Construction method and application of neutrophil development defect model
By using CRISPR/Cas9 to target and edit the tead1a gene, a stable and well-defined zebrafish neutrophil developmental defect model was constructed. This solved the problem that existing models could not stably inherit the genetic information and observe in vivo hematopoiesis, enabling efficient disease mechanism research and drug screening.
Patent Information
- Application Number
- CN202511137623.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-14
- Publication Date
- 2025-11-21
AI Technical Summary
Existing technologies, such as mouse models, cannot observe the in vivo hematopoietic process in real time, are complex to operate and have low throughput, and zebrafish gene knockdown technology has short-lived effects and cannot be stably inherited. There is a lack of accurate models that simulate human neutrophil developmental defects, which restricts the screening of targeted drugs.
By targeting and editing the zebrafish tead1a gene with CRISPR/Cas9 and using specific gRNA nucleotide sequences, a stable and well-defined zebrafish neutrophil developmental defect model with a clear phenotype was constructed.
A zebrafish neutrophil developmental defect model with a long lifespan has been developed, which is suitable for disease mechanism research and drug screening. The modeling cycle is short, the cost is reduced by 60%, and it can perform real-time in vivo imaging with high drug throughput.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of animal models, and particularly relates to a construction method of a neutrophil development deficiency model and application thereof. BACKGROUND
[0002] Neutrophils are a kind of white blood cells, also known as granulocytes. They are an important component of the immune system and are mainly responsible for resisting infection and inflammation. Neutrophils play an important role in the defense and protection functions of the human body and are the first line of defense against infection. Changes in the number of neutrophils in the blood can reflect the body's stress response to various diseases and conditions. Neutrophil deficiency-related diseases (such as severe congenital neutropenia and chemotherapy-induced neutropenia) have great clinical harm and lack effective treatment methods.
[0003] Animal models of neutrophil development deficiency can simulate and extrapolate human conditions. Animal models can accurately simulate the pathophysiological processes of human diseases and provide strong support for the study of disease mechanisms and the evaluation of drug efficacy. Existing animal models mainly rely on mouse gene knockout technology (such as VPS45 and CSF3R gene mutations), but mouse models cannot observe real-time hematopoiesis in vivo, and the operation is complex, the throughput is low, and there are problems such as long modeling period (> 6 months) and difficulty. Zebrafish, as a model organism that has been used for hematopoietic research for a long time, however, traditional zebrafish gene knockdown technology (such as morpholino oligonucleotides) has a short effect and cannot be stably inherited.
[0004] At present, there is still a lack of precise models that simulate human neutrophil development deficiency, which restricts the screening of targeted drugs. Therefore, it is of great significance to construct a humanized animal model of neutrophil deficiency for the study of disease mechanisms and clinical treatment of neutrophil deficiency-related diseases. SUMMARY
[0005] The purpose of the present application is to overcome the shortcomings of the prior art and provide a construction method of a neutrophil development deficiency model and application thereof.
[0006] To achieve the above-mentioned purpose, the technical solutions adopted by the present application are as follows:
[0007] In a first aspect, the present application provides a construction method of a neutrophil development deficiency model: a tead1a gene of zebrafish is targeted edited by CRISPR / Cas9; the nucleotide sequence of the gRNA for editing the tead1a gene is shown in SEQ ID NO: 1.
[0008] The application discloses a method for constructing a neutrophil development deficiency model, and is characterized in that a TEA domain transcription factor 1a (tead1a) gene is used to construct a neutrophil deficiency standardization model,
[0009] The application discloses a method for constructing a neutrophil development deficiency model, and is characterized in that a TEA domain transcription factor 1a (tead1a) gene is used to construct a neutrophil deficiency standardization model,
[0010] As a preferred embodiment of the method for constructing the neutrophil development deficiency model, the gRNA is mixed with the Cas9 protein and then injected into zebrafish embryos to obtain F0 generation embryos; the F0 generation embryos are bred into adult fish and then crossed with wild types to obtain F1 generation heterozygotes; the F1 generation heterozygotes are bred into adult fish and then self-crossed to obtain homozygous mutants.
[0011] As a further preferred embodiment of the method for constructing the neutrophil development deficiency model, the concentration of the gRNA is 40-60 ng / μL; the concentration of the Cas9 protein is 250-350 ng / μL; and the mass ratio of the gRNA to the Cas9 protein is 1:5-7.
[0012] As a further preferred embodiment of the method for constructing the neutrophil development deficiency model, the zebrafish embryos are single-cell stage zebrafish embryos.
[0013] As a further preferred embodiment of the method for constructing the neutrophil development deficiency model, the injection volume is 1-2 nL per embryo.
[0014] As a further preferred embodiment of the method for constructing the neutrophil development deficiency model, the primers with the nucleotide sequences as shown in SEQ ID NOs: 2 and 3 are used in the screening of the F1 generation heterozygotes.
[0015] As a further preferred embodiment of the method for constructing the neutrophil development deficiency model, the homozygous mutants are crossed with a transgenic line, F1 embryos are bred, and the homozygous mutant hematopoietic cell fluorescent transgenic fish line is screened after self-crossing of the adult fish.
[0016] The transgenic line is any one of Tg(gata1:DsRed), Tg(mpx:eEGFP) and Tg(mpeg1:eEGFP).
[0017] In a second aspect, the present application provides an evaluation method of the neutrophil development deficiency model constructed by the construction method of the first aspect, wherein the neutrophil development deficiency model is evaluated by at least one of the following methods: appearance, survival curve analysis, Sudan black staining, hematopoietic cell fluorescence transgenic hybrid fish line, flow cytometry fluorescence sorting neutrophil, hematopoietic development cell phenotype screening by whole-mount in situ hybridization technology, and hematopoietic tissue whole transcriptome sequencing analysis.
[0018] In a third aspect, the present application provides the use of the neutrophil development deficiency model constructed by the construction method of the first aspect, the construction method of the first aspect, and the evaluation method of the second aspect in screening drugs for treating and / or preventing diseases related to neutrophil development deficiency.
[0019] In a fourth aspect, the present application provides the use of the neutrophil development deficiency model constructed by the construction method of the first aspect, the construction method of the first aspect, and the evaluation method of the second aspect in preparing drugs for treating and / or preventing diseases related to neutrophil development deficiency, such as Notch pathway agonists.
[0020] Compared with the prior art, the present application has the following beneficial effects:
[0021] 1. The present application uses specific gRNA to target and edit tead1a gene by CRISPR / Cas9, and a neutrophil development deficiency model of zebrafish with stable inheritance, clear phenotype and long survival period is constructed, which can be applied to disease mechanism research and drug screening.
[0022] 2. The zebrafish model of the present application has a short modeling period of only 8 weeks (F2 generation homozygote); does not need to be edited and bred complicatedly, but directly uses embryo injection, with a cost reduction of 60%; does not need to be killed for sampling when observing the phenotype, but can be imaged in real time (fluorescent labeling) in vivo; has high drug throughput, and can be used for embryo screening by 96-well plates. BRIEF DESCRIPTION OF DRAWINGS
[0023] Figure 1 Figure 1 is a sequencing diagram of zebrafish tead1a homozygous mutant and wild type tead1a gene; in the figure, the red labeled sequence is the mutation target sequence.
[0024] Figure 2 Figure 2 is an electrophoresis diagram of Bts1Mut1 enzyme digestion identification and appearance of adult fish; in the figure, A: wild type, tead1a mutant heterozygote and tead1a mutant homozygote tail cutting extraction DNA, PCR amplification and Bts1Mut1 enzyme digestion diagram, it can be seen that the wild type can be cut by Bts1Mut1 (producing about 400bp+100bp two bands), while the homozygous mutant sequence (tead1a - / -) cannot be cut by restriction enzyme Bts1Mut1, about 400 bp single band; B: the final construction of homozygous mutation (tead1a - / - ) appearance of teleost.
[0025] Figure 3 Survival analysis of zebrafish tead1a homozygous mutant model; in the figure, 200 wild type and 50 tead1a homozygous mutant fish were taken for survival analysis, the results showed that compared with wild type, A: tead1a - / - mutant model had about 10% of the embryos died in the early embryonic development before 24hpf; B: tead1a - / - mutant model started to die at about 6 months, about 5%-7% per month, and there was a significant difference between the survival curves P<0.0001).
[0026] Figure 4 Sudan black staining model to verify zebrafish tead1a homozygous mutant model; in the figure, A: Sudan black staining photo; compared with wild type, zebrafish tead1a - / - mutant model had significantly reduced granulocyte in the kidney marrow area marked by yellow arrow and the tail hematopoietic area marked by red arrow at 5dpf; B: statistical chart of labeled cells; there was a significant difference between the wild type and mutant granulocyte hematopoietic development cells, *** P<0.001.
[0027] Figure 5 Fluorescent transgenic fish line model to verify hematopoietic development cells; in the figure, A: fluorescence detection chart; B: detection statistical chart; compared with wild type, the number of eGFP positive green fluorescent neutrophils marked by mpx decreased significantly at 72hpf after tead1a gene mutation, while the number of DsRed positive red fluorescent red blood cells marked by gata1 and eGFP positive green fluorescent monocyte macrophages marked by mpeg1 remained roughly the same, confirming that tead1a mutation specifically impaired neutrophil development and number.
[0028] Figure 6 Flow cytometry fluorescence sorting neutrophils verification; in the figure, A: flow cytometry sorting results of eGFP positive neutrophils dissociated from wild type Tg(mpx:eGFP) fish line at 3dpf; B: flow cytometry sorting results of eGFP positive neutrophils dissociated from tead1a homozygous mutant tead1a - / - / / Tg(mpx:eGFP) fish line at 3dpf; C: observation of neutrophils under microscope after single cell suspension dissociation in two fish lines; D: number statistics of neutrophils after flow cytometry analysis in two fish lines, the number of eGFP positive neutrophils dissociated from tead1a homozygous mutant tead1a- / - The number of neutrophils in the Tg(mpx:eGFP) fish strain was significantly reduced compared to the wild type. *** P < 0.001.
[0029] Figure 7 This was used to validate the early hematopoietic development phenotype of neutrophils in whole-embryo in situ hybridization; in the figure, A: WISH analysis graph; B: detection statistics graph; WISH analysis showed that the 22hpf phase tead1a homozygous mutant zebrafish model (tead1a - / - In this study, the number of L-plastin-labeled panleukocytes was significantly reduced. *** P < 0.001), the number of cells decreased was roughly equivalent to the number of mpx-labeled neutrophils, while the number of mfap4-labeled macrophages remained unchanged, suggesting that the tead1a homozygous mutant zebrafish model (tead1a - / - The white blood cells that are reduced in the cell count are mainly neutrophils.
[0030] Figure 8 Phenotypic validation of neutrophil-directed hematopoietic development during whole-embryo in situ hybridization; In the figure, A: WISH analysis graph; B: detection statistics graph; WISH analysis shows that the tead1a homozygous mutant zebrafish model (tead1a) is in the directed hematopoietic development phase. - / - In the study, the number of neutrophils labeled with mpx and lyz was significantly reduced. ** P < 0.01 or * P < 0.05), while the number of c-myb-labeled hematopoietic stem progenitor cells (HSPCs) was slightly reduced, indicating that some hematopoietic stem cells and stem progenitor cells that are directed to differentiate and develop into neutrophils may be damaged from the early stage of directed differentiation, resulting in a phenotypical and thorough neutrophil developmental defect.
[0031] Figure 9 Phenotypic screening of other hematopoietic developmental cells for whole embryo in situ hybridization; WISH or staining analysis revealed no significant differences in mafap4-positive macrophages, apoe-positive microglia, neutral red-positive phagocytes in the nervous system, or rag1-positive lymphoid cells in the tead1a homozygous mutant zebrafish model.
[0032] Figure 10 This is a phenotypic overview of whole transcriptome sequencing of the renal pulp; in the figure, A: summary of differentially expressed genes; B: analysis of the KEGG signaling pathway for genes with decreased expression.
[0033] Figure 11 These are genes with decreased expression as shown by whole transcriptome sequencing of the renal pulp.
[0034] Figure 12Phenotype validation for kidney marrow whole transcriptome sequencing; heat map showing differential expression of key genes in hematopoietic development tissue in kidney marrow caused by tead1a mutation in hematopoietic process. tead1a mutation causes expression changes of most hematopoietic stem / progenitor cell (HSPC) key genes (black arrows): key hematopoietic genes related to erythroid lineage are slightly elevated (red arrows), while expression of genes essential for neutrophil development is significantly reduced (green arrows). DETAILED DESCRIPTION
[0035] For the purpose of better illustrating the object, technical scheme and advantages of the present application, the present application will be further described in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present application, and are not used to limit the present application.
[0036] The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used are all commercially available unless otherwise specified.
[0037] Example 1: Construction of tead1a gene (TEA domain transcription factor 1a) mutant zebrafish model
[0038] (1) gRNA design and synthesis
[0039] Target design: Tead1a gene is located on the positive strand of chromosome 25 of zebrafish 17293517-17373206 (https: / / www.ncbi.nlm.nih.gov / datasets / gene / 405773 / #summary), its transcript NM_212847.3 is 6428 bases in length, 1368 bp in length, has 13 exons, and sgRNA is designed for the first exon region (Chr25: 17293526-17293669) of tead1a gene (https: / / www.ncbi.nlm.nih.gov / nuccore / NM_212847.3 / ) using online tool ZiFiT Targeter software (http: / / zifit.partners.org / ZiFiT), the target sequence is: 5'-GGAGCGGCAGTGAAAAGTGCC-3'(SEQ ID NO: 1).
[0040] Synthesis: The target sequence is connected to the pUC19-gRNA vector, and sgRNA is generated by in vitro transcription.
[0041] (2) Embryo microinjection
[0042] Injection combination: Mix sgRNA (50 ng / μL) with Cas9 protein (300 ng / μL);
[0043] Injection object: single-cell stage zebrafish embryos (AB strain), 300 embryos were injected in total;
[0044] Parameters: injection volume 1.5 nL / embryo.
[0045] (3) Mutant screening
[0046] F0 generation embryos were cultured to adult fish, and F1 generation was obtained by crossing with wild type;
[0047] Genotype identification: tead1a fragment was amplified using primer pair (FP: 5'-GCTGCTTCCCAGGTCAGT-3'; RP: 5'-ACTTGTCATCAACAAACCAAATTAGTGC-3'), and screened for heterozygotes (tead1a + / - ) by Bts1Mut1 enzyme digestion (wild type: about 300 bp+100 bp; mutant: about 400 bp);
[0048] After the heterozygotes were raised to adult fish, they were self-crossed to obtain homozygous mutant (tead1a - / - ), which is the tead1a gene mutant zebrafish model.
[0049] DNA was extracted from the tail clip, and after PCR amplification, Sanger sequencing was performed Figure 1 ). Using this DNA as substrate, after amplification by primers FP and RP, Bts1Mut1 enzyme digestion was performed as shown in the schematic diagram Figure 2 A), it can be seen that the wild type can be cut by Bts1Mut1 (producing about 400 bp+100 bp two bands), while the homozygous mutant sequence (tead1a - / - ) cannot be cut by the restriction enzyme Bts1Mut1, and is about 400 bp single band. The final constructed homozygous mutant (tead1a - / - ) adult fish appearance is as Figure 2 B.
[0050] (4) Construction and screening of homozygous mutant classic hematopoietic cell fluorescent transgenic fish lines
[0051] Homozygous mutant (tead1a - / - ) was crossed with Tg(gata1:DsRed), Tg(mpx:eEGFP) and Tg(mpeg1:eEGFP) transgenic lines, respectively. F1 embryos were cultured, and after the adult fish were raised, they were self-crossed and screened (the method is the same as above) to establish homozygous mutant classic hematopoietic cell fluorescent transgenic fish lines: tead1a - / - tg(gata1:DsRed), tead1a - / - tg(mpx:eEGFP), tead1a - / -tg(mpeg1:eEGFP) line.
[0052] Survival analysis of 200 embryos and 50 adult fish of each wild type and tead1a homozygous mutant was performed, and it was found that compared with the wild type, the tead1a homozygous mutant had a survival rate of 50% at 24 hpf and 6 months after fertilization. - / - The survival rate of the mutant model was only slightly worse than that of the wild type before 24 hpf and 6 months after fertilization. Figure 3 ).
[0053] Example 2: Phenotype verification of the tead1a gene mutant zebrafish model
[0054] (1) Sudan black staining verification
[0055] Sudan black (Sudan Black) is a fat-soluble azo dye that can dissolve in neutral lipids (such as phospholipids, cholesterol esters) and acidic lipids (such as phosphatidylserine) in cells, and stains lipid particles into dark black or blue-black by physical adsorption. Granulocytes become the main target cells of Sudan black staining because their cytoplasm contains a large number of azurophilic granules and specific granules. In zebrafish embryos, Sudan black staining mainly marks granulocyte progenitor cells (Granulocyte Progenitors) and mature granulocytes (Mature Granulocytes) in the primitive hematopoietic system (Primitive Hematopoiesis).
[0056] The Sudan black staining results showed that the granulocyte hematopoietic cells marked by the zebrafish tead1a gene mutant model constructed in Example 1 were significantly reduced after Sudan black staining. Figure 4 ).
[0057] (2) Fluorescent transgenic fish line model verification
[0058] The homozygous mutant (tead1a - / - ) was crossed with Tg(gata1:DsRed), Tg(mpx:eEGFP) and Tg(mpeg1:eEGFP) transgenic lines, respectively. F1 embryos were cultured, and after the adult fish were raised and self-crossed and screened (the method is the same as above), the homozygous mutant classic hematopoietic cell fluorescent transgenic fish lines were established: tead1a - / - tg(gata1:DsRed), tead1a - / - tg(mpx:eEGFP), tead1a - / - tg(mpeg1:eEGFP) line.
[0059] Results showed that in these lines, after tead1a gene mutation, the eGFP positive green fluorescent neutrophil granulocytes of tead1a homozygous mutant zebrafish embryos were significantly reduced at 72hpf, and the number of other hematopoietic cells had no obvious change; while the number of DsRed positive red fluorescent red blood cells and eGFP positive green fluorescent mononuclear macrophages remained roughly the same, confirming that tead1a mutation specifically impaired the development and number of neutrophils Figure 5 ).
[0060] (3) Flow cytometry fluorescence sorting neutrophil verification
[0061] Preparation of eGFP positive green fluorescent neutrophil granulocyte suspension marked by mpx using zebrafish model: collect intact embryos from two zebrafish lines (wild type Tg(mpx:eGFP) fish line and tead1a homozygous mutant / Tg(mpx:eGFP) fish line), dissociate by repeated blowing, trypsin digestion and tissue shearing, and store in 0.9x phosphate buffer solution (PBS) containing 2% fetal bovine serum on ice. Then place it on Falcon nylon cell screen and obtain single cell suspension by gravity and slight blowing. In order to sort eGFP positive (eGFP + ) neutrophil granulocytes, at least 400 embryos are needed for each group each time, and repeated up to four times to isolate the required cells for further analysis (including quantitative PCR, flow cytometry detection, RNA sequencing, etc.).
[0062] Flow cytometry was used to detect the wild type and homozygous mutant zebrafish models, and the results showed that compared with the control group, the eGFP positive neutrophil granulocytes in tead1a - / - / / Tg(mpx:eGFP) were significantly reduced by 71.4% at 72hpf Figure 6 ).
[0063] (4) Whole mount in situ hybridization technology for hematopoietic development cell phenotype screening and verification
[0064] Whole mount in situ hybridization (WISH): RNA probes labeled with digoxin (DIG) were prepared by T7, T3 or SP6 polymerase (Ambion) transcription. Probe labeling DIG (Roche), anti-digoxin Fab fragment antibody (Roche), 5-bromo-4-chloro-3-indolyl phosphate / nitro blue tetrazolium (BCIP / NBT) staining (Vector Laboratories) detection.
[0065] WISH neutrophil hematopoietic development phenotype verification: WISH analysis confirmed that the tead1a homozygous mutant zebrafish model (tead1a - / -Neutrophils were significantly reduced at 22 hpf. Figure 7 Further investigation revealed that tead1a - / - The zebrafish showed a significant decrease in neutrophils labeled with both mpx and lyz, confirming neutropenia syndrome. Figure 8 (Red arrow) and a slight reduction in c-myb-positive hematopoietic stem and progenitor cells (HSPCs) indicate that some hematopoietic stem cells and progenitor cells oriented towards neutrophil differentiation and development may be damaged from the early stages of directed differentiation, resulting in a very obvious and thorough neutrophil developmental defect. Figure 8 ).
[0066] WISH screening of other hematopoietic developmental cell phenotypes: WISH or staining analysis revealed no significant differences in mafap4-positive macrophages, apoe-positive microglia, neutrophil-positive phagocytes in the nervous system, or rag1-positive lymphoid cells in the tead1a homozygous mutant zebrafish model. Figure 9 The results showed that the remaining hematopoietic cells in the tead1a homozygous mutant zebrafish model did not show significant changes, and the phenotype of neutrophil developmental defects was highly specific.
[0067] (5) Validation by whole transcriptome sequencing analysis of hematopoietic tissue
[0068] After dissociation of the anterior renal medulla region of an adult fish model with homozygous tead1a mutation, whole transcriptome sequencing was performed to obtain a volcano map of differentially expressed genes. Figure 10 The study revealed that most genes showed decreased expression. Subsequent Kyoto Genetic and Genome Extensive Screening (KEGG) analysis of the enriched genes showed that the decreased-expressed genes were significantly enriched in complement activation and bacterial infection-related signaling pathways. Figure 10 ), consistent with the phenotype.
[0069] Further hematopoietic results from renal bone marrow RNA sequencing showed that neutrophil marker genes (such as coro1a, mpx / mpo, spi1 / pu.1, cebpα, cebp1, and lyz / lysozyme C) in the tead1a mutant were significantly downregulated, suggesting the presence of bone marrow-derived neutropenia. Figure 11 (Green arrow). Other genes with reduced expression include c-myb. Aberrant activation of the c-myb gene leads to abnormal granulocyte proliferation in zebrafish, similar to human myelodysplastic syndrome (MDS), suggesting that c-myb is a marker of myelogenous hematopoietic stem cell / progenitor (HSPC) proliferation. Figure 11 ).
[0070] After further analysis of hematopoietic-related genes, a heatmap was created to illustrate the differential expression of key genes during hematopoiesis. Figure 12). tead1a mutation leads to the expression change of most hematopoietic stem / progenitor cell (HSPC) key genes: the expression of key hematopoietic genes related to erythroid lineage is slightly increased, while the expression of neutrophil development essential genes is significantly reduced.
[0071] The present application constructs a stable genetic and phenotype-defined zebrafish neutrophil development deficiency model by CRISPR / Cas9 targeted editing of tead1a gene, which can be applied to the mechanism research and drug screening of neutrophil development deficiency diseases.
[0072] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the present application, and are not intended to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.
Claims
1. A method for constructing a neutrophil developmental defect model, characterized in that, The tead1a gene in zebrafish was edited using CRISPR / Cas9; the nucleotide sequence of the gRNA used to edit the tead1a gene is shown in SEQ ID NO:
1.
2. The method for constructing a neutrophil developmental defect model according to claim 1, characterized in that, The gRNA was mixed with Cas9 protein and injected into zebrafish embryos to obtain F0 generation embryos; the F0 generation embryos were raised to adulthood and then hybridized with wild-type fish to obtain F1 generation heterozygotes; the F1 generation heterozygotes were raised to adulthood and then self-crossed to obtain homozygous mutants.
3. The method for constructing a neutrophil developmental defect model according to claim 2, characterized in that, The concentration of the gRNA is 40-60 ng / μL; the concentration of the Cas9 protein is 250-350 ng / μL; and the mass ratio of the gRNA to the Cas9 protein is 1:5-7.
4. The method for constructing a neutrophil developmental defect model according to claim 2, characterized in that, The zebrafish embryos mentioned are single-cell stage zebrafish embryos.
5. The method for constructing a neutrophil developmental defect model according to claim 2, characterized in that, The injection volume is 1-2 nL / embryo.
6. The method for constructing a neutrophil developmental defect model according to claim 2, characterized in that, The F1 generation heterozygotes were screened using primers with nucleotide sequences as shown in SEQ ID NO:2 and 3.
7. The method for constructing a neutrophil developmental defect model according to claim 2, characterized in that, The homozygous mutant was hybridized with the transgenic line, F1 embryos were cultured, and after being raised to adulthood, they were self-crossed to screen for homozygous mutant hematopoietic cell fluorescent transgenic fish lines. The transgenic line is any one of Tg(gata1:DsRed), Tg(mpx:eEGFP), and Tg(mpeg1:eEGFP).
8. An evaluation method for a neutrophil developmental defect model constructed by the construction method according to any one of claims 1-7, characterized in that, The neutrophil developmental defect model was evaluated by at least one of the following methods: appearance, survival curve analysis, Sudan Black staining, fluorescent transgenic hybrid fish line of hematopoietic cells, flow cytometry fluorescent sorting of neutrophils, whole embryo in situ hybridization technology for screening hematopoietic developmental cell phenotypes, and whole transcriptome sequencing analysis of hematopoietic tissue.
9. The use of the neutrophil developmental defect model constructed by any one of the construction methods of claims 1-7, the construction method of any one of claims 1-7, and the evaluation method of claim 8 in screening drugs for the treatment and / or prevention of diseases related to neutrophil developmental defects.
10. The use of the neutrophil developmental defect model constructed by any one of the construction methods of claims 1-7, the construction method of any one of claims 1-7, and the evaluation method of claim 8 in the preparation of medicaments for the treatment and / or prevention of diseases related to neutrophil developmental defects.