CN116250509B

CN116250509B - Construction method and application of SIGLEC10 humanized mouse model

Info

Publication number: CN116250509B
Application number: CN202310432191.8A
Authority: CN
Inventors: 王腾; 汪慧怡; 范佳露; 王韬; 朱珠; 陈颖珊
Original assignee: Guangdong Yaokang Biotechnology Co ltd
Current assignee: Guangdong Yaokang Biotechnology Co ltd
Priority date: 2023-04-21
Filing date: 2023-04-21
Publication date: 2023-10-13
Anticipated expiration: 2043-04-21
Also published as: CN116250509A

Abstract

The invention relates to a construction method of a SIGLEC10 humanized mouse model, which comprises the following steps: (1) constructing a targeting vector for expressing the humanized SIGLEC 10; (2) Designing and obtaining sgrnas for the mouse Siglecg genes Exon2 to Exon11 portions; (3) Co-injecting or co-electrotransferring the targeting vector, sgRNA and Cas9 protein into cytoplasm or nucleus of fertilized egg of mouse, transplanting fertilized egg into pseudopregnant mouse, and carrying out genotype identification to pseudopregnant mouse, and screening positive F0 mouse successfully inserted into correct humanized fragment; (4) F1 mice are obtained by breeding F0 mice and background mice, and SIGLEC10 humanized mouse models are screened. The SIGLEC10 humanized mouse constructed by the invention has application value in the fields of oncology, immunology and the like.

Description

Construction method and application of SIGLEC10 humanized mouse model

Technical Field

The invention relates to the field of animal genetic engineering, in particular to a construction method and application of a SIGLEC10 humanized mouse model.

Background

Immune cells are activated or inhibited by binding of the corresponding antibodies to receptors on the surface of the immune cells, these activated or inhibited receptors being termed immune checkpoints. Normally, immune checkpoints will maintain autoimmunity and respond to pathogen infection to protect tissues from injury. In the case of tumors, the tumor releases corresponding antibodies to act on immune checkpoints, and immune cells are inhibited, so that tumor immune escape occurs, and in the process of tumor development, the tumor can be immunized against the tumor by developing various immune mechanisms. Thus, by exploring the mechanisms by which tumor cells escape immune surveillance, it is helpful to formulate important strategies for targeting tumor immune escape.

In recent years, scientists have realized significant success in the study of tumor immunotherapy strategies in view of the significant success of treatment of innate immune checkpoints associated with tumor immune escape. Recent studies indicate that CD24 may be the primary immune checkpoint in tumors, which is capable of interacting with sialic acid-binding immunoglobulin-like lectin 10 (SIGLEC 10) to promote tumor immune escape, and is expected to become a new target for tumor treatment.

Sialic acid binding immunoglobulin-like lectin 10 (SIGLEC 10) the corresponding gene in mice is Siglecg, which is widely expressed in B cells and a subset of macrophages. The lgV domain of SIGLEC10 on the surface of immune cells is capable of binding to sialic acid in the CD24 terminal region on the surface of tumor cells, resulting in the recruitment of the immune receptor tyrosine inhibitory motif (ITIM) within immune cells to phosphorylate Src family kinases and to recruit tyrosine phosphatases, such as SHP-1 and SHP-2, to reduce downstream signaling to promote tumor immune escape.

In the process of drug development, the important role played by the mouse experimental model is not replaceable. However, due to race variability, the potency of SIGLEC10 inhibitors screened in mouse experiments may vary from that of humans. Therefore, the construction of the SIGLEC10 humanized mouse model has higher application value in screening and evaluating SIGLEC10 target drugs.

At present, a construction method of a SIGLEC10 humanized mouse model and a related literature report of the SIGLEC10 humanized mouse model in the aspect of target drug application are not seen.

Disclosure of Invention

In order to solve the existing problems, a first aspect of the present invention provides a method for constructing a SIGLEC10 humanized mouse model, the method comprising the steps of:

(1) Constructing a targeting vector for expressing the humanized SIGLEC10, and inserting the humanized SIGLEC10 gene;

(2) Designing sgrnas for the Exon2 to Exon11 parts of the mouse Siglecg gene, and obtaining the sgrnas by using an in vitro transcription technology;

(3) Co-injecting or co-electrotransferring the targeting vector constructed in the step (1), the sgRNA obtained in the step (2) and the Cas9 protein into cytoplasm or nucleus of fertilized ovum of the mouse, transplanting the fertilized ovum into pseudopregnant mouse, carrying out genotype identification on pseudopregnant mouse, and screening positive F0 mouse successfully inserted into correct human fragment;

(4) F0 mice and background mice are bred to obtain F1 mice, and gene identification is carried out on the tail of the F1 generation, so that a SIGLEC10 humanized mouse model is screened.

Preferably, the step (1) includes the following steps: according to the structure and function of the human SIGLEC10, the signal peptide and extracellular region of the human SIGLEC10 are selected to replace the signal peptide and extracellular region sequence of the murine SIGLEC10 gene, the amino acid sequence of the selected human SIGLEC10 gene is shown as SEQ ID No.1, and the amino acid sequence of the replaced murine SIGLEC gene is shown as SEQ ID No. 2.

Preferably, the step (1) includes the following steps: exon1 to Exon9 of the humanized SIGLEC10 gene are selected, exon2 to Exon11 of the mouse Siglecg gene are replaced by using the homologous recombination technology, and the sequence of the selected humanized SIGLEC10 gene is shown as SEQ ID No. 3.

Preferably, the sequence of the targeting vector successfully constructed in the step (1) is shown as SEQ ID No. 4.

Preferably, the sgRNA in step (2) has the gene sequence of (a) SEQ ID NO.5 and SEQ ID NO.7, or (b) SEQ ID NO.6 and SEQ ID NO.8.

More preferably, the sgRNA in the step (2) has the gene sequences of SEQ ID NO.5 and SEQ ID NO.7.

Preferably, the strain of mice and pseudopregnant mice provided with fertilized eggs in step (3) is BALB/c.

Preferably, the 5 'end identification primer used for F0 mouse genotype identification in the step (3) is shown as SEQ ID NO.9 and SEQ ID NO.10, and the 3' end identification primer is shown as SEQ ID NO.11 and SEQ ID NO. 12.

Preferably, the PCR reaction system used for genotyping the F0 mice in the step (3) is as follows:

preferably, the PCR reaction conditions used for genotyping the F0 mice in the step (3) are as follows:

the second aspect of the invention provides the application of the mice obtained by the construction method in researching the related functions and action mechanisms of the SIGLEC10 genes.

Preferably, the use is for non-diagnostic and non-therapeutic purposes.

In a third aspect, the present invention provides an application of the mouse obtained by the above construction method in screening a drug for treating diseases related to SIGLEC10 gene.

Preferably, the use is for non-diagnostic and non-therapeutic purposes.

The invention has the beneficial effects that:

according to the invention, according to the comparison of the human SIGLEC10 protein functional domain and human and mouse homology, exon1 to Exon9 of the human SIGLEC10 gene are selected to replace Exon2 to Exon11 of the mouse Siglecg gene, and the mouse SIGLEC10 gene is successfully humanized to obtain the BALB/c-hSIGLEC10 mouse model. The model has application value in the fields of oncology, immunology and the like.

Drawings

FIG. 1 is an electrophoretogram of the 5 'and 3' gene identifications of SIGLEC10-KI-target F0 mice;

FIG. 2 is an electrophoretogram of the 5 'and 3' gene identifications of SIGLEC10-KI-target F1 mice;

FIG. 3 is a flow cytometry map of SIGLEC10 expression in peripheral blood B lymphocytes of BALB/c mice and BALB/c-hSIGLEC 10F 1 heterozygous mice;

FIG. 4 is a flow cytometry image of SIGLEC10 expression in peripheral blood macrophages of BALB/c mice and BALB/c-hSIGLEC 10F 1 heterozygous mice;

FIG. 5 is a flow cytometry image of SIGLEC10 expression in peripheral blood T/B/NK cells of BALB/c mice and BALB/c-hSIGLEC 10F 1 heterozygous mice.

Detailed Description

The present invention is further illustrated by way of examples, but the present invention is not limited to the following examples.

Test example 1, establishment of SIGLEC10 humanized mouse model

The invention uses CRISPR/Cas9 technology to replace a murine Siglecg (mSIGLEC 10) gene with a human SIGLEC10 gene on a mouse with BALB/c background, thereby constructing a mouse model capable of expressing the human SIGLEC10, and the specific method is as follows:

1. determination of human fragment substitution region and inserted human sequence

According to the structure and function of the human SIGLEC10, the signal peptide and extracellular region of the human SIGLEC10 are selected to replace the signal peptide and extracellular region sequence of the murine Siglecg. The amino acid sequence (Aa: 1-550) of the selected humanized SIGLEC10 gene is shown as SEQ ID No.1, and the amino acid sequence (Aa: 1-543) of the replaced murine Siglecg gene is shown as SEQ ID No. 2.

MLLPLLLSSLLGGSQAMDGRFWIRVQESVMVPEGLCISVPCSFSYPRQDWTGSTPAYGYWFKAVTETTKGAPVATNHQSREVEMSTRGRFQLTGDPAKGNCSLVIRDAQMQDESQYFFRVERGSYVRYNFMNDGFFLKVTALTQKPDVYIPETLEPGQPVTVICVFNWAFEECPPPSFSWTGAALSSQGTKPTTSHFSVLSFTPRPQDHNTDLTCHVDFSRKGVSAQRTVRLRVAYAPRDLVISISRDNTPALEPQPQGNVPYLEAQKGQFLRLLCAADSQPPATLSWVLQNRVLSSSHPWGPRPLGLELPGVKAGDSGRYTCRAENRLGSQQRALDLSVQYPPENLRVMVSQANRTVLENLGNGTSLPVLEGQSLCLVCVTHSSPPARLSWTQRGQVLSPSQPSDPGVLELPRVQVEHEGEFTCHARHPLGSQHVSLSLSVHYSPKLLGPSCSWEAEGLHCSCSSQASPAPSLRWWLGEELLEGNSSQDSFEVTPSSAGPWANSSLSLHGGLSSGLRLRCEAWNVHGAQSGSILQLPDKKGLISTAFSN(SEQ ID No.1)

MSLLLFLLSFLLDGPQGQMESYFLQVQRIVKAQEGLCIFVPCSFSSPEGKWLNRSPLYGYWFKGIRKPSLSFPVATNNKDKVLEWEARGRFQLLGDISKKNCSLLIKDVQWGDSTNYFFRMERGFERFSFKEEFRLQVEALTQKPDIFIPEVLEPGEPVTVVCLFSWTFNQCPAPSFSWMGDAVSFQESRPHTSNYSVLSFIPGLQHHDTELTCQLDFSRMSTQRTVRLRVAYAPRSLAISIFHDNVSVPDLHENPSHLEVQQGQSLRLLCTADSQPPATLSWVLEDQVLSWSSPVGSRTLALELPWVKAGDSGHYTCQAENRLGSQQHTLDLSVLYPPQDLRVTVSQANRTVLEILRNAISLPVLEGQSLCLVCVTYSNPPANVSWAWVTQTLIPIQSSEPGVLELPLVQREHEGEFTCAAQNPLGAQRISLSLSVHYPPQMSSPSCSWEAKGLHCNCSSRAWPAPSLRWRLGEGLLEGNSSNASFTVTFSSLGPWVNSSLSLLQELGPSLWLSCESWNTHGAQTTSVLLLPDKDSATAFSK(SEQ ID No.2)

2. Injection to obtain positive mice

The Exon1 to Exon9 portions of the humanized SIGLEC10 gene are replaced by the Exon2 to Exon11 portions of the mouse Siglecg gene by using a CRISPR Cas9 mode, and a SIGLEC10 gene humanized mouse model is established. BALB/c mice were used as background mice, and SIGLEC10 humanized mice model was successfully obtained.

1) Determination of human fragment substitution region and inserted human sequence

Based on the comparison of extracellular domain of human SIGLEC10 protein and human and mouse homology, the Exon1 to Exon9 portions of human SIGLEC10 gene replaced the Exon2 to Exon11 portions of mouse Siglecg gene, and the sequence of the selected human SIGLEC10 gene replacement was shown as SEQ ID No.3 (the underlined portion is the Exon sequence, and the non-underlined portion is the Intron sequence).

ATGCTACTGCCACTGCTGCTGTCCTCGCTGCTGGGCGGTGAGTGGGCCAAGGGCCGGAAAGGACCTGGGGCGGAGCTAGGGCTGGGGTTGCAGGTTGAGCCTCTGTCTCCCCACAGGGTCCCAGGCTATGGATGGGAGATTCTG GATACGAGTGCAGGAGTCAGTGATGGTGCCGGAGGGCCTGTGCATCTCTGTGCCCTGCTCTTTCTCCTACCCCCGA CAGGACTGGACAGGGTCTACCCCAGCTTATGGCTACTGGTTCAAAGCAGTGACTGAGACAACCAAGGGTGCTCCTG TGGCCACAAACCACCAGAGTCGAGAGGTGGAAATGAGCACCCGGGGCCGATTCCAGCTCACTGGGGATCCCGCCAA GGGGAACTGCTCCTTGGTGATCAGAGACGCGCAGATGCAGGATGAGTCACAGTACTTCTTTCGGGTGGAGAGAGGA AGCTATGTGAGATATAATTTCATGAACGATGGGTTCTTTCTAAAAGTAACAGGTATGGAATGGGGTGGGAACCCCTGCCTCTCACACTGGGGAGGGACCCTGGGGACAGCCTATGGGCTGAGCAGAGAGGGCTCTCAGGGACCCCTGCAGCACAAGAATCTCCCACCACGGTCTCTGTCCCAGCCCTGACTCAGAAGCCTGATGTCTACATCCCCGAGACCCTGGAGC CCGGGCAGCCGGTGACGGTCATCTGTGTGTTTAACTGGGCCTTTGAGGAATGTCCACCCCCTTCTTTCTCCTGGAC GGGGGCTGCCCTCTCCTCCCAAGGAACCAAACCAACGACCTCCCACTTCTCAGTGCTCAGCTTCACGCCCAGACCC CAGGACCACAACACCGACCTCACCTGCCATGTGGACTTCTCCAGAAAGGGTGTGAGCGCACAGAGGACCGTCCGAC TCCGTGTGGCCTGTGAGTGTGGCCTGGGAGGGTGGGGTGTGCAGACAGCCCCGGTGGGTGGGGAGGTGGAGGAGCCCAGTGGGACAGTGAGCGGCTCCCAGCTCAGGAGCATCCAGGGAGAGGAAGCTGTGGGGTCCCAGGATGCCGCCGCAGCCCTGGGAGGGGGATGGGAATGGCGTCTGGTCCACACACGTGAGCCCTGGCGCTGGTTGTCACTTGTCTTCCCGGGATAGTCCCACTTTCTTTTCCCTGAGGGAGTTTTTTCCAGGTGTGAGGAATAAATTGTCCCTCCCTGAAGCCAGCTCACAATCTTGTTGCAGATGCCCCCAGAGACCTTGTTATCAGCATTTCACGTGACAACACGCCAGGTACTGAGGGCCGTCGGGCTGGGGCTGGGCCAGTTGTCTTTAGATGAAAAGGCTTCAGGGGATGAGGGGATGTGCTCCTCCCTGTACCCCTTCTCTCTTTCTCCCTCCCTTTCTCTCTTTTTTTTTTTTCCTCCCACTCCAGCCCTGGAGCCCCAGCCCCAGGG AAATGTCCCATACCTGGAAGCCCAAAAAGGCCAGTTCCTGCGGCTCCTCTGTGCTGCTGACAGCCAGCCCCCTGCC ACACTGAGCTGGGTCCTGCAGAACAGAGTCCTCTCCTCGTCCCATCCCTGGGGCCCTAGACCCCTGGGGCTGGAGC TGCCCGGGGTGAAGGCTGGGGATTCAGGGCGCTACACCTGCCGAGCGGAGAACAGGCTTGGCTCCCAGCAGCGAGC CCTGGACCTCTCTGTGCAGTGTGAGTGTGCCCAGCAGGGGCCTGGAGTCCATTGGGAGGGCAGAGGGATACAGGGGCTGGGCTCAGGGTCCCAGAGCTGAGGGGGCCTGGAGCCCCAGGCCTCGGAGACCGACCTTCTTACCTGTGTAGACCCTCATGCAGTTTGTGTCTGGGACTCAGTGGGTGATTCTGCCCTGCCCTTCTATCCCACCCACTTCCCCCACCTCAGTGTCCAGGATGGTTCCCTTTACCCAGAGGGAAGCCCCTGGTCCGTCTAGAGCCGGTCCCCTGTCTCCATTTCAGAT CCTCCAGAGAACCTGAGAGTGATGGTTTCCCAAGCAAACAGGACAGGTAGGAAAGGGGACAGAGGAGCCAAGGCCTCTCAGTGCCGAATTGGGGGCCCAGGAGTCTGGAGGGTCCCCACGCAGGAGGGTCCCTGAGCCCTGAGCTGCTCATCGATTCTGCCTCTTCCTTCCCTAGTCCTGGAAAACCTTGGGAACGGCACGTCTCTCCCAGTACTGGAGGGCCAAAGC CTGTGCCTGGTCTGTGTCACACACAGCAGCCCCCCAGCCAGGCTGAGCTGGACCCAGAGGGGACAGGTTCTGAGCC CCTCCCAGCCCTCAGACCCCGGGGTCCTGGAGCTGCCTCGGGTTCAAGTGGAGCACGAAGGAGAGTTCACCTGCCA CGCTCGGCACCCACTGGGCTCCCAGCACGTCTCTCTCAGCCTCTCCGTGCACTGTGAGTGGGGGAAAGGGGACACCTGGGTCCCAGGAAGGGGACCCTGCTGAGTCCTGTCCTCCCTCCCCTCAGACTCCCCGAAGCTGCTGGGCCCCTCCT GCTCCTGGGAGGCTGAGGGTCTGCACTGCAGCTGCTCCTCCCAGGCCAGCCCGGCCCCCTCTCTGCGCTGGTGGCT TGGGGAGGAGCTGCTGGAGGGGAACAGCAGCCAGGACTCCTTCGAGGTCACCCCCAGCTCAGCCGGGCCCTGGGCC AACAGCTCCCTGAGCCTCCATGGAGGGCTCAGCTCCGGCCTCAGGCTCCGCTGTGAGGCCTGGAACGTCCATGGGG CCCAGAGTGGATCCATCCTGCAGCTGCCAGGTTAGGGGGCCGCCTGGATGCCAAGGTTCTTGGAGGAGAAGCTGACTGGGGCAAAGAGTAGGGTCCTGTGGTTGGAGCAGGAGCTTGGAGGGACACAGCAGACCCGTGGCATCTGTATGTAGACAGGAAAACACAATGGGAAGTCCTAACACCTGGGCCGGGGTGTGGCAGGGTTTCCTAAAAGTAAACAGAGGGGTTGCAGATACAATGTCCGCCATGGGAACCGGAAGCCTTAATGCCGAGTGATGAGCGGGCTTCGTGAGCCCTGCGTGGATGGGAAATGCTCACCTTGCCCTCTCCGTGTGCAGATAAGAAGGGACTCATCTCAACGGCATTCTCCAAC(SEQ ID No.3)。

2) Humanized targeting vector construction

Constructing Exon1 to Exon9 of a humanized SIGLEC10 gene into a targeting vector, replacing Exon2 to Exon11 of a murine Siglecg gene by using a homologous recombination technology, and constructing a successful targeting vector sequence as shown in SEQ ID No.4 (KI fragments are shown in italics).

3) Construction of sgRNA

(1) Respectively synthesizing upstream and downstream primers of the sgRNA, wherein the purification mode of the primers is PAGE;

(2) The upstream and downstream primers of the sgRNA are respectively diluted to 100 umol/mu l and evenly mixed according to the proportion of 1:1, and the mixture is automatically and slowly annealed at room temperature;

(3) The double strand formed by annealing is connected with Puc57-sgRNA-NEO-Amp (Bsa I) for 1h, transformed and coated with Amp+ plates;

(4) Selecting a monoclonal, and carrying out PCR identification;

(5) Further sequencing and confirming the PCR positive monoclonal, wherein the sequencing primer is pUC57-T7-F;

(6) Using the properly sequenced clone as a template, and then using a primer to amplify the sgRNA transcribed DNA product by PCR;

(7) The sgrnas were transcribed and further purified using the transcribed DNA product of the sgrnas as templates. The constructed targeting vector is then transcribed and then reverse transcribed to obtain ssDNAdonor which can be used for injection.

4) sgRNA screening prepared from SIGLEC10 humanized mice

2 groups of sgrnas were designed and synthesized (5s1+3s1 and 5s2+3s2, specific sequence information is shown in table 1), the 5 'end target site and the 3' target site were paired two by two, then the 2 pairs of sgrnas were incubated with Cas9 protein, injected into fertilized eggs for 0.5 day, cultured to blastula, and the KO positive rate of the mouse Siglecg gene was identified, thereby verifying the sgRNA cleavage activity.

The sgRNA cleavage experimental identification method comprises the following steps: the collected blasts were subjected to PCR amplification, the protocol of PCR is shown in tables 4 to 5, the amplified bands were subjected to second generation sequencing, and the results were compared with the WT bands, and the probability of mutation was counted (the identification results are shown in Table 2).

TABLE 1 sgRNA information

TABLE 2 sgRNA cleavage Activity

sgRNA name	Cutting efficiency
		GPT0X0019-01-Balbc-SIGLEC10-5S1	6/7＝85％
GPT0X0019-01-Balbc-SIGLEC10-5S2	6/10＝60％
		GPT0X0019-01-Balbc-SIGLEC10-3S1	20/20＝100％
GPT0X0019-01-Balbc-SIGLEC10-3S2	6/10＝60％

5) SIGLEC10 humanized mouse model establishment

The screened sgRNA (5S1+3s1) is designed and constructed to carry a human sequence ssDNAdonor, the ssDNA donor and Cas9/sgRNA system are injected into fertilized eggs of 0.5d mice, the fertilized eggs are transplanted into 0.5d pseudopregnant female mice, and after the mice are born, the mid-target mice (F0) are screened through genetic identification.

6) Genotyping of humanized F0 mice

The obtained rat tail genomic DNA of the F0 mouse was subjected to two-end PCR identification after mid-targeting using the two pairs of primers shown in table 3, and the PCR reaction conditions and reaction procedures are shown in tables 4 and 5, respectively. Primers GPT0X0019-01-mSIGLEC10-5tF1/GPT0X0019-01-hSIGLEC10-5tR1 are respectively positioned outside a 5 'end homology arm and in a humanized fragment of ssDNA donor, if the pair of primers are amplified to generate PCR products, the target donor is effectively inserted into the 5' end of a mouse genome; GPT0X0019-01-mSIGLEC10-3tF1/GPT0X0019-01-hSIGLEC10-3tR1 is respectively positioned in a human fragment of ssDNA donor and outside a 3 '-end homology arm, and if the pair of primers are amplified to generate PCR primers, the target donor is effectively inserted into the 3' -end of a mouse genome.

TABLE 3 F0 identification primers

TABLE 4 PCR reaction System

TABLE 5 PCR reaction conditions

In this test example, 51F 0 mice were obtained by co-injection, and positive F0 mice were detected using the above-described identification scheme. As shown in the PCR electrophoresis results of FIG. 1 (WT is BALB/cJGpt genomic DNA (negative control), N is negative blank control (no template control), M is DNAMaroker, TRANS 2K PLUS II band: 8000bp,5000bp, 2000bp,1000bp,750bp,500bp,250bp,100 bp), 39#, 41#, 42#, 54#, 63#,66#,69#, 77#, 78#, 79#, 80# and 81# mice were positive for 5 'and 3' end identification of the human SIGLEC10 gene. The gene identification results show that 54# mice, 63# mice, 66# mice and 80# mice have no base mutation of the inserted gene sequence, and can be bred in an attempt. Mice # 39, # 41, # 42, # 69, # 77, # 78, # 79 and # 81 have base mutations in the insert sequences and are therefore not available for subsequent breeding. In addition, other batches of F0 mice are identified by the identification method, so that a plurality of F0 positive mice which can only be bred are obtained.

F1 is obtained by breeding positive F0 mice and background mice, the tail genes of the F1 generation are identified, the identification results of the genes of the F1 generation mice are shown in figure 2, the 5 'and 3' end identifications of the humanized SIGLEC10 genes of the mice # 35, # 38, # 40, # 41 are positive, and the detection of the murine source is positive. Indicating that the obtained mice are heterozygous positive mice for correctly carrying out gene recombination. And F1 mice are subjected to mass propagation and then are matched with each other to obtain homozygous mice.

Test example 2 detection of hSIGLEC10 expression in BALB/c-hSIGLEC 10F 1 heterozygous mice

1. Test method

Flow cytometry experiments examined SIGLEC10 expression in peripheral blood of BALB/c mice and BALB/c-hSIGLEC 10F 1 heterozygous mice. Peripheral blood of BALB/c mice and BALB/c-hSIGLEC 10F 1 heterozygous mice was collected and expression of hSIGLEC10 in B lymphocytes and macrophages was examined using a flow cytometer. In addition, the clustering of immune cell T cells (mCD3+), B cells (mCD19+) and NK cells (mCD335+) cells in peripheral blood of BALB/c mice and BALB/c-hSIGLEC 10F 1 heterozygous mice was examined.

2. Test results

Flow cytometry was performed to examine the expression of B lymphocytes and macrophages SIGLEC10 in peripheral blood of BALB/c mice and BALB/c-hSIGLEC 10F 1 heterozygous mice. As shown in FIGS. 3 and 4, expression of hSIGLEC10 was detected in BALB/c-hSIGLEC 10F 1 heterozygous mice peripheral blood B cells and macrophages, whereas expression of hSIGLEC10 was not detected in BALB/c mice peripheral blood B cells and macrophages.

Flow cytometry examined the clustering of immune cell T cells (mCD3+), B cells (mCD19+) and NK cells (mCD335+) cells in peripheral blood of BALB/c mice and BALB/c-hSIGLEC 10F 1 heterozygous mice. As shown in FIG. 5, the T/B/NK cell distribution in the peripheral blood of the BALB/c-hSIGLEC 10F 1 heterozygote mice was substantially identical to the T/B/NK cell distribution in the peripheral blood of the BALB/c mice.

The test results show that the BALB/c-hSIGLEC10 mouse model is successfully constructed by replacing the humanized gene with the mouse Siglecg gene, which indicates that the model has wide application prospect in the fields of oncology, immunology and the like.

Although the method has been described in detail with respect to the steps, it will be apparent to those skilled in the art that modifications may be made to some of the parameters and aspects of the overall process within the scope of the invention. Therefore, the present invention is intended to cover all modifications, alternatives, and adaptations falling within the spirit and scope of the present invention.

Claims

1. A method for constructing a SIGLEC10 humanized mouse model, comprising the steps of:

(4) F0 mice are bred with background mice to obtain F1 mice, gene identification is carried out on the tail of the F1 mice, and SIGLEC10 humanized mouse models are screened out;

the step (1) comprises the following steps: exon1 to Exon9 of the humanized SIGLEC10 gene are selected, exon2 to Exon11 of the mouse Siglecg gene is replaced by utilizing the homologous recombination technology, and the sequence of the selected humanized SIGLEC10 gene is shown as SEQ ID No. 3;

the sequence of the targeting vector successfully constructed in the step (1) is shown as SEQ ID No. 4;

the gene sequences of the sgRNA in the step (2) are SEQ ID NO.5 and SEQ ID NO.7.

2. The construction method according to claim 1, wherein the strain of mice and pseudopregnant mice provided with fertilized eggs in the step (3) is BALB/c.

3. The construction method according to claim 1, wherein the 5 '-terminal identification primer used in the genotyping of the F0 mouse in the step (3) is shown in SEQ ID NO.9 and SEQ ID NO.10, and the 3' -terminal identification primer is shown in SEQ ID NO.11 and SEQ ID NO. 12.

4. Use of mice obtained by the construction method according to any one of claims 1-3 for studying functions and mechanisms of action associated with SIGLEC10 genes.

5. Use of mice obtained by the construction method of any one of claims 1-3 for screening for a medicament for treating diseases associated with SIGLEC10 gene.