Construction method and application of BDCA-2 transgenic mouse model

Abstract

The invention provides a construction method and application of a BDCA-2 transgenic mouse model, and belongs to the technical field of bioengineering. The invention provides a BDCA-2 transgenic mouse model, and the core strategy of the BDCA-2 transgenic mouse model is to integrate a human BDCA-2 gene locus in a mouse genome at a fixed point, and the human BDCA-2 gene locus comprises a complete human BDCA-2 promoter region, a coding region and a part of intron region, so as to realize the physiological expression of the gene in mouse pDCs. According to the invention, a C57BL / 6J mouse is taken as a genetic background, a human-derived BDCA-2 gene is accurately inserted into a Rosa26 safety site of the mouse through a CRISPR / Cas12a mediated homologous recombination technology, and meanwhile, an expression regulation element of an endogenous Rosa26 gene of the mouse is retained. The design can ensure the specific expression of the human-derived BDCA-2 in the pDCs.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for constructing and applying a BDCA-2 transgenic mouse model. Background Technology

[0002] Dendritic cells, as the most powerful professional antigen-presenting cells, serve as a "bridge" connecting innate and adaptive immunity. Among the many subpopulations of dendritic cells, plasmacytoid dendritic cells (pDCs) can produce type I interferon with extremely high efficiency, thereby directly inhibiting viral replication and activating various immune cells such as natural killer cells, T lymphocytes, and myeloid dendritic cells, coordinating the entire antiviral immune response. However, autoimmune diseases such as systemic lupus erythematosus and inflammatory diseases lead to the continuous activation of pDCs, causing pDC dysfunction and the production of large amounts of type I interferon, forming the "type I interferon signature," driving autoimmune responses and attacking the body's own tissues. Moreover, in specific tumor microenvironments, pDCs may exhibit functional tolerance or dysfunction, not only failing to effectively initiate anti-tumor immunity but also potentially promoting immunosuppression through the induction of regulatory T cells, thus facilitating tumor immune escape. Therefore, pDCs have become an important target for the treatment of autoimmune diseases, chronic inflammation, and cancer.

[0003] To achieve precise intervention on pDCs, identifying their highly specific surface molecules is crucial. Blood dendritic cell antigen 2 (BDCA-2, also known as CD303 or CLEC4C) is a member of the type II transmembrane C lectin family. It is a type II transmembrane glycoprotein composed of 213 amino acids, selectively expressed on the surface of pDCs in humans and non-human primates. BDCA-2 is also a functional immune receptor. When BDCA-2 is cross-linked with its specific antibody or ligand, it transmits an inhibitory signal into the pDCs. This signal, through its associated FcRγ chain, recruits molecules containing immunoreceptor tyrosine inhibitory motifs, thereby inhibiting the production of type I interferons and pro-inflammatory factors mediated by the TLR7 / 9 signaling pathway. Existing mouse models do not contain the human BDCA-2 homolog; their pDCs cannot express functionally active human BDCA-2 protein, and therefore cannot reproduce the human BDCA-2-mediated IFN-I inhibitory pathway and disease-related effects. This makes it difficult to assess target binding specificity, signal regulation efficiency, and in vivo efficacy in conventional mice for drug screening targeting BDCA-2, greatly limiting the accuracy of translational research. Furthermore, BDCA-2 humanized mice often employ transgenic protocols, and differences in the number of exogenous gene copies and insertion sites among individual mice lead to fluctuations in BDCA-2 expression levels, resulting in extremely poor reproducibility of drug response experiments and difficulty in generating stable data support.

[0004] Therefore, there is an urgent need to find a new method that can achieve functional expression on the surface of pDCs at the protein level. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention presents a method for constructing and applying a BDCA-2 transgenic mouse model, which enables functional expression of pDCs at the protein level.

[0006] To achieve the above objectives, the present invention provides the following technical solution to address the technical problem: In a first aspect, the present invention provides a method for constructing a BDCA-2 transgenic mouse model, the method comprising: introducing the hBDCA-2 gene into the Rosa26 site of a mouse, wherein the hBDCA-2 gene includes a human BDCA-2 gene and a human BDCA-2 promoter.

[0007] In this invention, the rat is from the family Fryridae. In one embodiment, the rat is selected from mice, rats, and hamsters. In one embodiment, the rat is selected from the families Cricetidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamsters, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny rats, crested rats), Madagascar Muridae (climbing mice, rock mice, tailed rats, Madagascar rats and mice), Dormicidae (e.g., spiny dormice), and Mole-like Muridae (e.g., Mole rats, bamboo rats, and mole rats).

[0008] In one embodiment, the genetically modified mouse is derived from a member of the Muridae family. In a particular embodiment, the mouse is selected from mice and rats.

[0009] In some embodiments, the transgenic mouse cells express the human BDCA-2 protein.

[0010] In some embodiments, the exon 2 nucleotide sequence of the human BDCA-2 gene begins with the start codon ATG and does not include the nucleotide sequence preceding ATG.

[0011] In some embodiments, the human BDCA-2 gene comprises the nucleotide sequence of exon 2, intron 2, exon 3, intron 3, exon 4, exon 5, exon 6, and exon 7 of human BDCA-2.

[0012] In some embodiments, the human BDCA-2 promoter is a nucleotide sequence 5509 bp upstream of the start codon ATG; and / or the hBDCA-2 gene comprises a nucleotide sequence as shown in SEQ ID NO: 20, or a nucleotide sequence having at least 90% sequence identity with it, or a nucleotide sequence comprising substitutions, deletions, and / or insertions of one or more nucleotides compared to it; and / or the transgenic mouse is a transgenic mouse.

[0013] In some embodiments, the method includes: inserting a mouse endogenous Rosa26 gene into a targeting vector at the Rosa26 site; the targeting vector of the hBDCA-2 gene comprises the human BDCA-2 gene and the human BDCA-2 promoter, a 3' homologous arm and a 5' homologous arm; and / or the substitution also uses a crRNA / cas12 system, wherein the crRNA's targeting site is located on the mouse Rosa26 gene; and / or the sequence of the crRNA comprises the sequence shown in SEQ ID NO: 1 (TTCAAGTTTTCATCACTTGAAACACATT).

[0014] In some embodiments, the method includes the following steps: (1) Determine the target site based on the sequence of the mouse Rosa26 gene; (2) Synthesize crRNA sequences based on the target sites determined in step (1); (3) Construct a linear targeting vector containing the hBDCA-2 gene; (4) The crRNA obtained in step (2) and the linear targeting vector obtained in step (3) were introduced into mouse embryonic stem cells to obtain human BDCA-2 protein expression; (5) The mouse embryonic stem cells obtained in step (4) are polymerized with tetraploid cells and injected into the uterine horn of a surrogate mouse to prepare a transgenic mouse.

[0015] In some embodiments, in step (2), the crRNA comprises a sequence as shown in SEQ ID NO: 1 and / or as shown in SEQ ID NO: 2; and / or in step (5), the transgenic mouse is prepared by tetraploid compensation technology.

[0016] In some embodiments, the hBDCA-2 gene comprises a nucleotide sequence as shown in SEQ ID NO: 20, or a nucleotide sequence having at least 90% sequence identity with it, or a nucleotide sequence comprising substitutions, deletions and / or insertions of one or more nucleotides compared to it.

[0017] Secondly, the present invention also provides a recombinant vector containing the aforementioned humanized gene.

[0018] In some embodiments, the targeting vector further comprises a 3' homologous arm and a 5' homologous arm, the 3' homologous arm being as shown in SEQ ID NO: 2 and the 5' homologous arm being as shown in SEQ ID NO: 3.

[0019] Thirdly, the present invention also provides the application of the BDCA-2 transgenic mouse model constructed by the above method and the above recombinant vector in the preparation of the following products (1)-(5): (1) Products of immune processes mediated by human plasmacytoid dendritic cells (pDCs); (2) Anti-BDCA-2 antibody; (3) Reagents targeting BDCA-2; (4) Disease models related to pDCs dysfunction; (5) Identify or screen human-targeted BDCA-2 drugs and their efficacy products.

[0020] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for establishing hBDCA-2 transgenic mice. Not only do these mice contain exons and introns for the first time, preserving the original gene structure, but introns 2 and 3 are also added. The resulting transgenic mice stably express human BDCA-2 protein, with high expression levels, and their cellular and pDC-mediated BDCA-2 molecular functional characteristics are highly consistent. This transgenic mouse is constructed by introducing an hBDCA-2 gene containing the human BDCA-2 gene and its promoter at the Rosa26 site. The human BDCA-2 gene contains complete functional exons and regulatory sequences, accurately mimicking the expression pattern and signal transduction function of human BDCA-2. This transgenic mouse can reproduce the human pDC-mediated immune response and can be applied to the development of anti-BDCA-2 antibodies and the evaluation of vaccine adjuvants. Attached Figure Description

[0021] To more clearly illustrate the technical solution of the present invention, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a schematic diagram of the targeting strategy for hBDCA-2 humanized mice in the embodiments of this disclosure.

[0023] Figure 2 This is a schematic diagram of the targeting vector for hBDCA-2 humanized mice in the embodiments of this disclosure and its sequencing results.

[0024] Figure 3 This is a linear schematic diagram of the 5' end gene (5' KI) targeting vector and a gel image of the cell identification results in the embodiments of this disclosure.

[0025] Figure 4 This is a linear schematic diagram of the 3' end gene (3' KI) targeting vector and a gel image of the cell identification results in the embodiments of this disclosure.

[0026] Figure 5 This is a gel image showing the cell identification results of homozygous KI in an embodiment of this disclosure.

[0027] Figure 6 This is a linear schematic diagram and a gel image of cell identification results of the targeting vector after knocking in the 3' end gene (3' KI) and deleting the resistance in the embodiments of this disclosure.

[0028] Figure 7 This is an image showing the SRY sex identification results in an embodiment of this disclosure.

[0029] Figure 8 This is a gel image showing the cell identification results after knocking in the 5' end (5' KI) and deleting the resistance in the embodiments of this disclosure.

[0030] Figure 9 This is a diagram showing the genotype identification results of hBDCA-2 humanized mice in the embodiments of this disclosure.

[0031] Figure 10 This is a PCR result diagram of the expression analysis of hBDCA-2 in the spleen, thymus and liver of hBDCA-2 humanized mice in the embodiments of this disclosure.

[0032] Figure 11 This is a FACS result diagram of protein expression analysis in the bone marrow and spleen of hBDCA-2 humanized mice in the embodiments of this disclosure.

[0033] Figure 12 This is a FACS result diagram of the analysis of hBDCA-2 humanized mouse-related immune cells in the embodiments of this disclosure. Detailed Implementation

[0034] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to specific examples. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0035] In this invention, unless otherwise specified, the conditions shall be performed according to conventional conditions or conditions recommended by the manufacturer; and unless the manufacturer of the reagents or instruments used is specified, they are all conventional products that can be purchased commercially.

[0036] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. For the purposes of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural forms, and vice versa.

[0037] In this invention, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0038] In this invention, the "sequence identity percentage" or "identity percentage" between two polynucleotide or polypeptide sequences refers to the number of identical matching positions shared by the sequences within a comparison window, taking into account additions or deletions (i.e., vacancies) that must be introduced for optimal alignment of the two sequences. A matching position is any position in which the same nucleotide or amino acid is present in both the target and reference sequences. Since vacancies are not nucleotides or amino acids, vacancies present in the target sequence are not counted. Similarly, vacancies present in the reference sequence are not counted because nucleotides or amino acids from the target sequence are counted, but nucleotides or amino acids from the reference sequence are not. The sequence identity percentage can be calculated by determining the number of positions in which the same amino acid residue or nucleic acid base appears in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the sequence identity percentage. The comparison of sequences and the determination of the sequence identity percentage between two sequences can be performed using software that is readily available online and downloadable. Suitable software programs are available from various sources for the alignment of protein and nucleotide sequences. A suitable program for determining the percentage of sequence identity is bl2seq, which is part of the BLAST program suite available from the National Center for Biotechnology Information's BLAST website (blast.ncbi.nlm.nih.gov). Bl2seq uses either the BLASTN or BLASTP algorithm to compare two sequences. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, Needle, Stretcher, Water, or Matcher, which are part of the EMBOSS suite of bioinformatics programs and are also available from the European Institute of Bioinformatics (EBI) at www.ebi.ac.uk / Tools / psa.

[0039] In this invention, the term "homologous arm" refers to the flanking sequences on the target vector that are completely identical to the genome sequence and are used to identify and allow recombination to occur.

[0040] BDCA-2 humanized mice are created by introducing a human BDCA-2 (CLEC4C / CD303) gene fragment using gene editing technology, enabling the mice to mimic the human BDCA-2 signaling pathway and immune regulation. The goal is to provide a precise in vivo model for research on the mechanisms of BDCA-2-related diseases, drug target validation, and efficacy evaluation, thereby accelerating the development of therapies for immune diseases and cancer.

[0041] The core value of BDCA-2 humanized mice is based on the biological functions of human BDCA-2: as a receptor specifically expressed on plasmacytoid dendritic cells (pDCs), BDCA-2 binds to the FcRγ-chain to form a signaling pathway. Upon triggering, it activates Syk, recruits SLP65, and regulates PLCγ2 activity. Through the PLCγ2-PKC pathway, it negatively regulates the NF-κB pathway, ultimately inhibiting the transcription and production of type I interferon (IFN-I). This regulatory role is crucial in autoimmune diseases such as systemic lupus erythematosus (SLE)—IFN-I secreted by pDCs is a core pathogenic factor in SLE, and BDCA-2 signaling can independently inhibit IFN-I production, making it a key target for disease intervention. Furthermore, BDCA-2 also participates in the pathological processes of chronic inflammation, infectious diseases such as EBV / HIV, and tumors such as primitive plasmacytoid dendritic cell tumors (BPDCN) and melanoma.

[0042] Example 1

[0043] I. Design and synthesis of crRNA Based on the mouse Rosa26 gene sequence (Gene ID: 14910), crRNA was designed and synthesized (designed by the website https: / / chopchop.cbu.uib.no / and synthesized by Genscript Biotech). The target sequence of crRNA on Rosa26 is shown in SEQ ID NO: 1.

[0044] UAAUUUCUACUCUUGUAGAUCCGTTCTTCAGCATTTGGATTTCTTTC (SEQ ID NO: 1).

[0045] II. Construction of the target carrier The target-firing strategy of the present invention is as follows: Figure 1As shown, the hBDCA-2 gene containing the human BDCA-2 gene and its promoter was introduced at the Rosa26 site.

[0046] The human BDCA-2 gene contains the nucleotide sequences of exon 2, intron 2, exon 3, intron 3, exon 4, exon 5, exon 6, and exon 7, which can accurately mimic the expression pattern and signal transduction function of human BDCA-2.

[0047] The serial numbers of each component of the target carrier are shown in Table 1 below. For the specific sequence, please refer to the sequence table.

[0048] Table 1 Serial Number Table of Target Carrier Components

[0049] Example 2: Obtaining humanized BDCA-2 mouse embryonic stem cells I. Stem Cell Targeting C57BL6 / J mouse embryonic stem cells were revived from a liquid nitrogen cryopreservation cell bank, specifically using mouse embryonic stem cells with a passage number of p10 or less. They were cultured for 3 days in 6cm culture dishes under the following conditions: 15% serum + LIF + 2i: Knockout DMEM + 15% FBS + NEAA + Gluamax + β-me + LIF + pD0325901 + chir99021 + feeder. Electroporation was performed using the Nucleofector™ IIs / 2b electroporator and the Mouse ES Cell Nucleofector® Kit (Lonza, VPH-1001) mouse embryonic stem cell electroporation kit, following the A023 program, with approximately 2 × 10⁻⁶ cells per cell. 6 Cells were electroporated in 100 μL of electroporation buffer containing the linearized targeting vector (4 μg), Cas12 protein (6 μg), and two crRNAs (2 μg each) prepared in Example 1. The transfected cells were seeded into three 6-well plates and allowed to recover for 36 hours. After recovery, 10 μg / mL blastomycin S (BSD) (Merck) was added to the cell culture medium. After 3 days of selection, puromycin-resistant mouse embryonic stem cell clones were picked and cultured in 96-well plates using a glass needle. The cultured cell clones were subjected to PCR to screen for positive clones.

[0050] PGK-BSD selection marker deletion: The mouse embryonic stem cells that tested positive for PCR were cultured to 6 cm plates and then electroporated using the Nucleofector™ IIs / 2b electroporator (using the A023 program) and the Mouse ES Cell Nucleofector® Kit (Lonza, VPH-1001) mouse embryonic stem cell electroporation kit, approximately 2 × 10⁻⁶ cells / cm². 6 Cells were electroporated in 100 μL of electroporation buffer containing the pPGK-FLPo plasmid (Addgene, 13793). The transfected cells were seeded into six wells of a 12-well plate. Three days later, mouse embryonic stem cell clones were picked using a glass needle and cultured in a 96-well plate. The next day, the clones were digested, passaged, and divided in two: one portion was selected for puromycin selection, and the other was cultured normally. If the PGK-BSD resistance selection marker was successfully deleted, the cells would die after BSD selection due to intolerance. The clones corresponding to BSD-intolerant cells were the ultimately successfully edited humanized BDCA-2 mouse embryonic stem cells.

[0051] II. Genotyping of humanized BDCA-2 mouse embryonic stem cells The successfully edited monoclonal cells obtained in step one were trypsinized and divided into two aliquots. One aliquot was lysed individually at 56 °C for 60 min in 10 μL of NP 40 lysis buffer (NP40 lysis buffer: 10 mL TE (20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA) + 0.5% NP40 + 10 μL 10 mg / mL proteinase K), followed by lysis at 95 °C for 10 min. The lysates were used as PCR screening templates for genotyping. PCR was performed using Phanta Max Super-Fidelity DNA Polymerase (Novizan) according to the manufacturer's instructions.

[0052] PCR analysis was performed on homologous recombination (HDR) with 5' and 3' homologous arms for targeted insertion into the mouse BDCA-2 locus. The genotyping protocol is shown in Table 2 below. The primers for targeted insertion and the identification results are as follows: Figures 3-4 As shown. If the recombinant vector insertion site is correct, a band identical to the expected one should appear. Based on the PCR gel image of the corresponding primers, it can be determined that: a successful 5'-KI result will show a 1353 bp band, and a successful 3'-KI result will show a 2029 bp (BSD) band; if it is homozygous KI, a 583 bp WT band should not appear. Figures 3-4The results showed that homozygous KI embryonic stem cell lines that met expectations, such as E1, B2, F2, G3, C4, and F4, were selected for further deletion of resistance. The FRT-PGK-BSD-polyA-FRT resistance expression cassette in the KI sequence was deleted using FLP recombinase, leaving only one FRT site.

[0053] Table 2 Primer Information for Genotype Identification

[0054] The full-length sequence of the successfully inserted nucleotide is shown in SEQ ID NO: 20, which is the sequence of hBDCA-2 successfully inserted, and it encodes the amino acid sequence shown in SEQ ID NO: 21: TPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGS HSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGYYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQ YAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDSPKAHVTHHPRSKGEVTLRCWALGFYPADITLTWQLNGEELTQDM ELVETRPAGDGTFQKWASVVVPLGKEQNYTCRVYHEGLPELPLTLRWEPPPSTDSYMVIVAVLGVLGAMAIIGAVVAFVMKRRRNTGGKGGDYALAPGSQSSEMSLRDCKA (SEQ ID NO: 21).

[0055] Example 3: Obtaining humanized BDCA-2 mice using tetraploid embryo compensation technology I. Tetraploid Compensation Techniques Embryo transfer using a tetraploid compensation experiment is performed as follows: 1. Administer 7.5 units of pregnant mare serum gonadotropin (PMSG) intraperitoneally to 4-10 week old B6C3F1 female mice. 48 hours later, administer human chorionic gonadotropin (hCG) and pair the mice with CD1 male mice. The next morning, examine the female mice for vaginal plugs and remove any female mice with vaginal plugs. Record the corresponding fertilization time of the female mice.

[0056] 2. The next day, the pregnant mice were euthanized by cervical dislocation. The abdomen of the mice was disinfected with 70% alcohol. The abdominal skin and muscle layers were cut open with auxiliary forceps and ophthalmic scissors to open the abdominal cavity. The upper part of one uterine horn was grasped with forceps, and a small incision was made in the membrane near the fallopian tube with scissors. The connection between the fallopian tube and the ovary was cut. The fallopian tube and the attached uterus were transferred to a 35 mm culture dish. The fimbriae of the fallopian tube were fixed with forceps. A flushing needle filled with M2 culture medium was gently inserted into the fimbriae, and the fallopian tube was flushed with 0.1 mL of M2 culture medium. The flushed embryos were collected using the transfer tube and washed three times with M2. E1.5 mouse 2-cell embryos were collected. For specific experimental procedures, please refer to Hogan, B. (1994). Manipulating the mouse embryo: a laboratory manual, 2nd edn (Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press).

[0057] 3. The collected mouse embryos were placed in 0.3M mannitol containing 0.1mM MgSO4, 0.1mM CaCl2, and 0.3% bovine serum albumin. Fusion was performed using a Cellfusion CF-150 / B electrofusion apparatus and a 250-um fusion tank (BLS Ltd., Budapest, Hungary) at 60V for 50 microseconds to obtain tetraploid embryos. These embryos were then placed in KSOM medium (Summers, MC, McGinnis, LK, Lawitts, JA, Raffin, M., and Biggers, JD (2000). IVF of mouse embryos in a simple x optimized medium supplemented with amino acids. HumReprod 15, 1791-1801.), after being cultured in a CO2 incubator for 24 hours, the zona pellucida was removed with acidic Tyrode's solution (Sigma-Aldrich, T1788), and then aggregated with embryonic stem cells (i.e., humanized hBDCA-2 mouse embryonic stem cells prepared in Example 3) to form chimeric embryos (Nagy, A., Rossant, J., Nagy, R., Abramow-Newerly, W., and Roder, JC (1993). Derivation of completely cell embryo-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci USA 90, 8424-8428.).

[0058] 4. The chimeric embryos were cultured overnight in a CO2 incubator and then transferred to the uterus of a pseudopregnant mouse at 2.5 days of gestation. After 17 days, the surrogate mouse was euthanized by cervical dislocation and then laparotomy was performed. The surviving and breathing newborn mice were placed in a surrogate mouse cage and weaned after 21 days. Transgenic mice derived entirely from embryonic stem cells were obtained, which are humanized hBDCA-2 genetically modified mice (hBDCA-2 humanized mice).

[0059] II. Mouse Genotype Identification: After lysis of mouse tails, they were used as templates for PCR identification. The identification protocol is shown in Table 3. The identification results for different mice are as follows: Figure 8-9 As shown, this demonstrates the successful construction of humanized hBDCA-2 genetically modified mice (hBDCA-2 humanized mice). These mice are F0 generation mice with a homozygosity of 100%. A large number of individuals can be obtained through subsequent tetraploid embryo transfer without the need for traditional fertilized eggs.

[0060] Table 3 Primer information for mouse genotyping

[0061] Example 4: RT-PCR detection of gene expression in organs of hBDCA-2 humanized mice Spleen, thymus, and liver from mice (n=3, 8 weeks old) and wild-type mice (n=3, 8 weeks old) were washed with PBS, ground in liquid nitrogen, and the powder was collected. Trizol was added for lysis, chloroform was added, and the mixture was centrifuged. The supernatant was transferred to pre-chilled isopropanol and centrifuged again. The supernatant was discarded, and the precipitate was washed with 75% ethanol, centrifuged, and dried. The precipitate was dissolved in water without RNase and used for reverse transcription. 1 μg of RNA was used in a Novizan reverse transcription kit (catalog number: R333-01) according to the manufacturer's instructions to obtain cDNA for qPCR experiments.

[0062] Using the primers shown in Table 4 and the Novizan qPCR kit (catalog number: Q312-02), and following the instructions, hBDC2 was quantified by qPCR. The results are as follows: Figure 10 The expression levels of hBDC2 in the spleen, thymus, and liver of humanized hBDC2 mice were significantly higher than those in wild-type mice.

[0063] Table 4 hBDC-2 qRCR Primer Information

[0064] Example 5: FACS detection of related gene expression in hBDCA-2 humanized mice I. Flow cytometry experimental procedure: Peripheral blood was obtained from male C57BL / 6 and humanized mice (n=3, 8 weeks old). Flow cytometry was used to analyze the cells to assess leukocyte subsets. The specific steps are as follows: (1) Obtaining single-cell suspension: Place 200 μL of peripheral blood sample into a 15 mL centrifuge tube, add 5 mL of ACK-gibco (erythrocyte lysis buffer), lyse at room temperature for 10 min, add 2 times the volume of PBS, mix well, and centrifuge at 10℃, 350 g for 5 min. (2) Cell counting: Discard the supernatant, resuspend the cells in 100-200 μL of FACS Buffer (2% FBS); perform cell counting between cells; take 5 × 10⁻⁶ cells for each reaction. 5 For approximately 100 cells, replenish FACS Buffer to 100 μL per reaction. (3) FC blocking: Add 3-5 μL of Fc blocking solution (Anti-Mo CD16 / CD32) according to the cell volume, block on ice for 15 min. The general volume ratio of Fc used is 1:100, and the stock solution concentration is 0.5 mg / mL. (4) Antibody staining: All antibodies used in the arrangement (all antibodies were purchased from Biolegend, including: human BDCA-2 antibody, catalog number 354205; mouse CD3 antibody, catalog number 100215; mouse CD19 antibody, catalog number 11519; mouse NK1.1 antibody, catalog number 156507; mouse B220 antibody, catalog number 103221; mouse CD317 antibody, catalog number 127009) were added to 1.5 mL EP tubes according to the color matching combination and mixed well. The human antibody was usually 5 μL / Test and the mouse antibody was usually 1 μL / Test. The tubes were stained on ice in the dark for 15 min, during which time the bottom of the tube was gently tapped 1-2 times to mix well for thorough staining. (5) Termination of staining: Add 1 mL of FACS Buffer to wash away unbound antibody-conjugated dye, invert to mix, and centrifuge at 10°C, 350 g for 5 min. Aspirate the supernatant using a pipette, leaving approximately 50-100 μL. (6) DAPI staining: Add 400 μL of DAPI staining solution, resuspend and mix well, stain for 1 min, then add 1 mL of FACS Buffer and mix well; centrifuge at 10℃, 350 g, for 5 min to wash away unbound DAPI. Aspirate the supernatant using a pipette, leaving approximately 50-100 μL. (7) Resuspension and flow cytometer: The BD LSR Fortessa SORP analytical flow cytometer was used. The sample was resuspended in 400 μL FACS Buffer (DPBS+2%FBS), then filtered through a 300-mesh sieve into the flow cytometer tube, and placed on ice to avoid light before flow cytometer detection.

[0065] II. Protein Expression Analysis in Spleen and Bone Marrow like Figure 11 , 12 As shown, in the bone marrow and spleen of wild-type C57BL / 6J mice (WT) and BDCA-2 humanized mice (hBDCA2), human BDCA-2 was detected only in the pDCs of BDCA-2 humanized mice, indicating successful expression of human BDCA-2. Simultaneously, the composition of immune cell subsets showed no significant difference from wild-type mice. Flow cytometry data from this example demonstrates successful BDCA-2 expression in BDCA-2 humanized mice. Therefore, the BDCA-2 humanized mouse was successfully prepared and has significant application value for mouse breeding.

[0066] Example 5: Obtaining BDCA-2 humanized mice This embodiment exemplarily prepared BDCA-2 humanized mice, wherein the promoter region, exon2, intron2, exon3, intron3, exon4, exon5, and exon6 are as shown in Table 1 above.

[0067] The prepared BDCA-2 humanized mice were subjected to genotyping. The primers used for identification are shown in Table 2. The mouse genotyping results are as follows: Figure 8-9 As shown.

[0068] In summary, this invention provides a humanized mouse model of BDCA-2, in which the human BDCA-2 gene locus is precisely integrated into the mouse genome, including the complete human BDCA-2 promoter region, coding region, and 3' regulatory region, to achieve physiological expression of this gene in mouse pDCs. This disclosure exemplarily uses C57BL / 6J mice as the genetic background and employs CRISPR / Cas12a-mediated homologous recombination technology to precisely insert the human BDCA-2 gene (exon 2, intron 2, exon 3, intron 3, exon 4, exon 5, exon 6, exon 7, corresponding to SEQ ID NO:20) into the safe site of the mouse Rosa26 gene, while preserving the expression regulatory elements of the mouse endogenous Rosa26 gene. This design ensures the specific expression of human BDCA-2 in pDCs.

[0069] The humanized mouse model of the present invention has the following characteristics: (1) At the molecular level, the human BDCA-2 gene is highly expressed in the relevant organs, and RT-PCR detection shows that its mRNA level is highly expressed in the spleen, thymus and liver; (2) At the cellular level, flow cytometry analysis confirms that the human BDCA-2 protein is located on the surface of mouse pDCs cell membranes and the expression rate is over 95%; (3) Functional immunophenotypic analysis shows that the composition of immune cell subsets in the model mice is not significantly different from that in wild-type mice, excluding the interference of gene insertion on the overall homeostasis of the immune system.

[0070] The humanized mouse model of this invention can be used to prepare drugs for autoimmune diseases such as systemic lupus erythematosus, as well as for preparing therapeutic or analytical products targeting BDCA-2, including monoclonal antibodies, ADC drugs, and small molecule inhibitors. It can also be used to prepare IFN-I level and disease-related phenotypic monitoring products, or to construct viral infection models such as HCV and HIV, thereby facilitating research on the role of BDCA-2 in viral immune escape and intervention strategies. The humanized mouse model of this invention can also be used to prepare immunotherapeutic products for hematological malignancies such as BPDCN, demonstrating significant application value and providing experimental evidence for BDCA-2-regulated tumor immunotherapy.

[0071] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for constructing a BDCA-2 transgenic mouse model, characterized in that, The method includes: introducing the hBDCA-2 gene into the Rosa26 site of a mouse, wherein the hBDCA-2 gene includes a human BDCA-2 gene and a human BDCA-2 promoter.

2. The method according to claim 1, characterized in that, The transgenic mouse cells expressed human BDCA-2 protein.

3. The method according to claim 1, characterized in that, The human BDCA-2 gene contains the nucleotide sequence of exon 2, intron 2, exon 3, intron 3, exon 4, exon 5, exon 6, and exon 7 of human BDCA-2.

4. The method according to claim 1, characterized in that, The human BDCA-2 promoter is a nucleotide sequence 5509 bp upstream of the start codon ATG; and / or the hBDCA-2 gene comprises a nucleotide sequence as shown in SEQ ID NO: 20, or a nucleotide sequence having at least 90% sequence identity with it, or a nucleotide sequence that includes substitutions, deletions, and / or insertions of one or more nucleotides compared to it; and / or the transgenic mouse is a transgenic mouse.

5. The method according to claim 1, characterized in that, The method includes: inserting a targeting vector at the Rosa26 site into the mouse endogenous Rosa26 gene; the targeting vector of the hBDCA-2 gene comprises the human BDCA-2 gene and the human BDCA-2 promoter, a 3' homologous arm and a 5' homologous arm; and / or the substitution also uses a crRNA / cas12 system, wherein the target site of the crRNA is located on the mouse Rosa26 gene; and / or the sequence of the crRNA comprises the sequence shown in SEQ ID NO:

1.

6. The method according to claim 1, characterized in that, The method includes the following steps: (1) Determine the target site based on the sequence of the mouse Rosa26 gene; (2) Synthesize crRNA sequences based on the target sites determined in step (1); (3) Construct a linear targeting vector containing the hBDCA-2 gene; (4) The crRNA obtained in step (2) and the linear targeting vector obtained in step (3) were introduced into mouse embryonic stem cells to obtain human BDCA-2 protein expression; (5) The mouse embryonic stem cells obtained in step (4) are polymerized with tetraploid cells and injected into the uterine horn of a surrogate mouse to prepare a transgenic mouse.

7. The method according to claim 6, characterized in that, In step (2), the crRNA includes the one shown in SEQ ID NO:1; and / or in step (5), the transgenic mouse is prepared by tetraploid compensation technology.

8. A recombinant vector, characterized in that, The recombinant vector comprises the humanized gene and its promoter as described in claims 4 and 5.

9. The recombinant vector according to claim 8, characterized in that, The recombinant vector further comprises a 3' homologous arm and a 5' homologous arm, the 3' homologous arm being as shown in SEQ ID NO: 2, and the 5' homologous arm being as shown in SEQ ID NO:

3.

10. The use of the BDCA-2 transgenic mouse model constructed by the method according to any one of claims 1-7, and the recombinant vector according to any one of claims 8-9, in the preparation of the following products (1)-(5): (1) Products of human plasmacytoid dendritic cell-mediated immune processes; (2) Anti-BDCA-2 antibody; (3) Reagents targeting BDCA-2; (4) Disease models related to pDCs dysfunction; (5) Identify or screen human-targeted BDCA-2 drugs and their efficacy products.

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