Hairless severe combined immunodeficiency mouse model as well as construction method and application thereof

Abstract

The invention relates to the technical field of animal genetic engineering, and particularly discloses a hairy severe combined immunodeficiency mouse model as well as a construction method and application thereof. The construction method of the hair-free severe combined immunodeficiency mouse model, disclosed by the invention, comprises the following steps: introducing a hair-free gene Foxn1 mutation into an NTG mouse, and constructing a homologous introduction inbred line. According to the technical scheme provided by the invention, multiple methods such as multi-gene breeding genotype identification, construction of a homologous introduction inbred line, a molecular marker-assisted selection method and IVF propagation are combined, so that a method for rapidly, accurately and directionally breeding the hairy severe combined immunodeficiency animals is established, and an experimental animal strain with genetic stability and phenotype stability is obtained.

Description

Technical Field

[0001] This application relates to the technical field of animal genetic engineering, specifically to a hairless mouse model with severe combined immunodeficiency, its construction method, and its application. Background Technology

[0002] Laboratory animal science, a newly emerging independent and comprehensive discipline in the 1950s, is not only a "living experimental material" used to evaluate and monitor the effectiveness and safety of products, but also a raw material for biomedicine and an indispensable supporting condition for life science research. Immunodeficient animals are animals with congenital genetic mutations or deficiencies in one or more components of the immune system induced by artificial methods. They are widely used in medical and biological research, especially in oncology and immunology. In scientific research experiments, different strains of immunodeficient animal models are selected according to the experimental objectives. Based on the method of their creation, immunodeficient animals are classified into congenital immunodeficient animals and secondary immunodeficient animals; based on the type of immune cell deficiency, they are classified into T lymphocyte-deficient, B lymphocyte-deficient, NK lymphocyte-deficient, and combined immunodeficient animals. Mice are undoubtedly the most commonly used experimental animal models in biology and medicine; however, mice are not humans and cannot completely replace humans. The advent of immunodeficient mice made it possible to construct human-mouse chimeric models by transplanting human immune tissues or cells into them, resulting in humanized immune system mice (humanized mice). Due to their similarity to defects in the human immune system, humanized mouse models have become important model tools in fields such as human hematopoietic function, innate and adaptive immunity, autoimmunity, infectious diseases, cancer biology, and regenerative medicine.

[0003] In 1966, British scientists first discovered that mutations in the Foxn1 gene in the C57BL / 6 strain led to abnormal development of thymic epithelial cells in mice, resulting in hairlessness, a lack of T cells in the blood, and low immunity. This hairless phenotype led to their being named nude mice. Nude mice are the starting point for immunodeficient animal research and remain the most classic immunodeficient animal. Due to their hairless phenotype, they are highly favored by researchers in the field of tumor research. However, because their immunodeficiency is relatively mild, they are not suitable for experiments involving the reconstruction of a humanized immune system. Over the past 80 years, research has discovered and created various immunodeficient animals with varying degrees of immunodeficiency. With the continuous development of gene editing technology, the types of immunodeficient mouse strains are constantly increasing. In research, the impact of mutations in a specific gene on the immune response is usually considered, and the gene is edited specifically to achieve a higher degree of immunodeficiency. The NSG, BRG, and NTG series of immunodeficient mice are the most widely used model animals in tumor and immunological research and are recognized as suitable animals for the reconstruction of a humanized immune system. Then, the subsequent creation and discovery of these severely combined immunodeficient mouse models were all hairy mice, which greatly limited their observation and imaging in tumor-bearing experiments. Summary of the Invention

[0004] To address the aforementioned technical issues, this application provides a hairless, severely immunodeficient mouse model, its construction method, and its application.

[0005] This application provides a method for constructing a hairless severe combined immunodeficiency mouse model, wherein the method involves introducing a mutation in the hairless gene Foxn1 into a homologous inbred line of NTG mice.

[0006] Preferably, the method for constructing the hairless severe combined immunodeficiency mouse model specifically includes the following steps: Original strain selection: female NTG mice from the inbred basal population with the least residual immune cells and hairless appearance, and CD3 + Genetic testing was performed on 46 SNP samples from the base population of inbred lines with the fewest T cell residues. Female NTG mice and male BALB / c-Nu mice whose results at each locus matched the target were selected for further screening. One NTG female and one NTG male, which were from the same littermate as the original F0 generation NTG female and passed genetic testing, were selected for inbreeding to serve as the NTG selection population for backcrossing. F1 generation selection: Original female NTG mice were crossed with original male BALB / c-Nu mice to obtain F1 female offspring mice that were heterozygous for all three genes and heterozygous for some SNP sites. F2-F9 generation selection: female mice from the previous generation were crossed with male NTG mice. The male NTG mice were selected from a pair of NTG populations selected from the original species. The results of all SNP site detection were as consistent as possible with those of NTG mice until there were no heterozygous mutations. F10 generation selection: The F10 generation selects female and male species with homozygous knockout of Prkdc and Il2rg genes, and Foxn1 as heterozygote. All SNP sites must be completely identical to NTG nucleotide types. The F11 generation is obtained by mating. F11 generation selection: Hairless female and hairless male mice were selected for genotyping and genetic consistency testing; F12 generation selection: Among the offspring selected from the F11 generation IVF propagation, the best three pairs are selected to establish and propagate the inbred core population; thereafter, each generation of core breeding animals undergoes genotyping, hairless phenotype determination, genetic testing, and flow cytometry detection of immunodeficient cells.

[0007] Preferably, in the selection of the original species: the original female NTG mice are selected based on the minimum residual lymphocytes and the resulting F1 generation of NTG mice with balanced sex ratios; the original male BALB / c-Nu mice are selected based on CD3... + The mice with the fewest residual T cells and the cleanest thymus.

[0008] Preferably, the F1 generation mice and the female animals retained for breeding in each subsequent generation are subjected to genotyping and genetic consistency testing. The genotyping identification includes Prkdc genotyping, Il2rg genotyping, and Foxn1 genotyping: The nucleotide sequences of the primers used for identifying the Prkdc genotype are shown in SEQ ID NO: 1-4; heterozygotes are 400bp, 270bp, and 183bp; homozygotes are 400bp and 270bp. The nucleotide sequences of the primers used for Il2rg genotyping are shown in SEQ ID NO: 5-8; heterozygotes are 350bp and 660bp; homozygotes are 350bp. The nucleotide sequences of the primers used for Foxn1 genotyping are shown in SEQ ID NO: 9-12; The genetic consistency test is a SNP locus genetic identification, including the following loci and their corresponding identification primers: The nucleotide sequences of the SNP site rs3022883 and its identification primers are shown in SEQ ID NO: 10-11. The nucleotide sequences of SNP site rs13478622 and its identification primers are shown in SEQ ID NO: 12-13. The nucleotide sequences of the SNP site rs3023203 and its identification primers are shown in SEQ ID NO: 14-15. The nucleotide sequences of the SNP site rs29315008 and its identification primers are shown in SEQ ID NO: 16-17. The nucleotide sequences of the SNP site rs3023380 and its identification primers are shown in SEQ ID NO: 18-19. The nucleotide sequences of SNP site rs3023436 and its identification primers are shown in SEQ ID NO: 20-21. The nucleotide sequences of SNP site rs3089349 and its identification primers are shown in SEQ ID NO: 22-23. The nucleotide sequences of the SNP site rs3022953 and its identification primers are shown in SEQ ID NO: 24-25. The nucleotide sequences of the SNP site rs3089984 and its identification primers are shown in SEQ ID NO: 26-27. The nucleotide sequences of the SNP site rs3023382 and its identification primers are shown in SEQ ID NO: 28-29. The nucleotide sequences of the SNP site rs3023442 and its identification primers are shown in SEQ ID NO: 30-31.

[0009] Preferably, the selection method for the F2-F9 generation female hybrid animals is as follows: (1) Genotype identification: Genotype identification of three genes was performed on each female hybrid offspring. The Prkdc and Il2rg genes were homozygous knocked out, and the female species with Foxn1 as heterozygote were subjected to genetic testing. (2) Genetic testing: SNP locus testing is performed on females that have been screened by genotype identification. Individuals that are closer to the reference nucleotide type of NTG mice at the locus are selected for further breeding. Full locus genetic testing is required for F4 and F7 generations.

[0010] Preferably, the F11 generation selection method is as follows: (1) Genotyping: The selected individuals were subjected to Prkdc genotyping, Il2rg genotyping, and Foxn1 genotyping to confirm that all three genes were homozygous; (2) Genetic testing: Genetic testing was performed on 46 SNP sites to confirm that the nucleotide types of all SNP sites in the selected seeds were completely consistent with NTG.

[0011] Preferably, the F11 generation selection further includes flow cytometry screening and IVF propagation: After hairless F11 male mice reached 9 weeks of age, their sperm were frozen, and blood was collected for flow cytometry to detect T cell remnants and autopsy to detect thymus remnants. CD3 was selected. + Subsequent experiments were conducted using mouse sperm with the fewest residual T cells and the cleanest thymus. Hairless F11 female mice underwent superovulation, and whole blood was collected for T, B, and NK lymphocyte flow cytometry analysis. Selected frozen sperm were used for IVF to obtain offspring. Offspring with the fewest residual lymphocytes and the best F12 generation were selected for further breeding.

[0012] Preferably, in the F12 generation selection, if the F11 generation did not use Foxn1 heterozygotes for subsequent breeding experiments, then the F12 generation will start using surrogate mother mice.

[0013] Secondly, this application provides a hairless mouse model of severe combined immunodeficiency, which is constructed by the above method.

[0014] Thirdly, this application provides the application of the above-mentioned hairless severe combined immunodeficiency mouse model in the preparation of tumor immune animal models.

[0015] This application aims to establish a hairless mouse model of severe combined immunodeficiency. The classic hairless mouse, BALB / c-Nu, is a recessive mutant gene on chromosome 11 of the BALB / c mouse: Foxn1 (Forkhead box protein N1). A 1bp deletion in exon 3 of the Foxn1 gene leads to a frameshift mutation and premature introduction of a stop codon. This application aims to create a severely combined immunodeficient NTG mouse model derived from NOD SCID mice by knocking out the Il2rg (interleukin 2 receptor subunit gamma chain) gene on chromosome X. The mutant genes are Il2rg and the Prkdc (Proteinkinase, DNA activated, catalytic polypeptide) gene on chromosome 16. Establishing a hairless mouse model of severe combined immunodeficiency essentially involves creating a homologous inbred line by introducing the hairless gene (Foxn1 gene mutation) into NTG mice. However, traditional homologous inbred line establishment protocols and the establishment of homologous inbred lines under microsatellite DNA monitoring are limited in this approach and are time-consuming. The technical solution of this application aims to provide a method for rapidly and accurately obtaining phenotypically and genetically stable hairless, severely combined immunodeficient animals by combining multiple technologies.

[0016] Homologous inbred lines are new inbred lines created by introducing a gene into an inbred line through hybridization, reciprocal crossing, or backcrossing. These lines differ from the original inbred lines only in a very small segment of a chromosome; they are simply called homologous inbred lines (or similar lines). Mendel's theory of inheritance posits that approximately half of an animal's genetic material is passed on to its offspring during reproduction. Theoretically, after 10 backcrosses, over 99.9% of the genome should be identical to the original line.

[0017] In summary, the technical solution of this application has the following effects: This application provides a method for constructing hairless, severely combined immunodeficient mice and constructs solid immunodeficient animals, which can be applied to scientific research fields such as tumor research and humanized immune system reconstruction.

[0018] The technical solution provided in this application combines multiple methods such as multi-gene breeding genotype identification, construction of homologous inbred lines, molecular marker-assisted selection, and IVF propagation to establish a rapid, precise, and targeted method for breeding hairless animals with severe combined immunodeficiency, thereby obtaining genetically and phenotypic stable experimental animal strains. Attached Figure Description

[0019] Figure 1 The results of genotyping identification of the F1 generation selected in Example 1; wherein, Figure 1 A represents the Prkdc genotype identification result; Figure 1 B represents the Il2rg genotype identification result; Figure 1 C represents the Sanger sequencing results of the Foxn1 gene.

[0020] Figure 2 This is a breeding pattern diagram from Example 1; in which, pure black mice refer to NTG mice, light gray mice refer to BALB / c mice, and dark gray mice refer to hybrid offspring in breeding; black mice with white spots are hairless NTG mice / NTG nude mice; X refers to mating, the mouse on the left is a female mouse, and the mouse on the right is a male mouse.

[0021] Figure 3 This is a comparative illustration of hairless and hairy mice in Example 1.

[0022] Figure 4 This is a flow cytometry result of mouse immune cells in Example 1. Detailed Implementation

[0023] The present application will be further described in detail below with reference to embodiments, comparative examples and performance test results. These embodiments should not be construed as limiting the scope of protection claimed in this application. Example

[0024] The classic hairless BALB / c-Nu mouse is a recessive mutant gene on chromosome 11 of the BALB / c mouse: Foxn1 (Forkhead box protein N1). A 1bp deletion in exon 3 of the Foxn1 gene leads to a frameshift mutation and premature introduction of a stop codon. This application aims to create a severely combined immunodeficient NTG mouse derived from NOD SCID mice by knocking out the Il2rg (interleukin 2 receptor subunit gamma chain) gene on chromosome X. The mutant genes are Il2rg and the Prkdc (Protein kinase, DNA activated, catalytic polypeptide) gene on chromosome 16. Establishing a hairless severely combined immunodeficient mouse essentially involves creating a homologous inbred line by introducing the hairless gene (Foxn1 gene mutation) into NTG mice. However, traditional homologous inbred line establishment protocols and the establishment of homologous inbred lines under microsatellite DNA monitoring are limited in this approach and are time-consuming. The technical solution of this application aims to provide a method for rapidly and accurately obtaining phenotypically and genetically stable hairless, severely combined immunodeficient animals by combining multiple technologies. Example 1

[0025] I. Selection of Original Animal Species

[0026] Classic inbred lines are developed through at least 20 consecutive generations of full-sibling mating. All individuals within the line can be traced back to a common ancestor originating in the 20th generation or later. As the definition of an inbred line indicates, the original population has a crucial impact on the genetic quality of subsequent inbred lines; therefore, the method provided in this application involves specific selection of the F0 generation primordial population. Considering that female BALB / c-Nu mice are non-lactating and NTG mice... Il2rg The gene is located on the X chromosome. The F0 generation of the original strain consisted of one female NTG mouse and one male BALB / c-Nu mouse.

[0027] (1) First, genetic testing was performed on 46 SNP samples from the female NTG mice and male BALB / c-Nu mice of the best-looking inbred lineage. Ten female NTG mice and five male BALB / c-Nu mice whose test results were consistent with those of all points in Table 1 were selected for further screening. Note that among the selected NTG females, one NTG female and one NTG male that passed the genetic testing were inbred together to be used as NTG breeding stock for backcrossing.

[0028] Table 1 Genetic testing standards for seed selection

[0029] (2) Selection of male BALB / c-Nu mice: Five 9-week-old male BALB / c-Nu mice with clean fur, rosy skin, and good condition were selected, numbered, and their sperm were frozen. Blood samples were collected for flow cytometry to detect T cell remnants, and thymus remnants were examined by dissection. CD3 cells were selected. + Subsequent experiments were conducted using mouse sperm with the fewest remaining T cells and the cleanest thymus.

[0030] (3) Selection of female NTG mice: Ten NTG mice were superovulated, and whole blood was collected for flow cytometry detection of T, B and NK lymphocytes. The selected BALB / c-Nu mice were used for IVF fertilization to obtain offspring. The offspring of NTG mice with the least residual lymphocyte complex and the F1 generation with balanced sex were selected for further breeding.

[0031] II. F1 generation selection

[0032] Three female F1 generation mice from the NTG mouse lineage were selected for genotyping and genetic testing. Subsequent generations of female mice used for breeding were subjected to genotyping and genetic consistency testing using the same methods. The F1 generation consisted of female mice that were heterozygous for all genes and all SNP loci.

[0033] (1) Genotyping (1.1) Prkdc genotyping ( Figure 1 A): The identification primer is: Prkdc-FI-T:5'-TGGTATCCACAACATAAAATACGCCAT-3' (SEQ ID NO: 1); Prkdc-FI-A:5'-TGGCCCCTGCTAACTTTCTCTTAACT-3' (SEQ ID NO: 2); Prkdc-FO-F:5'-TATGTGCCATGTCAGTGTCTGACTAGAA-3' (SEQ ID NO: 3); Prkdc-FO-R:5'-GCCCTAAGAGTCACTTTCTCCATCTACA-3' (SEQ ID NO: 4).

[0034] Heterozygotes were 400bp, 270bp, and 183bp; homozygotes were 400bp and 270bp.

[0035] Genotyping of extracted rat tail genome samples was performed, containing 10 μL of 2×PCR Mix, 0.5 μL of each of the four primers (100 pmol / μL), 1 μL of genomic DNA, and 6 μL of purified water (ddH2O). The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; annealing at 65℃ for 30 s; extension at 72℃ for 45 s; 30 cycles; further extension at 72℃ for 7 min; and storage of the amplified product at 4℃. 5 μL of the PCR product was subjected to 2% agarose gel electrophoresis at 150 V for 1 h; the electrophoresis results were analyzed using a gel imaging system. The size and number of bands corresponding to each primer group were used to determine whether the Prkdc gene in the sample was heterozygous or homozygous.

[0036] Figure 1 A represents the Prkdc genotype identification result; three bands indicate heterozygotes, and the top two bands indicate homozygotes.

[0037] (1.2) Il2rg genotyping ( Figure 1 B): The identification primer is: IL2rg-F1: 5'-CTGCTCAGAATGCCTCCAATTCC-3' (SEQ ID NO: 5); IL2rg-R1: 5'-GATCCAGATTGCCAAGGTGAGTAG-3' (SEQ ID NO: 6); IL2rg-R2: 5'-CCTGCGTGCAATCCATCTTGTTCAAT-3' (SEQ ID NO: 7); Heterozygotes are 350bp and 660bp; homozygotes are 350bp.

[0038] Genotyping of extracted rat tail genome samples was performed, containing 10 μL of 2×PCR Mix, 0.5 μL of each of the three primers (100 pmol / μL), 1 μL of genomic DNA, and 6.5 μL of purified water (ddH2O). The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; annealing at 60℃ for 30 s; extension at 72℃ for 30 s; 30 cycles; further extension at 72℃ for 7 min; and storage of the amplified product at 4℃. 5 μL of the PCR product was subjected to 2% agarose gel electrophoresis at 150 V for 1 h; the electrophoresis results were analyzed using a gel imaging system. The size of the bands was used to determine whether the Il2rg gene in the sample was heterozygous or homozygous.

[0039] Figure 1 B represents the Il2rg genotype identification result.

[0040] (1.3) Foxn1 genotyping ( Figure 1 C): The identification primer is: Foxn1-mus-GI-F: 5'-TGGAATCCCAAGAGCAGGTTG-3' (SEQ ID NO: 8); Foxn1-mus-GI-R: 5'-GGCTGGGGTAAGGGAGTTTA-3' (SEQ ID NO: 9).

[0041] Genotyping of the extracted rat tail genome was performed, containing 10 μL of 2×PCR Mix, 0.5 μL each of two primers (100 pmol / μL) per set, 1 μL of genomic DNA, and 7 μL of pure water (ddH2O). The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; annealing at 60℃ for 30 s; extension at 72℃ for 30 s; 30 cycles; and a final extension at 72℃ for 7 min. The amplified products were then sequenced using Sanger sequencing. Figure 1 C-analysis sequencing results.

[0042] Figure 1 C represents the Sanger sequencing results of the Foxn1 gene, which is consistent with the preset wild type. A double peak indicates heterozygote, and a 1bp deletion indicates homozygote.

[0043] (1.4) Genetic testing According to Table 2, the extracted rat tail genome was genetically identified for each SNP locus. The mixture contained 10 μL of 2×PCR Mix, 0.5 μL of each of the two primers (100 pmol / μL) for each group, 1 μL of genomic DNA, and 7 μL of pure water (ddH2O). The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s; annealing at 60℃ for 30 s; extension at 72℃ for 30 s; 30 cycles; and a final extension at 72℃ for 7 min. The amplified products were analyzed using Sanger sequencing.

[0044] Table 2 Primers and standards for genetic testing in breeding

[0045] III. F2-F9 generation selection and breeding

[0046] like Figure 2 The diagram shown is a breeding model diagram of the technical solution of this application.

[0047] From F2 to F9, female hybrid animals were crossed with male NTG mice. Male NTG mice were selected from a pair of NTG mice selected from the original strain, requiring that all SNP loci detected were consistent with those of the NTG mice, and that there were no heterozygous mutations. The selection method for female hybrid animals in F2 to F10 generations is as follows: (1) Genotyping according to Figure 1 The results showed that the genotypes of three genes were identified for each female hybrid offspring (Prkdc and Il2rg were only homozygous in the F3 generation, but genotype identification was still performed). Homozygous knockout of the Prkdc and Il2rg genes was selected, and genetic testing was performed on females that were heterozygous for Foxn1.

[0048] (2) Genetic testing Females selected through genotyping and screening were subjected to SNP locus testing according to Table 2. Individuals with nucleotide types more closely resembling the NTG mouse reference nucleotide type at the selected loci were retained for further breeding. Note that the F4 and F7 generations underwent full locus genetic testing according to Table 1.

[0049] IV. F10 Generation Selection

[0050] In the F10 generation, homozygous knockout of the Prkdc and Il2rg genes was selected, and Foxn1 was heterozygous. Females and males that underwent full locus genetic testing according to Table 1 and whose nucleotide types at all SNP loci were completely consistent with NTG were mated to obtain the F1 generation.

[0051] V. F11 generation selection

[0052] (1) Selection of hairless phenotype Hairless females and males were selected for further testing.

[0053] (2) Genotyping The selected individuals underwent genotyping of three types, confirming that all three genes were homozygous.

[0054] (3) Genetic testing Genetic testing was performed on 46 SNP sites according to Table 1 to confirm that the nucleotide types of all SNP sites in the selected seeds were completely consistent with NTG.

[0055] (4) Flow cytometry screening and IVF propagation Sperm was frozen from hairless F11 male rats after they reached 9 weeks of age. Blood samples were collected for flow cytometry analysis to detect residual T cells, and thymus tissue was examined for residual T cells. CD3+ was selected. +Subsequent experiments were conducted using sperm from mice with the fewest residual T cells and the cleanest thymus. Hairless F11 female mice underwent superovulation, and whole blood was collected for T, B, and NK lymphocyte counts by flow cytometry. Selected frozen sperm were used for IVF to obtain offspring. Offspring with the fewest residual lymphocytes and the best F12 generation (with balanced sex ratio) were selected for further breeding.

[0056] VI. F12 generation selection

[0057] From the offspring selected through F11 generation IVF propagation, the three best pairs were chosen for the establishment and propagation of the inbred core population. Subsequently, each generation of the core breeding animals underwent genotyping, hairless phenotype determination, genetic testing, and flow cytometry analysis of immunodeficient cells.

[0058] Note that if the F11 generation does not use Foxn1 heterozygotes (hairy) for subsequent breeding experiments, then the F12 generation will start using surrogate mothers.

[0059] VII. Population Phenotypic Detection

[0060] (1) Phenotypes of hairless and hairy mice like Figure 3 The figure shows a comparison between hairless mice and hairy mice; the hairless mouse on the left in the figure refers to the hairless severe combined immunodeficiency mouse model constructed in this application; the hairy mouse on the right in the figure refers to the hairy NTG mouse.

[0061] (2) Flow cytometry detection of immunodeficient cells like Figure 4 The image shown is a flow cytometry result of mouse immune cells; CD3 + Refers to mature T cells, B220 + Refers to B cells, CD49b + The term "NK cells" refers to the hairless, severely immunodeficient mouse model constructed in this application; "NTG mice" refers to hairy NTG mice; and "WT" stands for Wild Type, representing immunocompetent mice used as control animals. As shown in the figure, the NTG naked mice constructed in this application lack T cells, B cells, and NK cells. Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for constructing a hairless severe combined immunodeficiency mouse model, characterized by, The construction method is to introduce a hairless gene Foxn1 mutation into NTG mice to construct a homologous introduction inbred line.

2. The method for constructing the hairless severe combined immunodeficiency mouse model according to claim 1, characterized in that, Specifically includes the following steps: Founder selection: The inbred female NTG mice with the least immune cell residues and the inbred male BALB / c-Nu mice with the least T cell residues were selected as the founder population. The female NTG mice and the male BALB / c-Nu mice with the same genotype as the target were selected for the next step of screening. One female NTG mouse and one male NTG mouse with the same genotype as the F0 generation of the founder NTG female were selected for inbreeding, and the NTG selected population was used for backcrossing. + Genetic detection of 46 SNP samples was performed on the inbred male BALB / c-Nu mice with the least T cell residues, and the female NTG mice and the male BALB / c-Nu mice with the same detection results as the target were selected for the next step of screening. One female NTG mouse and one male NTG mouse with the same genotype as the F0 generation of the founder NTG female were selected for inbreeding, and the NTG selected population was used for backcrossing. F1 generation selection: the original female NTG mice are crossed with the original male BALB / c-Nu mice to obtain F1 female mice with three genes being heterozygotes and part of the SNP sites being heterozygotes; F2-F9 generation selection: the female mice of the previous generation are crossed with male NTG mice, the male NTG mice are selected from the original selection of a pair of NTG populations, and all SNP site detection results are as consistent as possible with the NTG mice until the complete absence of heterozygous mutations; F10 generation selection: F10 generation selects Prkdc and Il2rg gene homozygous knockout, Foxn1 is a heterozygote, and female and male species mating is required to obtain F11 generation, which requires full-site genetic detection of all SNP sites nucleotide types consistent with NTG; F11 generation selection: select hairless female mice and hairless male mice for genotype identification and genetic consistency detection; F12 generation selection: among the offspring selected by F11 generation IVF expansion, the best three pairs are selected for inbred line core group establishment and expansion; thereafter, the core breeding animals are subjected to genotype identification, hairless phenotype determination, genetic detection and immune deficiency cell flow detection.

3. The method for constructing the hairless, severely immunodeficient mouse model according to claim 1, characterized in that, In the selection of the original strain, the selection method of the original female NTG mouse is to obtain the NTG mouse with the least comprehensive residual lymphocytes and balanced F1 generation of male and female; the selection method of the original male BALB / c-Nu mouse is to obtain the mouse with the least number of CD3 + T cells and the cleanest thymus.

4. The method of claim 1, wherein the mouse model of severe combined immunodeficiency without hair is constructed by, The female animals of the F1 generation mice and each generation of the breeding animals are subjected to genotype identification and genetic consistency detection; The genotype identification includes Prkdc genotype identification, Il2rg genotype identification and Foxn1 genotype identification: The nucleotide sequence of the identification primer for the Prkdc genotype identification is shown in SEQ ID NO: 1-4; the heterozygote is 400bp, 270bp and 183bp; and the homozygote is 400bp and 270bp; The nucleotide sequence of the identification primer for the Il2rg genotype identification is shown in SEQ ID NO: 5-7; the heterozygote is: 350bp and 660bp; and the homozygote is: 350bp; The nucleotide sequence of the identification primer for the Foxn1 genotype identification is shown in SEQ ID NO: 8-9; The genetic consistency detection is SNP site genetic identification, including the following sites and the corresponding identification primers: The nucleotide sequence of SNP site rs3022883 and its identification primer is shown in SEQ ID NO: 10-11, The nucleotide sequence of SNP site rs13478622 and its identification primer is shown in SEQ ID NO: 12-13, The nucleotide sequence of SNP site rs3023203 and its identification primer is shown in SEQ ID NO: 14-15, The nucleotide sequence of SNP site rs29315008 and its identification primer is shown in SEQ ID NO: 16-17, The nucleotide sequence of SNP site rs3023380 and its identification primer is shown in SEQ ID NO: 18-19, The nucleotide sequence of SNP site rs3023436 and its identification primer is shown in SEQ ID NO: 20-21, The nucleotide sequence of SNP site rs3089349 and primers for identifying the same are shown as SEQ ID NO: 22-23, The nucleotide sequence of SNP site rs3022953 and primers for identifying the same are shown as SEQ ID NO: 24-25, The nucleotide sequence of SNP site rs3089984 and primers for identifying the same are shown as SEQ ID NO: 26-27, The nucleotide sequence of SNP site rs3023382 and primers for identifying the same are shown as SEQ ID NO: 28-29, The nucleotide sequence of SNP site rs3023442 and primers for identifying the same are shown as SEQ ID NO: 30-31.

5. The method for constructing a hairless, severely immunodeficient mouse model according to claim 1, characterized in that, The F2-F9 generation female crossbred animal selection method is as follows: (1) Genotype identification: genotype identification of three genes is performed on each female crossbred offspring, and female species with Prkdc and Il2rg gene homozygous knockout and Foxn1 heterozygote are selected for genetic detection; (2) Genetic detection: SNP site detection is performed on the female species screened by genotype identification, and individuals closer to the NTG mouse reference nucleotide type at the site are selected for further breeding; F4 generation and F7 generation need to be subjected to whole site genetic detection.

6. The method for constructing a hairless, severely immunodeficient mouse model according to claim 1, characterized in that, The F11 generation selection method is as follows: (1) Genotype identification: Prkdc genotype identification, Il2rg genotype identification, and Foxn1 genotype identification are performed on the selected individuals, and it is confirmed that the three genes are all homozygotes; (2) Genetic detection: genetic detection is performed on 46 SNP sites, and it is confirmed that the nucleotide types of all SNP sites of the selected seeds are completely consistent with NTG.

7. The method for constructing a hairless, severely immunodeficient mouse model according to claim 1, characterized in that, The F11 generation selection also includes flow screening and IVF propagation: The sperm of the hairless F11 male mice was frozen when they reached 9 weeks old, blood was collected for flow cytometry to detect T cell residues, and the thymus was dissected to detect residues. The CD3 + The sperm of the mice with the least T cell residues and the cleanest thymus were used for subsequent experiments; the hairless F11 female mice were superovulated, whole blood was collected for flow cytometry to detect T, B, and NK lymphocytes, and the selected frozen sperm was used for IVF fertilization to obtain offspring animals. The offspring with the least comprehensive lymphocyte residues and the F12 generation with balanced male and female offspring were selected for further breeding.

8. The method of claim 1, wherein the mouse model of severe combined immunodeficiency without hair is constructed by, In the F12 generation selection, if the F11 generation does not use Foxn1 heterozygote for subsequent breeding experiments, the F12 generation starts to use foster mother mice.

9. A hairless severe combined immunodeficiency mouse model, characterized in that, Constructed by the method of any one of claims 1-8.

10. The use of the hairless severe combined immunodeficiency mouse model of claim 9 in the preparation of a tumor immune animal model.

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