Construction method and application of neutropenia model

Abstract

The invention discloses a construction method and application of a neutropenia model. The model constructed by the method disclosed by the invention can be used for researching various related targets for neutropenia and other neutrophil differentiation and development disorder diseases, has in-vivo simulation and integrity, is obvious in phenotype, focuses on specific knockout of bone marrow myeloid cells, and has a good application prospect. The gene expression of other cell tissues is not influenced, the normal survival and reproduction state of animals is ensured while the significant phenotype is ensured, and the development of a long-period experiment is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a method for constructing a neutropenia model and an application thereof. Background Art

[0002] Neutrophils are differentiated from bone marrow hematopoietic stem cells and are the most important defense cells of the body. Their main function is to phagocytose and kill bacteria, including adhesion, chemotaxis, phagocytosis and release, and they play an important role in the immune system. Neutropenia is a disease characterized by a large decrease in the number of neutrophils in the blood. It is one of the most common and serious complications of cancer patients after cytotoxic chemotherapy and radiotherapy.

[0003] Laboratory animals are indispensable tools in biomedical research. They play a very important role in exploring the basic functions of genes, exploring the pathogenesis of diseases, and preclinical screening of applied drugs. Mice have great similarities with humans in development and metabolism, and their genes are also highly similar to human genes (99% of mouse genes can be found in the human genome). They have a short life cycle and are considered to be ideal model organisms among mammals. Therefore, it is very necessary to provide a method for constructing a neutropenia model for basic and clinical research on neutropenia. Summary of the invention

[0004] To make up for the deficiencies of the prior art, the present invention provides a method for constructing a neutropenia model and its application.

[0005] To achieve the above purpose, the present invention adopts the following technical solution.

[0006] A first aspect of the present invention provides a method for constructing a neutropenia model, the method comprising constructing a neutropenia model by conditionally knocking out the Hax1 gene.

[0007] Furthermore, the conditional knockout is conditional knockout of the Hax1-201 transcript of the Hax1 gene.

[0008] Furthermore, the conditional knockout position of the Hax1-201 transcript is exon 2 and 3 of the Hax1-201 transcript.

[0009] Furthermore, the conditional knockout is selected from conditional knockout based on a site-specific recombinase system.

[0010] Furthermore, the system used for conditional knockout of the site-specific recombinase system is the Cre / LoxP system.

[0011] Furthermore, the method specifically comprises: 1) Construction of a targeting vector for conditional knockout of the Hax1 gene; 2) The targeting vector in step 1) is transfected into ES cells; 3) The positive ES cells from step 2) are injected into the donor animal; 4) The donor animal in step 3) is crossed with the Flp animal; 5) The positive animals in step 4) are crossed with animals that specifically express the Cre enzyme to obtain a neutropenia model.

[0012] Furthermore, the method for constructing the targeting vector in step 1) is an Infusion method.

[0013] Furthermore, the transfection method in step 2) is electroporation.

[0014] Furthermore, in step 4), the animal that specifically expresses the Cre enzyme is an animal that specifically expresses the Cre enzyme in myeloid cells.

[0015] Furthermore, the animal is selected from mammals.

[0016] Furthermore, the mammals are selected from rodents.

[0017] Furthermore, the rodent is selected from mice.

[0018] Furthermore, the rodent is selected from mice.

[0019] Furthermore, in step 3), the donor animal is selected from C57BL / 6J strain mice.

[0020] The second aspect of the present invention provides a neutropenia model, which is constructed by the method described in the first aspect of the present invention.

[0021] The third aspect of the present invention provides the use of the method described in the first aspect of the present invention in constructing a neutropenia model.

[0022] The fourth aspect of the present invention provides a method for screening candidate drugs for preventing / treating neutropenia, the method comprising administering the drug to be tested to the neutropenia model constructed by the method described in the first aspect of the present invention or the model described in the second aspect of the present invention, and selecting the drug to be tested that can improve the symptoms of neutropenia.

[0023] The fifth aspect of the present invention provides the use of the model constructed by the method described in the first aspect of the present invention or the model described in the second aspect of the present invention in screening drugs for preventing / treating neutropenia.

[0024] The sixth aspect of the present invention provides the use of the model constructed by the method described in the first aspect of the present invention or the model described in the second aspect of the present invention in the study of the disease mechanism / disease target of neutropenia.

[0025] Advantages and beneficial effects of the present invention: The model constructed by the method of the present application can be used to explore a variety of research targets related to neutropenia and other neutrophil differentiation and developmental disorders. It has in vivo simulation and integrity. The model of the present application has a significant phenotype, focusing on the specific knockout of bone marrow myeloid cells, and does not affect the gene expression of other cell tissues. It ensures the normal survival and reproduction of animals while ensuring a significant phenotype, which is conducive to conducting long-term experiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a schematic diagram of the sgRNA insertion site in the Hax1 gene knockout mouse model; Figure 2 The figure shows the construction and identification of the Hax1 knockout mouse model, where 2A shows the construction of the Hax1 knockout mouse model using the CRISPR / Cas9 system, 2B shows the DNA sequence information of the Hax1 gene location and the missing fragment information of the knockout mouse, 2C shows the sequencing results of the knockout fragment, and 2D shows the genotype identification of the Hax1 knockout mouse; Figure 3 It is a survival diagram of the Hax1 gene knockout mouse model, where 3A is a diagram of the difference in mouse body shape, 3B is a diagram of the difference in weight between 6-week-old and 8-week-old mice, and 3C is a diagram of the survival curves of Hax1 knockout mice and wild mice; Figure 4 It is a schematic diagram of the construction of Hax1 conditional knockout mouse model; Figure 5 It is a diagram of the genetic mouse model for identifying each hybridization result at the DNA level; Figure 6 This is a verification diagram of Cre enzyme activation at the DNA level; Figure 7 is a graph of Hax1 protein levels in bone marrow; Figure 8 It is a graph of the proportion of neutrophils in the bone marrow and peripheral blood. Among them, 8A-B is a graph of the proportion of neutrophils in lymphocytes in the bone marrow and peripheral blood, and 8C-D is a graph of the proportion of neutrophil subtypes at different developmental levels. DETAILED DESCRIPTION

[0027] The definitions of some terms used in this specification are provided below. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0028] The present invention provides a method for constructing a neutropenia animal model, which comprises constructing the neutropenia model by conditionally knocking out the Hax1 gene.

[0029] In some embodiments, conditional knockout of a gene is a technique for knocking out a specific gene in a specific cell type, developmental stage, or tissue organ. The main methods for conditional knockout of a gene include conditional knockout based on a site-specific recombinase system, conditional knockout based on an inducible promoter, and conditional knockout based on gene trap technology.

[0030] In a preferred embodiment, the conditional knockout is selected from a conditional knockout based on a site-specific recombinase system.

[0031] In some embodiments, the system used for conditional knockout based on site-specific recombinase system includes but is not limited to Cre / LoxP system, FLP / FRT system, Gin / Gix system, R / RS system.

[0032] In a specific embodiment, the system used for conditional knockout based on the site-specific recombinase system is the Cre / LoxP system.

[0033] The method specifically comprises: 1) Construction of a targeting vector for conditional knockout of the Hax1 gene; 2) The targeting vector in step 1) is transfected into ES cells; 3) The positive ES cells from step 2) are injected into the donor animal; 4) The donor animal in step 3) is crossed with the Flp animal; 5) The positive animals in step 4) are crossed with animals that specifically express the Cre enzyme to obtain a neutropenia model.

[0034] The animal is selected from mammals.

[0035] In some embodiments, the mammal is a non-human mammal. The non-human mammal includes primates (such as rhesus monkeys, bear monkeys, red-faced stump-tailed monkeys, crab-eating monkeys, etc.), rodents (such as mice, rats, squirrels, beavers, marmots, gophers, voles, woodchucks, hamsters, guinea pigs, agouti, etc.), lagomorphs (such as rabbits), and ungulates (such as cattle, sheep, horses, etc.).

[0036] In a preferred embodiment, the non-human mammal is selected from rodents.

[0037] In some embodiments, the rodents include but are not limited to mice, squirrels, and other rodents.

[0038] In a more preferred embodiment, the rodent is selected from murines.

[0039] In some embodiments, the rodents include but are not limited to mice, rats, hamsters, chinchillas, and guinea pigs.

[0040] In a specific embodiment, the rodent is selected from a mouse.

[0041] The present invention provides a method for screening candidate drugs for preventing / treating neutropenia, the method comprising administering a drug to be tested to a neutropenia model constructed by the above method or to the above model, and selecting a drug to be tested that can improve the symptoms of neutropenia.

[0042] In some embodiments, the specific method includes: (1) administering the drug to be tested to the neutropenia model prepared by the above method; (2) detecting whether the neutropenia model has changes in abnormal symptoms related to neutropenia after the administration of the drug to be tested; (3) when the drug to be tested improves the symptoms of neutropenia / has a therapeutic effect, the drug to be tested is identified as a candidate drug capable of treating / preventing neutropenia.

[0043] In some embodiments, the drug to be tested may be a low molecular weight compound, a protein (e.g., an antibody), DNA, RNA, a low molecular weight interfering RNA, or an antisense oligonucleotide. The drug to be tested may be, for example, a drug for treating a disease other than neutropenia. The test drug may be, for example, a mixture of one or more than two. The test drug is preferably a substance.

[0044] In some embodiments, treatment refers to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity and / or incidence of, one or more symptoms or features of a particular disease, disorder, and / or condition. Treatment can be applied to subjects who do not show signs of the disease and / or only show early signs of the disease, with the goal of reducing the risk of developing a condition associated with the disease.

[0045] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0046] Example 1 Full knockout mouse model First, sgRNA was designed at exons E2-E6 for Hax1 (HCLS1 associated X-1) [Mus Gene ID: 23897] using CRISPR-Cas9 technology. After annealing, the Hax1-sgRNA expression vector was successfully constructed by connecting with the linearized pUC57-sgRNA expression vector. The transcribed sgRNA and Cas9 vector were combined and injected into C57BL / 6J mouse fertilized eggs to obtain a C57BL / 6J mouse model with Hax1 genome-wide knockout. The schematic diagram of the sgRNA insertion site is shown in the figure. Figure 1 shown.

[0047] According to the PCR results, wild-type and heterozygous mice had a band at 596 bp using the WT identification primer, heterozygous and homozygous knockout mice had a band at 314 bp using the KO identification primer, and the deletion fragment was 2340 bp ( Figure 2 ).

[0048] During the breeding process, it was found that compared with wild-type mice, Hax1 knockout homozygous mice grew slowly, were thin, and had reduced activity ( Figure 3 A), whose body weight was significantly lower than that of wild-type mice ( Figure 3 B), and the survival rate of Hax1 knockout homozygous mice decreased, and they died within 3 to 9 weeks after birth ( Figure 3 C).

[0049] Example 2 Conditional knockout mouse model In order to overcome the problem of low survival rate, this application adopts ES cell targeting to perform flox modification before and after exons 2 and 3 of the transcript of Hax1 gene Hax1-201 (ENSMUST00000079724.9) to construct Hax1 fl / fl C57BL / 6J mice were then transfected with Lyz2 that specifically expresses the Cre enzyme gene in myeloid cells (monocytes, mature macrophages, and granulocytes). - Cre + Hax1 fl / fl ;lyz2 - Cre + The target model for myeloid-specific knockout of Hax1, the main construction diagram is shown in Figure 4 .

[0050] This project uses the principle of homologous recombination and ES cell targeting to perform flox modification on the Hax1 gene. The brief process is as follows: an ES cell targeting vector was constructed by the Infusion method, which contains a 2.9kb 5' homology arm, a 1.1 kb flox region, PGK-Neo-polyA, a 3.0kb 3' homology arm, and an MC1-TK-polyA negative screening marker. After linearization, the vector was electroporated into JM8A3 ES cells. After long-fragment PCR identification, a total of 1 positive clone with correct homologous recombination was obtained. After amplification, the positive ES cell clones were injected into the blastocysts of C57BL / 6J mice to obtain chimeric mice. High-proportion chimeric mice were mated with Flp mice to obtain 3 positive F1 generation Neo-de-heterozygous mice.

[0051] Hax1 fl / fl The primer sequences for identifying flox gene insertion in mice are shown in Table 1.

[0052] Table 1 Primers for identification of flox gene insertion

[0053]

[0054] Hax1 fl / fl The PCR reaction system for identifying the insertion of flox genes in mice is shown in Table 2.

[0055] Table 2 PCR reaction system for flox gene insertion

[0056]

[0057] Hax1 fl / fl The procedure for the PCR reaction for identifying the insertion of the flox gene in mice is shown in Table 3.

[0058] Table 3 PCR reaction procedure for flox gene insertion

[0059]

[0060] Example 3 Validation of conditional knockout mouse model Experiment 1 1. Experimental Materials The reagents and consumables required for the experiment are shown in Table 4, and the instruments are shown in Table 5.

[0061] Table 4 Reagents and consumables

[0062]

[0063] Table 5 Instruments

[0064]

[0065] 2. Experimental methods and results 1) Tissue digestion: Within seven days, cut the toes of mice, number them, and incubate them with the protease mixture of the PCR kit at 55℃ for 15 minutes to release genomic RNA. Incubate them at 95℃ for 5 minutes to inactivate the protease, and use the supernatant as a PCR template.

[0066] 2) Electrophoresis: After electrophoresis for 30 min on 2% agarose at 120V, the band type of each hybridization result gene mouse was identified at the DNA level ( Figure 5 ) and verified Cre enzyme activation at the DNA level ( Figure 6 ), indicating that Hax1 in mouse bone marrow tissue was successfully knocked out at the DNA level.

[0067] Experiment 2 The experimental materials are shown in Table 6.

[0068] Table 6 Experimental materials

[0069]

[0070] 2. Experimental methods Cells and mouse tissues were lysed on ice for 30 min using RIPA buffer and a 100× cocktail of protease and phosphatase inhibitors. After sonication, the samples were centrifuged at 13,500 rpm for 15 min, and the supernatant was collected and prepared for protein loading. After SDS-PAGE electrophoresis separation, PVDF membrane transfer, and 5% skim milk blocking, the protein samples were incubated with primary and secondary antibodies in sequence. The washed membranes were imaged at 700 and 800 nm using the Odyssey CLx (LI-COR Biosciences, USA) near-infrared imaging system. Western Blot was used to verify the conditional knockout effect of Hax1 in mouse bone marrow compared with other tissues at the protein level.

[0071] 3. Experimental results At the protein level, the expression of Hax1 in the bone marrow of the cKO group showed a significant decrease compared with other major organs with no significant differences ( Figure 7 ).

[0072] Experiment 3 1. Experimental Materials The experimental materials are shown in Table 7.

[0073] Table 7 Experimental materials

[0074]

[0075] 2. Experimental methods Fresh single-cell suspensions from peripheral blood mononuclear cells (PBMCs) and bone marrow (BM) were analyzed and cell sorted on a BD Biosciences FACSAria II (BD Bioscience, USA). Data were analyzed using FlowJo_v10.6.2 software.

[0076] 3. Experimental results The proportion of granulocytes in the bone marrow and peripheral blood showed a significant decrease, while the proportion of lymphocytes in the bone marrow and peripheral blood showed a significant increase ( Figure 8 A, B). The subtypes of neutrophils at different developmental stages were analyzed, and the results showed that the neutrophil population in the bone marrow of Hax1-deficient mice showed an overall downward trend, with a significant increase in immature neutrophils and a decrease in mature neutrophils, suggesting that the maturation and transformation of immature neutrophils may have been disturbed ( Figure 8 C, D).

[0077] The description of the above embodiments is only used to understand the method and core idea of ​​the present invention. It should be pointed out that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principle of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. A method for constructing a neutropenia model, characterized in that: The method comprises constructing a neutropenia model by conditionally knocking out the Hax1 gene.

2. The method according to claim 1, characterized in that The conditional knockout is conditional knockout of the Hax1-201 transcript of the Hax1 gene; Preferably, the conditional knockout position of the Hax1-201 transcript is exon 2 and 3 of the Hax1-201 transcript.

3. The method according to claim 2, characterized in that The conditional knockout is selected from conditional knockout based on a site-specific recombinase system; Preferably, the system used for conditional knockout of the site-specific recombinase system is the Cre / LoxP system.

4. The method according to claim 3, characterized in that: The method specifically comprises: 1) Construction of a targeting vector for conditional knockout of the Hax1 gene; 2) The targeting vector in step 1) is transfected into ES cells; 3) The positive ES cells from step 2) are injected into the donor animal; 4) The donor animal in step 3) is crossed with the Flp animal; 5) The positive animals in step 4) are crossed with animals that specifically express the Cre enzyme to obtain a neutropenia model.

5. The method according to claim 4, characterized in that The method for constructing the targeting vector in step 1) is the Infusion method; Preferably, the transfection method in step 2) is electroporation; Preferably, in step 4), the animal that specifically expresses the Cre enzyme is an animal that specifically expresses the Cre enzyme in myeloid cells; Preferably, the animal is selected from mammals; Preferably, the mammal is selected from rodents; Preferably, the rodent is selected from mice; Preferably, the rodent is selected from mice; Preferably, in step 3), the donor animal is selected from C57BL / 6J strain mice.

6. A neutropenia model, characterized in that: The model is constructed by the method described in any one of claims 1-5.

7. Use of the method according to any one of claims 1 to 5 in constructing a neutropenia model.

8. A method for screening candidate drugs for preventing / treating neutropenia, characterized in that: The method comprises administering a drug to be tested to the neutropenia model constructed by the method described in any one of claims 1 to 5 or the model described in claim 6, and selecting a drug to be tested that can improve the symptoms of neutropenia.

9. Use of the model constructed by the method according to any one of claims 1 to 5 or the model according to claim 6 in screening drugs for preventing / treating neutropenia.

10. Use of the model constructed by the method described in any one of claims 1 to 5 or the model described in claim 6 in the study of the disease mechanism / disease target of neutropenia.

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