpm for 5 min at 4 °C. The supernatant was discarded, leaving approximately 50 μL of residual liquid. The cells were resuspended in 200 μL of BD Cytofix / Perm Buffer and the cells were permeabilized at 4 °C for 20 min. After cell membrane perforation and fixation, 800 μL of Perm Wash Buffer was added to terminate the perforation reaction. The cells were centrifuged at 3500 rpm for 8 min at 4 °C, and the supernatant was discarded, leaving approximately 100 μL of cells for resuspending. An appropriate amount of intracellular factor antibody mixture was added, and the cells were incubated at 4 °C in the dark for 1.5 h, with the cells shaken every 15 min to improve staining efficiency. After staining, 800 μL of Perm Wash Buffer was added to terminate the staining, and the cells were centrifuged at 3500 rpm for 8 min at 4 °C, and the supernatant was discarded. Finally, 300 μL of FACS Buffer was added to resuspend the cells, and the cells were analyzed by flow cytometry.

[0078] The results are as follows Figure 7 As shown, Dyrk1a-cKO mice exhibited significantly increased dysfunction of tumor-infiltrating T cells, specifically decreased levels of secreted cytokines IFNγ, TNFα, and Granzyme B. These results indicate that DYRK1A in cDC1s plays a crucial anti-tumor immune role by regulating the secretion of tumor-killing cytokines by tumor-infiltrating T cells.

[0079] Example 4: Phenotypic Analysis of Subcutaneous Melanoma in cDC1 Cell-Specific Dyrk1a Gene Knockout Mice

[0080] To evaluate the universal function of Dyrk1a in tumor microenvironments of different natures, a B16F10 melanoma subcutaneous xenograft model was established in this embodiment, specifically following the method in Example 3. Eight-week-old, same-sex Dyrk1a xenografts prepared in Example 1 were used.flox / flox XCR1-Cre+ (Dyrk1a-cKO), Dyrk1a flox / flox Seven XCR1-Cre-(WT) mice were used, and each mouse was subcutaneously injected with 0.5 × 10⁻⁶ ozontally on its right back. 6 B16F10 melanoma cells were used, and tumor growth kinetics were dynamically monitored starting from day 7 after inoculation. Endpoint sampling and immunoassay were performed on day 14. The treatment process is as follows:

[0081] (1) Real-time monitoring of mouse tumors

[0082] Tumor growth in mice was monitored starting on day 7 after inoculation with B16F10 melanoma cells. The length and width of subcutaneous tumor tissue were measured at fixed times each day to calculate the tumor burden. Monitoring continued for about 7 days. On day 14 after tumor cell inoculation, tumor tissue was collected and weighed.

[0083] The results are as follows Figure 8 As shown, after tumor cell inoculation, the tumor growth rate of Dyrk1a-cKO mice was significantly faster than that of WT mice, exhibiting a marked immune escape phenotype. By the experimental endpoint (day 14), the tumor volume and weight of the knockout group mice were significantly higher than those of the control group. Long-term survival monitoring indicated that the absence of Dyrk1a in cDC1 significantly shortened the survival of tumor-bearing mice. These results demonstrate that DYRK1A is a key molecule essential for the host to maintain its anti-melanoma immune surveillance capacity, and that this function is highly conserved across tumor models (melanoma and colon cancer).

[0084] (2) Flow cytometry staining of cell surface

[0085] Consistent with Example 3, flow cytometry was used to systematically analyze immune cells infiltrating tumor tissue. The results are as follows: Figure 9 As shown, the expression levels of key surface co-stimulatory molecules (CD80, CD86, CD40) and antigen-presenting molecules (MHCI, MHCII) were significantly downregulated. This indicates that DYRK1A deficiency systematically weakens cDC1-induced anti-tumor immune responses, and these results are consistent with the phenotype in MC38 colon cancer.

[0086] (3) Flow cytometry detection of intracellular factors

[0087] Consistent with Example 3, intracellular factor staining was performed on tumor-infiltrating lymphocytes. The results are as follows: Figure 10As shown, Dyrk1a-cKO mice exhibited reduced levels of secreted cytokines IFNγ, TNFα, and Granzyme B. This suggests that the loss of DYRK1A in cDC1 leads to dysfunction of effector T cells by blocking the effective transmission of antigen presentation and co-stimulatory signals, results consistent with the phenotype observed in MC38 colon cancer.

[0088] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for constructing a type I classical dendritic cell-specific knockout Dyrk1a gene mouse model, characterized by: Includes the following steps: (1) Dyrk1a flox / + Donor mice were crossed with XCR1-Cre positive tool mice, and the genotype Dyrk1a was obtained through screening. flox / + XCR1-Cre+ and Dyrk1a flox / + F1 hybrid offspring of XCR1-Cre-; (2) The F1 generation hybrid offspring obtained in step (1) are crossbred with siblings or backcrossed to obtain the F2 generation population; (3) Identify and screen for genotype Dyrk1a from the F2 generation population obtained in step (2). flox / flox Individuals with XCR1-Cre+ are the type I classic dendritic cell-specific knockout Dyrk1a gene mouse model.

2. The construction method as described in claim 1, characterized in that: The Dyrk1a flox / + The donor mice were constructed by inserting loxP sites upstream of exon 5 and downstream of exon 6 of the Dyrk1a gene.

3. The construction method as described in claim 2, characterized in that: The Dyrk1a flox / + The donor mice were constructed by using a PGK-Neo selection box with FRT sites on the flanks, which was then inserted downstream of exon 6 of the Dyrk1a gene in conjunction with the downstream loxP site. Another loxP site was precisely inserted upstream of exon 5.

4. The construction method as described in claim 1, characterized in that: The identification and screening in step (3) are as follows: genomic DNA of mouse tissues from the F2 generation population is extracted as a template, and PCR amplification is performed using Dyrk1a gene primers and XCR1-Cre recombinase gene primers, respectively. Individuals that meet the following requirements are the type I classical dendritic cell-specific knockout Dyrk1a gene mouse models: homozygous Dyrk1a gene amplification product containing the loxP site, Cre recombinase transgene amplification product, and amplification product containing the internal reference.

5. The construction method as described in claim 4, characterized in that: The individual that meets the following requirements is further defined as: using Dyrk1a as shown in SEQ ID NO. 01 and 02 respectively. flox / flox Upstream and downstream primers amplified only a single band, 232 bp in size; and using XCR1-Cre primers 1, 2 and 3 as shown in SEQ ID NO.03, 04 and 05 respectively, two bands of 160 bp and 287 bp were amplified.

6. A type I classical dendritic cell-specific knockout Dyrk1a gene mouse model, characterized in that: It is constructed using the construction method described in any one of claims 1 to 4.

7. The type I classical dendritic cell-specific knockout Dyrk1a gene mouse model as described in claim 6, characterized in that: Its strain background is selected from C57BL / 6J, C57BL / 6N or their substrains.

8. The use of the type I classical dendritic cell-specific knockout Dyrk1a gene mouse model as described in claim 6 or 7 in screening or preparing drugs that regulate the immune function of cDC1 cells.

9. The application as described in claim 8, characterized in that: It can serve as an in vivo pharmacodynamic evaluation model for DYRK1A agonists, or as a research platform for assessing the immune escape mechanism caused by impaired cDC1 antigen cross-presentation function, or in the preparation of immunomodulatory drugs for the treatment of malignant solid tumors.

10. The application as described in claim 9, characterized in that: The malignant solid tumors were selected from melanoma and colon cancer.

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