CN114600837B - Animal model for granulocytopenia, construction method thereof and application of ikzf1 and cmyb in construction model - Google Patents

Abstract

The invention relates to a disease animal modelThe technical field of types, in particular to a granulocyte deficiency animal model, a construction method thereof and application of ikzf1 and cmyb in constructing the model. The zebra fish model construction method comprises the following steps in sequence: obtaining F0 generation zebra fish; the F0 generation zebra fish comprises ikzf1 ^+/‑ Strain and cmyb ^+/‑ Strain; mating the two strains of the F0 generation to obtain a heterozygous strain of the double-gene mutation of the zebra fish of the F1 generation; then selfing the heterozygous strain to obtain F2 generation zebra fish; the F2-generation zebra fish includes three lines of granulocyte defect models, and the extent of granulocyte depletion correlates with the in vivo doses of ikzf1 and cmyb. The technical problem that a disease model related to severe congenital granulocyte deficiency is lacking in the prior art can be solved, and the method can be used for researching pathogenesis of granulocyte deficiency related diseases and can also be used for drug screening and drug effect testing.

Description

Animal model for granulocytopenia, construction method thereof and application of ikzf1 and cmyb in construction model

Technical Field

The invention relates to the technical field of disease animal models, in particular to a granulocytopenia animal model, a construction method thereof and application of ikzf1 and cmyb in construction of the model.

Background

Severe congenital granulocytopenia is a disease affecting bone marrow cell production, resulting in severe congenital neutropenia. The development of granulocytes is stopped at the stage of promyelocytes or promyelocytes, so that the quantity of mature granulocytes in peripheral blood is reduced, other organ malformations are not usually accompanied, the granulocytes are autosomal dominant or recessive inheritance, and 60 to 80 percent of the granulocyte elastase gene ELA2 and other various mutations are present. Although severe congenital granulocytopenia has been listed in the first few diseases catalog and has received extensive attention from society, the prior art still lacks animal models of severe congenital granulocytopenia, resulting in intensive studies on the pathogenesis and molecular mechanisms of the disease, and drug screening and efficacy testing for the disease, which have not been performed effectively.

Zebra fish (Danio rerio) is a common tropical fish, and cell marking technology, tissue transplanting technology, mutation technology, haploid breeding technology, transgenic technology, gene activity inhibition technology and the like of the zebra fish are mature, and thousands of zebra fish embryo mutants are excellent resources for researching embryo development molecular mechanisms, and some zebra fish embryo mutants can be used as human disease models. Since the similarity of zebra fish genes and human genes reaches 87%, this means that the results obtained by performing a drug experiment on zebra fish genes are also applicable to human body in most cases; the embryos of zebra fish are transparent, so biologists can easily observe the effect of the drug on their organs in the body; female zebra fish can spawn 200, and embryos can develop and form within 24 hours, so that biologists can perform different experiments on the same generation of fish, further study the pathological evolution process and find the cause. How to use the advantages of zebra fish as an animal model of diseases to establish an animal model of severe congenital granulocyte deficiency, further to deeply study the pathogenic mechanism of the diseases and conduct drug screening and drug effect testing is a problem to be solved urgently at present.

Disclosure of Invention

The invention aims to provide a construction method of an animal model of granulocytopenia, which aims to solve the technical problem that a disease model related to severe congenital granulocytopenia is lacking in the prior art.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a method for constructing an animal model of granulocytopenia, comprising the following steps in sequence:

s1: obtaining F0 generation zebra fish; the F0 generation zebra fish comprises heterozygote ikzf1 with single gene mutation ^+/- Heterozygote cmyb of strain and single gene mutation ^+/- Strain;

s2: single gene mutated ikzf1 ^+/- Strain and monogenic mutant cmyb ^+/- Mating the strain to obtain F1 generation zebra fish; the F1 generation zebra fish comprises heterozygote ikzf1 with double gene mutation ^+/- cmyb ^+/- Strain;

S3：ikzf1 ^+/- cmyb ^+/- the strain is selfed to obtain F2 generation zebra fish; the F2 generation zebra fish comprises kzf1 ^+/- cmyb ^-/- Strain, ikzf1 ^-/- cmyb ^+/- Strain and ikzf1 ^-/- cmyb ^-/- Strain;

wherein, + represents a wild type allele, -represents a mutant allele; mutant alleles were obtained from wild type alleles after gene knockout.

The present solution also provides a zebra fish model obtained according to a method of constructing an animal model of granulocytopenia.

The scheme also provides application of the ikzf1 gene and the cmyb gene in constructing a zebra fish model for the granulocytopenia.

The principle and the advantages of the scheme are as follows:

by heterozygous mutant ikzf1 gene of zebra fish (ikzf 1 ^+/- Strain) and cmyb gene mutant heterozygotes zebra fish (cmyb) ^+/- Strain) were mated to obtain a zebra fish mutant heterozygous strain (ikzf 1) with simultaneous defects of ikzf1 gene and cmyb gene ^+/- cmyb ^+/- Strain). Then obtaining kzf1 by inbreeding in strain ^+/- cmyb ^-/- Strain, ikzf1 ^-/- cmyb ^+/- Strain and ikzf1 ^-/- cmyb ^-/- The F2 zebra fish in the strain shows that the granulocytes disappear in a large quantity or completely in the juvenile fish of the strain, and can be used as a model for researching the granulocyte deficiency and used for practical operations such as pathogenicity mechanism research, drug screening and the like. ikzf1 ^-/- cmyb ^-/- The granulocytopenia phenomenon of the strain is most remarkable. At ikzf1 ^-/- On the background, cmyb functions are missing half, or cmyb ^-/- On the background, half of the ikzf1 function is lost, and more serious granulocyte deficiency phenomenon can occur.

The research of the inventor shows that the Ikzf1 gene and the cmyb gene have the function of synergistically regulating the development, differentiation and functions of granulocytes, which is not reported in the prior research. Wherein, the ikzf1 protein and the cmyb protein can cooperatively form a complex to regulate the generation of granulocytes, and when two factors are simultaneously deleted, the granulocytes cannot generate. The extent of granulocyte depletion (severity of the disease) is related to the in vivo dose of these two factors. Therefore, by using the mutants of the ikzf1 gene and the cmyb gene for hybridization, a series of young zebra fish with two factors simultaneously defective and different defect degrees can be obtained, so that a disease animal model with serious and different severe congenital granulocytopenia can be created to adapt to various research requirements.

In the prior art, the number of disease models related to severe congenital granulocyte deficiency is very small, the technical scheme fills the defects in the field, solves the technical problem of lack of related disease models in the prior art, can be used for researching pathogenesis of granulocyte deficiency related diseases, and can also be used for drug screening and drug effect testing.

Wherein "+" and "-" in the upper right hand corner of the gene represent wild type allele and mutant allele, respectively; mutant alleles were obtained from wild type alleles after gene knockout. In specific embodiments, the monogenic mutant heterozygote ikzf1 ^+/- The strain is ikzf1 ^+/Δ4+3 Strain; heterozygote cmyb of single gene mutation ^+/- The strain is cmyb ^+/hkz3 Strain; heterozygote ikzf1 of double gene mutation ^+/- cmyb ^+/- The strain is ikzf1 ^+/Δ4+3 cmyb ^+/hkz3 Strain; kzf1 ^+/- cmyb ^-/- Strain, ikzf1 ^-/- cmyb ^+/- Strain and ikzf1 ^-/- cmyb ^-/- Lines are kzf respectively ^+/Δ4+3 cmyb ^hkz3/hkz3 Strain, ikzf1 ^{Δ4+3/Δ4+ 3} cmyb ^+/hkz3 Strain and ikzf1 ^Δ4+3/Δ4+3 cmyb ^hkz3/hkz3 Strain.

Further, the F2 generation zebra fish is ikzf1 ^-/- cmyb ^-/- Strain.

ikzf1 ^-/- cmyb ^-/- The strain was a homozygous strain with double gene mutations, showing complete loss of granulocytes of young fish starting 1.5 days after fertilization.

Further, the cDNA sequence of the wild type allele of the ikzf1 gene is shown as SEQ ID NO.1, and the cDNA sequence of the wild type allele of the cmyb gene is shown as SEQ ID NO. 2.

Both ikzf1 gene and cmyb gene are two of the relevant regulatory genes in the process of granulocyte development. Through a co-localization experiment and an immune co-precipitation experiment, the fact that strong interaction exists between protein products of the two genes can be combined with each other in the same cell to form a complex so as to ensure granulocyte generation, and the defect degree of the two genes directly influences the defect degree of granulocyte.

Further, the cDNA sequence of the mutant allele of the ikzf1 gene is shown as SEQ ID NO.3, and the cDNA sequence of the mutant allele of the cmyb gene is shown as SEQ ID NO. 4.

In particular embodiments of the present protocol, the mutant allele of the ikzf1 gene and the mutant allele of the cmyb gene are obtained by conventional CRISPR/Cas9 techniques and ENU chemically induced mutation, respectively. Both mutant genes express biologically inactive truncated proteins, thus achieving gene knockout. The knockout of the target gene can be achieved by screening using both of the above techniques, but it is not meant that the inactivation of the expression products (proteins) of the ikzf1 gene and the cmyb gene can be achieved only by the specific form of mutation of the two genes.

Further, one or both alleles of the ikzf1 gene are knocked out, and both alleles of the cmyb gene are knocked out; or one or both alleles of the cmyb gene are knocked out, and both alleles of the ikzf1 gene are knocked out.

By adopting the technical scheme, the double mutant (ikzf 1 ^+/Δ4+3 ；cmyb ^hkz3/hkz3 )、(ikzf1 ^Δ4+3/Δ4+3 ；cmyb ^+/hkz3 ) And (ikzf 1) ^Δ4+3/Δ4+3 ；cmyb ^hkz3/hkz3 ) The embryo is normal in development, the granulocyte number is in a low-level state, and the animal model of the agranulocytosis is successfully constructed.

Further, all alleles of the ikzf1 gene and all alleles of the cmyb gene are knocked out.

Both ikzf1 gene and cmyb gene are two of the relevant regulatory genes in the process of granulocyte development. After the present inventors tried on various granulocyte development-related genes, they found that experimental results in which granulocytes could not be detected at all could be achieved without constructing double mutant homozygotes of any granulocyte development-related genes. Some homozygotes of the double gene mutant can be in abnormal development, and some homozygotes of the double gene mutant can be in a condition that granulocyte positive signals can still be detected. Only in the case of complete knockout of ikzf1 gene and cmyb gene, granulocytes in zebra fish completely disappeared and embryo development was normal. Clinical diagnosis of severe congenital neutropenia SCN usually has only neutropenia with mononucleosis, no other cosmetic malformations, and normal hemoglobin and platelet counts. In double mutation (ikzf 1 ^Δ4+3/Δ4+3 ；cmyb ^hkz3/hkz3 ) With an increase in macrophage/monocyte numbers, the phenotype is highly similar to severe congenital granulocytopenia.

Drawings

FIG. 1 is a representative image of a Sudan black B staining experiment at 3dpf of example 1.

FIG. 2 shows statistics of positive cells from Sudan black B staining experiments at 3dpf of example 1.

FIG. 3 is a typical image of an mpx in situ hybridization experiment at 3dpf of example 1.

FIG. 4 is a graph showing the statistics of mpx in situ hybridization experiments positive cells at 3dpf of example 1.

FIG. 5 is a representative image of Sudan black B staining experiments at 36hpf of example 1.

FIG. 6 shows statistics of positive cells of Sudan black B staining experiments at 36hpf of example 1.

FIG. 7 is a typical image of an mpx in situ hybridization assay at 36hpf of example 1.

FIG. 8 shows statistics of mpx in situ hybridization experiments at 36hpf of example 1.

FIG. 9 is a typical image of an in situ hybridization experiment of the csf1r at 28hpf of example 1.

FIG. 10 shows statistics of the positive cells of the in situ hybridization assay of csp 1r at 28hpf of example 1.

FIG. 11 shows the results of experiments for co-localization studies of the transcription product of the ikzf1 gene and the transcription product of the cmyb gene of example 2.

FIG. 12 shows the results of the co-immunoprecipitation experiment of the Ikzf1 protein and the cMyb protein of example 2.

Detailed Description

The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto. Unless otherwise indicated, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used are all commercially available. The technical means used in the following examples are conventional means well known to those skilled in the art unless otherwise indicated; the experimental methods used are all conventional methods (including gene knockout based on CRISPR/Cas9 technology, N-ethyl-N-nitrosourea (ENU) chemical induced mutation and screening for organisms to obtain inactivation of the gene of interest) and can be accomplished according to the described recombinant, mutation techniques, etc. (e.g., molecular cloning laboratory Manual, 2 nd edition, cold spring harbor laboratory Press, cold spring harbor, N.Y.).

Example 1:

the construction process of the granulocyte deficiency zebra fish model is specifically as follows:

(1) Acquisition of the ikzf1 mutant heterozygotes (ikzf 1) ^+/Δ4+3 ) And cmyb mutant heterozygotes (cmyb) ^+/hkz3 )

The preparation of mutants of Ikzf1 (IKAROS family zinc finger, gene ID:30177, see SEQ ID NO.1 for wild-type Gene cDNA sequence, see SEQ ID NO.12 for protein sequence), see literature (Youkui Huang, ikzf1regulates embryonic T lymphopoiesis via Ccr9& Irf4 in zebrafish, journal of Biological Chemistry,2019, nov 1;294 (44): 16152-16163.), cmyb (Gene ID: 3059, see SEQ ID NO.2 for wild-type Gene cDNA sequence, see SEQ ID NO.13 for protein sequence), see literature (Zhang Y, cMyb regulates hematopoietic stem/progenitor cell mobilization during zebrafish minerals. Blood.2011Oct 13;118 (15): 4093-101.).

Wherein the ikzf1 mutant is constructed using prior art conventional CRISPR/Cas9 technology (Chang Nannan, genome editing with RNA-guide Cas9 nuclease in zebrafish embryos. Cell res.2013, 23, 465-472). Knocking out the ikzf1 gene by CRISPR/Cas9 technology construction, screening to obtain a mutant allele of the ikzf1 gene, wherein the mutant allele causes 1bp deletion (specifically the original deletion of 4bp nucleotide and the insertion of additional 3bp nucleotide) on the third exon of the wild type allele, and causes the early appearance of a stop codon on the coding sequence of the gene by frame shift mutation, thereby obtaining the ikzf1 gene ^Δ4+3 The mutant gene encodes a non-functional truncated ikzf1 protein (see, youkui Huang,2019, figure 1A, 1B, figure S1D-F, and the text of the "Embryonic T lymphopoiesis is abolished in ikzf variants" section). ikzf1 mutant heterozygotes (ikzf 1) ^+/Δ4+3 ) Refers to two alleles of the ikzf1 gene of zebra fish, one is a wild-type gene and the other is ikzf1 ^Δ4+3 Mutant gene (see SEQ ID NO.3 for sequence and SEQ ID NO.14 for protein sequence). In the case of obtaining the mutant gene of ikzf1, a stably inherited mutant heterozygote of ikzf1 (ikzf 1) ^+/Δ4+3 )。

The cmyb mutant is obtained by forward genetics method of chemically inducing mutation by N-ethyl-N-nitrosourea (ENU) ^hkz3 The strain, the mutation is due to the eighth intron in the genome of cmyb inserted into a base A, the mutation of the splice site between the eighth exon and the ninth exon from the base A to the base T leads to a competitive splice acceptor, so that the eighth exon and the ninth exon are inserted into 13bp nucleotides due to the existence of the competitive splice acceptor in the transcription process of cmyb, and the premature appearance of a stop codon of the coding sequence of the gene is caused, cmyb ^hkz3 The protein structure in the mutant is truncated in the trans-activating domain (TAD) and translated into a nonfunctional truncated cMyb protein (see Zhang Y,2011, figure S1-S2 and "Failure of larval and adult definitive hematopoiesis in the cMyb) ^hkz3 zebrafish mutant line "partial text description). cmyb mutant heterozygotes (cmyb) ^+/hkz3 ) Refers to two alleles of the cmyb gene of zebra fish, one is a wild-type gene and the other is cmyb ^hkz3 Mutant gene (sequence is shown in SEQ ID NO.4, protein sequence is shown in SEQ ID NO.15, the protein has no biological function, and corresponds to wild type cmyb gene being knocked out). Under the condition of obtaining cmyb gene mutant gene, the cmyb mutant heterozygote (cmyb) with stable inheritance can be obtained by the conventional hybridization means in the prior art ^+/hkz3 )。

The above is a general description of a method for obtaining two F0 generation single mutants, in which CRISPR/Cas9 technology is used to knock out the gene and ENU chemical induction technology is used to obtain the mutant with inactivated target gene (protein product), which are conventional means in the prior art, and the above two technologies are used to achieve the knockout of the target gene by screening. ikzf1 ^Δ4+3 Mutant gene and cmyb ^hkz3 The mutant gene was a mutant gene which has been obtained in the prior art literature, so in subsequent experiments we used the ikzf1 mutant heterozygote (ikzf 1 ^+/Δ4+3 ) And cmyb mutant heterozygotes (cmyb) ^+/hkz3 ) It is not meant to be limiting that only the specific forms of mutation of the two genes achieve the goal of inactivating the expression products (proteins) of the ikzf1 gene and the cmyb gene.

The ikzf1 mutant heterozygotes were specifically prepared and screened as follows:

(1) inquiring the genome DNA sequence of the zebra fish ikzf1 gene at https:// asia. Ensembl. Org/danio_rerio/Info/Index website, designing a target site at the third exon of ikzf1 according to the CRISPR/Cas9 knockout principle, and designing a pair of SNP primers around the target site: f-ikzf1:5'-CACTATGGCATATGGTGTAG-3' (SEQ ID NO. 5), R-ikzf1:5'-GCAAAAGCTGAAGATGCGAGT-3' (SEQ ID NO. 6).

(2) The template for in vitro transcription of the PCR amplified gRNA, the forward primer contained three parts, the T7 promoter sequence (17 bp), the target sequence (20 bp) and the gRNA scaffold sequence, namely F-gRNA:5'-TAATACGACTCACTATAGGATGCTAATGAAGGCGGAGGTTTTAGAGCTAGAAATAGC-3' (SEQ ID NO. 7), the reverse primer is a universal primer: R-gRNA:5'-AGCACCGACTCGGTGCCACT-3' (SEQ ID NO. 8).

(3) The gRNA was transcribed in vitro (10. Mu.L) using an RNA in vitro transcription kit, purified with 75% pre-chilled ethanol, and resuspended in an appropriate amount of ultra-pure water.

(4) Purified gRNA and Cas9 protein cocktail 1:1, mixing and co-introducing the zebra fish embryo, mixing 1 μl Cas9 protein with 0.5 μl 10 XBuffer, adding 3.5 μl ddH, and mixing the mixture of protein gRNA with final concentration of 50 ng/. Mu. L, cas9 ₂ O, standing at normal temperature for 5min for standby, and performing microinjection at the embryonic cell stage of the zebra fish with the injection quantity of 1nL.

(5) Screening and identifying F0 zebra fish, and feeding target working embryo to adult, and performing F0 screening. Adult F0 generation was mated with AB wild type zebra fish, the resulting embryos were lysed at 1.5dpf and the cleaved templates were sequenced using SNP primer PCR amplification. Offspring with detected cover peaks were bred to adult, resulting in adult F1.

(6) Screening and identifying F1 zebra fish mutants, cutting tail fins of each F1 adult zebra fish, cracking in proteinase K, sequencing by SNP primer PCR amplification cracking template, and obtaining the hybrid of the ikzf1 mutant (ikzf 1 ^+/Δ4+3 )。

The cmyb mutant heterozygotes were specifically prepared and screened as follows:

(1) cmyb mutant heterozygotes (cmyb) ^+/hkz3 ) Obtained by N-ethyl-N-nitrosourea (ENU) screening, cmyb ^hkz3 The mutant is a mutant existing in the prior art, and is described in detail in the foregoing.

(2) Identification of cmyb mutant heterozygote adult fish: for specific detection of aberrant splice forms of cmyb WT or hkz3, the following primer sets were used: common forward primer: f-cmyb:5'-CACTGCTGCTATCCAGAGACACTAC-3' (814_FP,SEQ ID NO.9); reverse primer R-WT:5'-AGCCGTTCATGGAGATTGGTAG-3' (947_wt_RP,SEQ ID NO.10) and R-hkz3:5'-CATGGAGATCTGGGTGGGGG-3' (947_hkz3_RP,SEQ ID NO.11) is specific for the cmyb WT form and the hkz aberrant splice form, respectively. Amplification was performed according to the following PCR conditions: pre-denaturation at 95℃for 3min; denaturation at 95℃for 30s, annealing at 60℃for 30s, extension at 72℃for 30s for 35 cycles; and then at 72 ℃ for 10min. And (5) performing gel electrophoresis identification on the PCR product.

(2) Breeding the ikzf1 mutant heterozygote and the cmyb mutant heterozygote into adult fish according to a zebra fish breeding method conventional in the prior art, hybridizing the ikzf1 mutant heterozygote adult fish and the cmyb mutant heterozygote adult fish, and screening to obtain double mutant heterozygote (ikzf 1) ^+/Δ4+3 ；cmyb ^+/hkz3 ) Designated ikzf1 ^+/Δ4+3 cmyb ^+/hkz3 Strain (F1 generation).

(3) Double mutant heterozygotes (ikzf 1) ^+/Δ4+3 ；cmyb ^+/hkz3 ) The conventional zebra fish breeding method in the prior art is used for breeding adult fish, and then the heterozygote adult fish is subjected to selfing to obtain zebra fish strains (F2 generation) with different mutation types, wherein the method comprises the following steps: double mutant (kzf 1) ^+/Δ4+3 ；cmyb ^hkz3/hkz3 )、(ikzf1 ^Δ4+3/Δ4+3 ；cmyb ^+/hkz3 )、(ikzf1 ^Δ4+3/Δ4+3 ；cmyb ^hkz3/hkz3 ) Respectively named kzf1 ^+/Δ4+3 cmyb ^hkz3/hkz3 Strain, ikzf1 ^Δ4+3/Δ4+3 cmyb ^+/hkz3 Strain and ikzf1 ^Δ4+3/Δ4+3 cmyb ^{hkz3 /hkz3} Strain. Markers such as mpx (myeloid-specific peroxidase ), sudan black B (SB), etc., of granulocytes were detected by in situ hybridization and staining, respectively, and the experimental results are shown in fig. 1. Positive products of sudan black B staining were black or blue-black particles localized in the cytoplasm. Except for the primary granulocytes, positive particles can be seen in granulocytes, and other blood cells, such as lymphocytes, young erythrocytes, megakaryocytes, platelets and the like, all react negatively.

FIG. 1 is a photomicrograph of typical Sudan black B staining (including whole-body, head and tail hematopoietic tissues (CHT) of zebra fish) at 3dpf (3 days post fertilization). FIG. 2 is a statistical plot of the number of Sudan black B positive cells at 3dpf for each strain (statistics of positive signals for whole zebra fish, mean+ -SEM, n=7, 23, 3, 6, 7, in order from left to right: ikzf1 ^+/+ cmyb ^+/+ Strain, ikzf1 ^+/Δ4+3 cmyb ^+/hkz3 Strain, ikzf1 ^+/+ cmyb ^hkz3/hkz3 Strain, ikzf1 ^Δ4+3/Δ4+3 cmyb ^+/+ Strain, ikzf1 ^Δ4+3/Δ4+3 cmyb ^hkz3/hkz3 Strain). FIG. 3 is a typical mpx in situ hybridization micrograph (CHT) of 3 dpf. FIG. 4 shows the number of mpx positive cells at 3dpf for each strain (statistics of positive signals for whole zebra fish, mean+ -SEM, n=6, 26, 5, 8, 7, in order from left to right: ikzf1 ^+/+ cmyb ^+/+ Strain, ikzf1 ^+/Δ4+3 cmyb ^+/hkz3 Strain, ikzf1 ^+/+ cmyb ^hkz3/hkz3 Strain, ikzf1 ^Δ4+3/Δ4+3 cmyb ^+/+ Strain, ikzf1 ^Δ4+3/Δ4+3 cmyb ^hkz3/hkz3 Strain). In FIGS. 1 and 3, the lower left corner of the left image represents ikzf1 ^+/+ cmyb ^hkz3/hkz3 Strain and ikzf1 ^Δ4+3/Δ4+3 cmyb ^+/+ The line, because the granulocyte numbers of both genotypes are at similar levels, is represented by a single plot.

From the experimental results of FIGS. 1-4, the parent ikzf1 is known ^+/Δ4+3 cmyb ^+/hkz3 Among the self-bred filial generations of the strain, filial generation ikzf1 ^+/+ cmyb ^+/+ The whole body of strain (i.e. wild type AB strain) contains a large number of granulocytes, ikzf1 ^+/Δ4+3 cmyb ^+/hkz3 The number of granulocytes in the line (double gene mutant heterozygote) was reduced relative to the wild type, whereas ikzf1 ^+/+ cmyb ^hkz3/hkz3 Strain and ikzf1 ^Δ4+3/Δ4+3 cmyb ^+/+ The lines are homozygotes of single-gene mutations, and the number of granulocytes of both lines is further reduced relative to the number of double-gene mutant heterozygotes. In the homozygote of the double gene mutation (ikzf 1) ^Δ4+3/Δ4+3 cmyb ^hkz3/hkz3 Strain), granulocytes completely disappeared at 3 dpf. The two genes are completely knocked out at the same time, so that the development process of granulocytes is obviously influenced, and the embryo-derived granulocyte precursor cells can be ensured not to be bred into mature granulocytes any more, and the establishment of the granulocyte deficiency animal model is successful. However, the above effect cannot be achieved by completely knocking out only the ikzf1 gene or the cmyb gene. The inventors have found that experimental junction in which granulocytes are not completely detected can be realized without constructing double mutant homozygotes of granulocyte development-related genes after trying a plurality of granulocyte development-related genes because of more genes affecting granulocyte developmentAnd (5) fruits. Some homozygotes of the double gene mutant can be in abnormal development, and some homozygotes of the double gene mutant can be in a condition that granulocyte positive signals can still be detected. Only in the homozygote of the double gene mutation (ikzf 1 ^{Δ4+3/Δ4+ 3} cmyb ^hkz3/hkz3 Strain) shows no detectable granulocytes and normal embryo development, and homozygotes with double gene mutations (ikzf 1) ^Δ4+3/Δ4+3 cmyb ^hkz3/hkz3 Strain) survived normally for 1-2 weeks, and the model can be used for research of disease mechanism and screening of drugs.

Clinical diagnosis of severe congenital neutropenia SCN usually has only neutropenia with mononucleosis, no other cosmetic malformations, and normal hemoglobin and platelet counts. Double mutant (ikzf 1) ^+/Δ4+3 ；cmyb ^hkz3/hkz3 )、(ikzf1 ^Δ4+3/Δ4+3 ；cmyb ^+/hkz3 ) And (ikzf 1) ^Δ4+3/Δ4+3 ；cmyb ^hkz3/hkz3 ) Embryo development is normal, granulocyte numbers are in a low level state, and in double mutations (ikzf 1 ^Δ4+3/Δ4+3 ；cmyb ^hkz3/hkz3 ) With an increase in macrophage/monocyte numbers, the phenotype is highly similar to severe congenital granulocytopenia. The double homozygote showed complete loss of granulocytes of young fish starting 1.5 days after fertilization (36 hpf), and the experimental results are shown in fig. 5, 6, 7 and 8. FIG. 5 is a typical mpx in situ hybridization micrograph (whole body) of 36 hpf; fig. 6 is a statistical plot of the number of mpx positive cells at 36hpf for each strain (statistics for positive signals of zebra fish whole body, mean±sem, n=10, 26, 7, 8, 17, 8); FIG. 7 is a photomicrograph of a typical Sudan black B stain of 36hpf (including head and CHT); fig. 8 is a statistical plot of the number of sudan black B positive cells at 36hpf for each strain (statistics for positive signals of zebra fish whole body, mean±sem, n=13, 28, 12, 11, 21, 10). The results of the macrophage/monocyte assay are shown in figures 9 and 10.CSF1R is a single pass tyrosine kinase transmembrane receptor belonging to the class iii protein tyrosine kinase receptor family, which is expressed primarily on the surface of monocytes and macrophages and controls the growth, function and differentiation of macrophages by its activation. The proposal is thatThe number of macrophages/monocytes in zebra fish was detected by in situ hybridization of csp 1 r. Fig. 9 is a typical in situ hybridization micrograph (head and CHT) of csf1r at 28 hours post fertilization (28 hpf), and fig. 10 is a statistical plot of the number of csf1r positive cells at 28hpf for each strain (statistics on positive signals of zebra fish as a whole, mean±sem, n=8, 25, 9, 13). From the experimental results of FIGS. 9 and 10, it is understood that double mutation (ikzf 1 ^Δ4+3/Δ4+3 ；cmyb ^hkz3/hkz3 ) In the group, the increase of the number of macrophages/monocytes is accompanied, and the clinical diagnosis and adjustment of severe congenital neutrophilic granulocyte deficiency are met, so that the model is successfully constructed.

In conclusion, with the present solution, the severity of granulocytopenia depends on the severity of functional defects of the ikzf1 gene and the cmyb gene. The zebra fish model with double gene mutations can be used as a disease model and is also helpful for researching the occurrence mechanism of diseases.

Example 2: research on cooperative relationship between ikzf1 gene and cmyb gene

The present inventors have further studied the synergistic interaction of ikzf1 gene and cmyb gene during the development of zebra fish granulocyte. By RNA staining the transcription product of the ikzf1 gene and the transcription product of the cmyb gene, the results suggest that these two factors are co-localized simultaneously in granulocyte precursor (see FIG. 11 for experimental results). Co-immunoprecipitation (Co-IP) by transfected cells suggests a strong interaction of these two factors, and the experimental results are shown in FIG. 12. FIG. 12A is an experiment of the interaction of fusion proteins GFP-Ikzf1 and Flag-c-Myb in the HEK293T cell line, which shows the detection of the band of interest (Flag-c-Myb) by precipitation of GFP-Ikzf1, indicated by the arrow. FIG. 12B shows the detection of the band of interest (GFP-Ikzf 1) by precipitation of Flag-c-Myb, the band of interest indicated by the arrow. This suggests that there is a strong interaction between the Ikzf1 protein and the cMyb protein, and that it can be combined with each other in the same cell to form a complex to ensure granulogenesis. Therefore, the defect degree of the two can directly influence the granulocyte defect degree, so that the young fish of the ikzf1/cmyb double mutant zebra fish can be used as a disease animal model of severe congenital granulocyte deficiency.

The foregoing is merely exemplary of the present invention, and specific technical solutions and/or features that are well known in the art have not been described in detail herein. It should be noted that, for those skilled in the art, several variations and modifications can be made without departing from the technical solution of the present invention, and these should also be regarded as the protection scope of the present invention, which does not affect the effect of the implementation of the present invention and the practical applicability of the patent. The protection scope of the present application shall be subject to the content of the claims, and the description of the specific embodiments and the like in the specification can be used for explaining the content of the claims.

SEQUENCE LISTING

<110> Runkang biological medicine (Suzhou) Co., ltd

<120> an animal model for agranulocytosis, method for constructing the same, and ikzf1 and cmyb in constructing the model

Application of

<130> 2022.4.12

<160> 15

<170> PatentIn version 3.5

<210> 1

<211> 1614

<212> DNA

<213> Danio rerio

<400> 1

atggagactg aggaggcaca ggaaatgtcc cagataacag gaagagacag cccgatgaat 60

gctaatgaag gcggagagga tcaagatgag gccatgcctg ttcctgaaga cttgtcagca 120

agcactggcc tccaacacaa caatcgcaca gataaaccac tggcctgtaa tataaaagtt 180

gaggctcgga gtgacgagga aaacggtctg tcctgtgaga tgaatggaga ggcagaggaa 240

tgtgcagctg aggacttgcg catactcgat ggctctgggg ccaaagtgaa cggctcccac 300

gcaggccccg acagcaagcc ggccgcctac cccacagccg ggggcatccg cctccccaac 360

gggaagctga agtgcgatat ctgtgggata gtttgcattg ggcccaatgt gttgatggtt 420

cacaagcgaa gtcacactga agaaagaaag tcagttttgg aacaacaaaa aggtgaaagg 480

ccattccagt gcaatcaatg tggtgcttca ttcactcaga agggtaacct gctccgacac 540

atcaaacttc actctggcga gaaacctttc aaatgtcacc tgtgcaacta tgcttgccgc 600

cgcagagacg ctctcactgg acatctgcgc actcattcgg ttggaaagcc ccataagtgt 660

gcatattgcg gacgcagtta caagcagcgg agctcactgg aggaacataa ggagagatgt 720

cacaactact tgcagtgcat gggccttcag aacagcattt atacagtgaa ggaagagaac 780

agccagaatg agcagaggga ggacatgcct gcatctgaga gggccttggt gctagacagg 840

atagctaaca atgtagctaa gcgtaagagc tctatgccac agaggtttgt gggagagaat 900

cgtctgtcag agctatcttt cgagagtggc tcaggtgagc tgatgcagcc ccatgtgatt 960

gatcaggcca tcaacagtgc aattagctat ctgggtgcag agtccttgcg gcctctggtt 1020

cagacctctc ctgggtccgc cgacatggtg gtcagccctc tatacaacct gcacaagtca 1080

caaacagctg aaggcaatgg cgtttctgct aaagacagcg ccgcagagca ccttctctta 1140

ctctctaagt ccaaatccgc ctctgttgac aaagacggtt cccccagtcc cagcgggcag 1200

gattccactg acactgagag caacaatgag gagcgttcag ccggggtaag cggaacagca 1260

gccacaggtg gtctcatcta cctgaccaac cacatggctc caggtatgag aaatggaggc 1320

ctgccagggg tgaaggaaga acaacagcgg cattttgagg ctttgcgagc agcaggaatg 1380

gatttgagta tagcgtcatc agaaggattt aaggtgctga gtggagatgg agaagaactg 1440

agggcgtacc gctgtatcca ctgcagagtt ttgttcctgg atcatgtcat gtacaccatc 1500

cacatgggct gtcatggctt ccgagacccc tttgagtgca acctatgcgg gtaccgcagt 1560

caggaccgtt atgagttctc atcgcacatc acacgtggag agcaccgcat ctga 1614

<210> 2

<211> 1926

<212> DNA

<213> Danio rerio

<400> 2

atggcgaggc ggcacagaca cagtgtttac agtagcgacg aagatgatga tgatgtggag 60

atttatgatc atgactatga tggtcctcat gccaagacag gaaaacgcca cctcggcaag 120

acacgctgga cccgtgagga ggatgaaaag ttaaagaggc tggtagagca tcatggttct 180

gaagactgga aagtcatcgc cagctttcta ccgaatcgaa cagatgttca atgccagcat 240

cgttggcaga aagtcctcaa ccctgaactc attaaaggac catggactaa agaagaagac 300

caacgggtga ttgagttggt gcagaagtac ggccccaaac gttggtcagt aatcgcaaag 360

catttaaagg ggcgaatcgg aaagcagtgt agagagcgct ggcacaacca tctaaaccct 420

gaggtgaaga agacgtcctg gactgaggaa gaagatcaaa tcatttacca ggcgcacgag 480

aaacttggaa accgatgggc cgagattgcc aagttacttc cgggaagaac cgataatgcc 540

atcaagaatc actggaactc cactatgcgg cggaaagtgg agcaagaagg ttatttgcag 600

cacgctgcta aagtcagccc aactccgcta aacaacagct attctaaacc tcacctcctg 660

aactacaatc acacaccaag caacacatcc atgcctgcct cctccatgag caatcagtat 720

ccatactaca ctgaatcatc acgggtgcca tttccacttg ctctccagtt aaacatcttg 780

aactttccac aacatggcac tgctgctatc cagagacact acagcgacga ggaccccgaa 840

aaagaaaaga gggtgaaaga aatcgagatg ctgttgatgt caacagaaaa cgagctgaag 900

ggacaacagg cactaccaat ctccatgaac ggctacggtg gctggaacag aggctctttg 960

gcggacagtt cggttggcat tgttgtttca gtcccggctc catccctaga gcaaggatgc 1020

cttcctgaag aaagcgcaca tggcaacaca aacagcccaa ataacgactc cagctccact 1080

ttaccagagt ttatcgatgc tattgattcg gccccatcag tctgggccaa caaacaccct 1140

aaaacggagt gccaaaccgg tcagggttcg cctgcaaaca acggcaacag aagtgccccc 1200

acttttcttt gctcaacgcc caaatattca tccgtcagac acctgccttt ttctcccact 1260

cagttcttta atgcttcagt gagtcccgat tcagacagtc aaaatatacc agtcacgcca 1320

tcgccggtct gctcccagaa agcactgcag caagaccttg ctcttcgacc tcagaaggaa 1380

aatgagctgt tccgaactcc caacctgagg cgttcgatca tggagtgttc tcctcgcaca 1440

cccactccat tcaaatccac actgacaatg caggacagca aatacggacc actgaagagg 1500

gtccacagtc cttcgctgga ctctggagtg gtcggcacga ttaaacaaga gccccaggag 1560

tgtgagatca gtgttggagg ggtgcatcta gaccagcccc ctctgaaaaa gatcaaacag 1620

gaggtggagt cagtgtgtct tcagtgggag gggcaagatc tccacactca gctgttccct 1680

tctaatggcc ccgcccacga catgcctgat ctgctgacta gctctgtgct gatgcttccc 1740

aacacagaga agactgagga cggacacaaa gctgcccagt tgccccgcag acccatagga 1800

agtcctttgc agcagttgaa cacatgggaa caggtgcttt gtgggaagac ggaagagcag 1860

acgattccct ctgaagccac gcacaaatac ctcagcaatt attcttcgcg agctctggtc 1920

atatga 1926

<210> 3

<211> 1613

<212> DNA

<213> Danio rerio

<400> 3

atggagactg aggaggcaca ggaaatgtcc cagataacag gaagagacag cccgatgaat 60

gctaatgaag gcggagagga tggcatgagg ccatgcctgt tcctgaagac ttgtcagcaa 120

gcactggcct ccaacacaac aatcgcacag ataaaccact ggcctgtaat ataaaagttg 180

aggctcggag tgacgaggaa aacggtctgt cctgtgagat gaatggagag gcagaggaat 240

gtgcagctga ggacttgcgc atactcgatg gctctggggc caaagtgaac ggctcccacg 300

caggccccga cagcaagccg gccgcctacc ccacagccgg gggcatccgc ctccccaacg 360

ggaagctgaa gtgcgatatc tgtgggatag tttgcattgg gcccaatgtg ttgatggttc 420

acaagcgaag tcacactgaa gaaagaaagt cagttttgga acaacaaaaa ggtgaaaggc 480

cattccagtg caatcaatgt ggtgcttcat tcactcagaa gggtaacctg ctccgacaca 540

tcaaacttca ctctggcgag aaacctttca aatgtcacct gtgcaactat gcttgccgcc 600

gcagagacgc tctcactgga catctgcgca ctcattcggt tggaaagccc cataagtgtg 660

catattgcgg acgcagttac aagcagcgga gctcactgga ggaacataag gagagatgtc 720

acaactactt gcagtgcatg ggccttcaga acagcattta tacagtgaag gaagagaaca 780

gccagaatga gcagagggag gacatgcctg catctgagag ggccttggtg ctagacagga 840

tagctaacaa tgtagctaag cgtaagagct ctatgccaca gaggtttgtg ggagagaatc 900

gtctgtcaga gctatctttc gagagtggct caggtgagct gatgcagccc catgtgattg 960

atcaggccat caacagtgca attagctatc tgggtgcaga gtccttgcgg cctctggttc 1020

agacctctcc tgggtccgcc gacatggtgg tcagccctct atacaacctg cacaagtcac 1080

aaacagctga aggcaatggc gtttctgcta aagacagcgc cgcagagcac cttctcttac 1140

tctctaagtc caaatccgcc tctgttgaca aagacggttc ccccagtccc agcgggcagg 1200

attccactga cactgagagc aacaatgagg agcgttcagc cggggtaagc ggaacagcag 1260

ccacaggtgg tctcatctac ctgaccaacc acatggctcc aggtatgaga aatggaggcc 1320

tgccaggggt gaaggaagaa caacagcggc attttgaggc tttgcgagca gcaggaatgg 1380

atttgagtat agcgtcatca gaaggattta aggtgctgag tggagatgga gaagaactga 1440

gggcgtaccg ctgtatccac tgcagagttt tgttcctgga tcatgtcatg tacaccatcc 1500

acatgggctg tcatggcttc cgagacccct ttgagtgcaa cctatgcggg taccgcagtc 1560

aggaccgtta tgagttctca tcgcacatca cacgtggaga gcaccgcatc tga 1613

<210> 4

<211> 1939

<212> DNA

<213> Danio rerio

<400> 4

atggcgaggc ggcacagaca cagtgtttac agtagcgacg aagatgatga tgatgtggag 60

atttatgatc atgactatga tggtcctcat gccaagacag gaaaacgcca cctcggcaag 120

acacgctgga cccgtgagga ggatgaaaag ttaaagaggc tggtagagca tcatggttct 180

gaagactgga aagtcatcgc cagctttcta ccgaatcgaa cagatgttca atgccagcat 240

cgttggcaga aagtcctcaa ccctgaactc attaaaggac catggactaa agaagaagac 300

caacgggtga ttgagttggt gcagaagtac ggccccaaac gttggtcagt aatcgcaaag 360

catttaaagg ggcgaatcgg aaagcagtgt agagagcgct ggcacaacca tctaaaccct 420

gaggtgaaga agacgtcctg gactgaggaa gaagatcaaa tcatttacca ggcgcacgag 480

aaacttggaa accgatgggc cgagattgcc aagttacttc cgggaagaac cgataatgcc 540

atcaagaatc actggaactc cactatgcgg cggaaagtgg agcaagaagg ttatttgcag 600

cacgctgcta aagtcagccc aactccgcta aacaacagct attctaaacc tcacctcctg 660

aactacaatc acacaccaag caacacatcc atgcctgcct cctccatgag caatcagtat 720

ccatactaca ctgaatcatc acgggtgcca tttccacttg ctctccagtt aaacatcttg 780

aactttccac aacatggcac tgctgctatc cagagacact acagcgacga ggaccccgaa 840

aaagaaaaga gggtgaaaga aatcgagatg ctgttgatgt caacagaaaa cgagctgaag 900

ggacaacagg cactaccatt cccccaccca gatctccatg aacggctacg gtggctggaa 960

cagaggctct ttggcggaca gttcggttgg cattgttgtt tcagtcccgg ctccatccct 1020

agagcaagga tgccttcctg aagaaagcgc acatggcaac acaaacagcc caaataacga 1080

ctccagctcc actttaccag agtttatcga tgctattgat tcggccccat cagtctgggc 1140

caacaaacac cctaaaacgg agtgccaaac cggtcagggt tcgcctgcaa acaacggcaa 1200

cagaagtgcc cccacttttc tttgctcaac gcccaaatat tcatccgtca gacacctgcc 1260

tttttctccc actcagttct ttaatgcttc agtgagtccc gattcagaca gtcaaaatat 1320

accagtcacg ccatcgccgg tctgctccca gaaagcactg cagcaagacc ttgctcttcg 1380

acctcagaag gaaaatgagc tgttccgaac tcccaacctg aggcgttcga tcatggagtg 1440

ttctcctcgc acacccactc cattcaaatc cacactgaca atgcaggaca gcaaatacgg 1500

accactgaag agggtccaca gtccttcgct ggactctgga gtggtcggca cgattaaaca 1560

agagccccag gagtgtgaga tcagtgttgg aggggtgcat ctagaccagc cccctctgaa 1620

aaagatcaaa caggaggtgg agtcagtgtg tcttcagtgg gaggggcaag atctccacac 1680

tcagctgttc ccttctaatg gccccgccca cgacatgcct gatctgctga ctagctctgt 1740

gctgatgctt cccaacacag agaagactga ggacggacac aaagctgccc agttgccccg 1800

cagacccata ggaagtcctt tgcagcagtt gaacacatgg gaacaggtgc tttgtgggaa 1860

gacggaagag cagacgattc cctctgaagc cacgcacaaa tacctcagca attattcttc 1920

gcgagctctg gtcatatga 1939

<210> 5

<211> 20

<212> DNA

<213> artificial sequence

<400> 5

cactatggca tatggtgtag 20

<210> 6

<211> 21

<212> DNA

<213> artificial sequence

<400> 6

gcaaaagctg aagatgcgag t 21

<210> 7

<211> 57

<212> DNA

<213> artificial sequence

<400> 7

taatacgact cactatagga tgctaatgaa ggcggaggtt ttagagctag aaatagc 57

<210> 8

<211> 20

<212> DNA

<213> artificial sequence

<400> 8

agcaccgact cggtgccact 20

<210> 9

<211> 25

<212> DNA

<213> artificial sequence

<400> 9

cactgctgct atccagagac actac 25

<210> 10

<211> 22

<212> DNA

<213> artificial sequence

<400> 10

agccgttcat ggagattggt ag 22

<210> 11

<211> 20

<212> DNA

<213> artificial sequence

<400> 11

catggagatc tgggtggggg 20

<210> 12

<211> 537

<212> PRT

<213> Danio rerio

<400> 12

Met Glu Thr Glu Glu Ala Gln Glu Met Ser Gln Ile Thr Gly Arg Asp

1 5 10 15

Ser Pro Met Asn Ala Asn Glu Gly Gly Glu Asp Gln Asp Glu Ala Met

20 25 30

Pro Val Pro Glu Asp Leu Ser Ala Ser Thr Gly Leu Gln His Asn Asn

35 40 45

Arg Thr Asp Lys Pro Leu Ala Cys Asn Ile Lys Val Glu Ala Arg Ser

50 55 60

Asp Glu Glu Asn Gly Leu Ser Cys Glu Met Asn Gly Glu Ala Glu Glu

65 70 75 80

Cys Ala Ala Glu Asp Leu Arg Ile Leu Asp Gly Ser Gly Ala Lys Val

85 90 95

Asn Gly Ser His Ala Gly Pro Asp Ser Lys Pro Ala Ala Tyr Pro Thr

100 105 110

Ala Gly Gly Ile Arg Leu Pro Asn Gly Lys Leu Lys Cys Asp Ile Cys

115 120 125

Gly Ile Val Cys Ile Gly Pro Asn Val Leu Met Val His Lys Arg Ser

130 135 140

His Thr Glu Glu Arg Lys Ser Val Leu Glu Gln Gln Lys Gly Glu Arg

145 150 155 160

Pro Phe Gln Cys Asn Gln Cys Gly Ala Ser Phe Thr Gln Lys Gly Asn

165 170 175

Leu Leu Arg His Ile Lys Leu His Ser Gly Glu Lys Pro Phe Lys Cys

180 185 190

His Leu Cys Asn Tyr Ala Cys Arg Arg Arg Asp Ala Leu Thr Gly His

195 200 205

Leu Arg Thr His Ser Val Gly Lys Pro His Lys Cys Ala Tyr Cys Gly

210 215 220

Arg Ser Tyr Lys Gln Arg Ser Ser Leu Glu Glu His Lys Glu Arg Cys

225 230 235 240

His Asn Tyr Leu Gln Cys Met Gly Leu Gln Asn Ser Ile Tyr Thr Val

245 250 255

Lys Glu Glu Asn Ser Gln Asn Glu Gln Arg Glu Asp Met Pro Ala Ser

260 265 270

Glu Arg Ala Leu Val Leu Asp Arg Ile Ala Asn Asn Val Ala Lys Arg

275 280 285

Lys Ser Ser Met Pro Gln Arg Phe Val Gly Glu Asn Arg Leu Ser Glu

290 295 300

Leu Ser Phe Glu Ser Gly Ser Gly Glu Leu Met Gln Pro His Val Ile

305 310 315 320

Asp Gln Ala Ile Asn Ser Ala Ile Ser Tyr Leu Gly Ala Glu Ser Leu

325 330 335

Arg Pro Leu Val Gln Thr Ser Pro Gly Ser Ala Asp Met Val Val Ser

340 345 350

Pro Leu Tyr Asn Leu His Lys Ser Gln Thr Ala Glu Gly Asn Gly Val

355 360 365

Ser Ala Lys Asp Ser Ala Ala Glu His Leu Leu Leu Leu Ser Lys Ser

370 375 380

Lys Ser Ala Ser Val Asp Lys Asp Gly Ser Pro Ser Pro Ser Gly Gln

385 390 395 400

Asp Ser Thr Asp Thr Glu Ser Asn Asn Glu Glu Arg Ser Ala Gly Val

405 410 415

Ser Gly Thr Ala Ala Thr Gly Gly Leu Ile Tyr Leu Thr Asn His Met

420 425 430

Ala Pro Gly Met Arg Asn Gly Gly Leu Pro Gly Val Lys Glu Glu Gln

435 440 445

Gln Arg His Phe Glu Ala Leu Arg Ala Ala Gly Met Asp Leu Ser Ile

450 455 460

Ala Ser Ser Glu Gly Phe Lys Val Leu Ser Gly Asp Gly Glu Glu Leu

465 470 475 480

Arg Ala Tyr Arg Cys Ile His Cys Arg Val Leu Phe Leu Asp His Val

485 490 495

Met Tyr Thr Ile His Met Gly Cys His Gly Phe Arg Asp Pro Phe Glu

500 505 510

Cys Asn Leu Cys Gly Tyr Arg Ser Gln Asp Arg Tyr Glu Phe Ser Ser

515 520 525

His Ile Thr Arg Gly Glu His Arg Ile

530 535

<210> 13

<211> 641

<212> PRT

<213> Danio rerio

<400> 13

Met Ala Arg Arg His Arg His Ser Val Tyr Ser Ser Asp Glu Asp Asp

1 5 10 15

Asp Asp Val Glu Ile Tyr Asp His Asp Tyr Asp Gly Pro His Ala Lys

20 25 30

Thr Gly Lys Arg His Leu Gly Lys Thr Arg Trp Thr Arg Glu Glu Asp

35 40 45

Glu Lys Leu Lys Arg Leu Val Glu His His Gly Ser Glu Asp Trp Lys

50 55 60

Val Ile Ala Ser Phe Leu Pro Asn Arg Thr Asp Val Gln Cys Gln His

65 70 75 80

Arg Trp Gln Lys Val Leu Asn Pro Glu Leu Ile Lys Gly Pro Trp Thr

85 90 95

Lys Glu Glu Asp Gln Arg Val Ile Glu Leu Val Gln Lys Tyr Gly Pro

100 105 110

Lys Arg Trp Ser Val Ile Ala Lys His Leu Lys Gly Arg Ile Gly Lys

115 120 125

Gln Cys Arg Glu Arg Trp His Asn His Leu Asn Pro Glu Val Lys Lys

130 135 140

Thr Ser Trp Thr Glu Glu Glu Asp Gln Ile Ile Tyr Gln Ala His Glu

145 150 155 160

Lys Leu Gly Asn Arg Trp Ala Glu Ile Ala Lys Leu Leu Pro Gly Arg

165 170 175

Thr Asp Asn Ala Ile Lys Asn His Trp Asn Ser Thr Met Arg Arg Lys

180 185 190

Val Glu Gln Glu Gly Tyr Leu Gln His Ala Ala Lys Val Ser Pro Thr

195 200 205

Pro Leu Asn Asn Ser Tyr Ser Lys Pro His Leu Leu Asn Tyr Asn His

210 215 220

Thr Pro Ser Asn Thr Ser Met Pro Ala Ser Ser Met Ser Asn Gln Tyr

225 230 235 240

Pro Tyr Tyr Thr Glu Ser Ser Arg Val Pro Phe Pro Leu Ala Leu Gln

245 250 255

Leu Asn Ile Leu Asn Phe Pro Gln His Gly Thr Ala Ala Ile Gln Arg

260 265 270

His Tyr Ser Asp Glu Asp Pro Glu Lys Glu Lys Arg Val Lys Glu Ile

275 280 285

Glu Met Leu Leu Met Ser Thr Glu Asn Glu Leu Lys Gly Gln Gln Ala

290 295 300

Leu Pro Ile Ser Met Asn Gly Tyr Gly Gly Trp Asn Arg Gly Ser Leu

305 310 315 320

Ala Asp Ser Ser Val Gly Ile Val Val Ser Val Pro Ala Pro Ser Leu

325 330 335

Glu Gln Gly Cys Leu Pro Glu Glu Ser Ala His Gly Asn Thr Asn Ser

340 345 350

Pro Asn Asn Asp Ser Ser Ser Thr Leu Pro Glu Phe Ile Asp Ala Ile

355 360 365

Asp Ser Ala Pro Ser Val Trp Ala Asn Lys His Pro Lys Thr Glu Cys

370 375 380

Gln Thr Gly Gln Gly Ser Pro Ala Asn Asn Gly Asn Arg Ser Ala Pro

385 390 395 400

Thr Phe Leu Cys Ser Thr Pro Lys Tyr Ser Ser Val Arg His Leu Pro

405 410 415

Phe Ser Pro Thr Gln Phe Phe Asn Ala Ser Val Ser Pro Asp Ser Asp

420 425 430

Ser Gln Asn Ile Pro Val Thr Pro Ser Pro Val Cys Ser Gln Lys Ala

435 440 445

Leu Gln Gln Asp Leu Ala Leu Arg Pro Gln Lys Glu Asn Glu Leu Phe

450 455 460

Arg Thr Pro Asn Leu Arg Arg Ser Ile Met Glu Cys Ser Pro Arg Thr

465 470 475 480

Pro Thr Pro Phe Lys Ser Thr Leu Thr Met Gln Asp Ser Lys Tyr Gly

485 490 495

Pro Leu Lys Arg Val His Ser Pro Ser Leu Asp Ser Gly Val Val Gly

500 505 510

Thr Ile Lys Gln Glu Pro Gln Glu Cys Glu Ile Ser Val Gly Gly Val

515 520 525

His Leu Asp Gln Pro Pro Leu Lys Lys Ile Lys Gln Glu Val Glu Ser

530 535 540

Val Cys Leu Gln Trp Glu Gly Gln Asp Leu His Thr Gln Leu Phe Pro

545 550 555 560

Ser Asn Gly Pro Ala His Asp Met Pro Asp Leu Leu Thr Ser Ser Val

565 570 575

Leu Met Leu Pro Asn Thr Glu Lys Thr Glu Asp Gly His Lys Ala Ala

580 585 590

Gln Leu Pro Arg Arg Pro Ile Gly Ser Pro Leu Gln Gln Leu Asn Thr

595 600 605

Trp Glu Gln Val Leu Cys Gly Lys Thr Glu Glu Gln Thr Ile Pro Ser

610 615 620

Glu Ala Thr His Lys Tyr Leu Ser Asn Tyr Ser Ser Arg Ala Leu Val

625 630 635 640

Ile

<210> 14

<211> 57

<212> PRT

<213> Danio rerio

<400> 14

Met Glu Thr Glu Glu Ala Gln Glu Met Ser Gln Ile Thr Gly Arg Asp

1 5 10 15

Ser Pro Met Asn Ala Asn Glu Gly Gly Glu Asp Gly Met Arg Pro Cys

20 25 30

Leu Phe Leu Lys Thr Cys Gln Gln Ala Leu Ala Ser Asn Thr Thr Ile

35 40 45

Ala Gln Ile Asn His Trp Pro Val Ile

50 55

<210> 15

<211> 346

<212> PRT

<213> Danio rerio

<400> 15

Met Ala Arg Arg His Arg His Ser Val Tyr Ser Ser Asp Glu Asp Asp

1 5 10 15

Asp Asp Val Glu Ile Tyr Asp His Asp Tyr Asp Gly Pro His Ala Lys

20 25 30

Thr Gly Lys Arg His Leu Gly Lys Thr Arg Trp Thr Arg Glu Glu Asp

35 40 45

Glu Lys Leu Lys Arg Leu Val Glu His His Gly Ser Glu Asp Trp Lys

50 55 60

Val Ile Ala Ser Phe Leu Pro Asn Arg Thr Asp Val Gln Cys Gln His

65 70 75 80

Arg Trp Gln Lys Val Leu Asn Pro Glu Leu Ile Lys Gly Pro Trp Thr

85 90 95

Lys Glu Glu Asp Gln Arg Val Ile Glu Leu Val Gln Lys Tyr Gly Pro

100 105 110

Lys Arg Trp Ser Val Ile Ala Lys His Leu Lys Gly Arg Ile Gly Lys

115 120 125

Gln Cys Arg Glu Arg Trp His Asn His Leu Asn Pro Glu Val Lys Lys

130 135 140

Thr Ser Trp Thr Glu Glu Glu Asp Gln Ile Ile Tyr Gln Ala His Glu

145 150 155 160

Lys Leu Gly Asn Arg Trp Ala Glu Ile Ala Lys Leu Leu Pro Gly Arg

165 170 175

Thr Asp Asn Ala Ile Lys Asn His Trp Asn Ser Thr Met Arg Arg Lys

180 185 190

Val Glu Gln Glu Gly Tyr Leu Gln His Ala Ala Lys Val Ser Pro Thr

195 200 205

Pro Leu Asn Asn Ser Tyr Ser Lys Pro His Leu Leu Asn Tyr Asn His

210 215 220

Thr Pro Ser Asn Thr Ser Met Pro Ala Ser Ser Met Ser Asn Gln Tyr

225 230 235 240

Pro Tyr Tyr Thr Glu Ser Ser Arg Val Pro Phe Pro Leu Ala Leu Gln

245 250 255

Leu Asn Ile Leu Asn Phe Pro Gln His Gly Thr Ala Ala Ile Gln Arg

260 265 270

His Tyr Ser Asp Glu Asp Pro Glu Lys Glu Lys Arg Val Lys Glu Ile

275 280 285

Glu Met Leu Leu Met Ser Thr Glu Asn Glu Leu Lys Gly Gln Gln Ala

290 295 300

Leu Pro Phe Pro His Pro Asp Leu His Glu Arg Leu Arg Trp Leu Glu

305 310 315 320

Gln Arg Leu Phe Gly Gly Gln Phe Gly Trp His Cys Cys Phe Ser Pro

325 330 335

Gly Ser Ile Pro Arg Ala Arg Met Pro Ser

340 345

Claims (4)

1. A method for constructing an animal model of granulocytopenia, comprising the steps of, in order:

s1: obtaining F0 generation zebra fish; the F0 generation zebra fish comprises heterozygotes of single gene mutationikzf1 ^+/- Heterozygotes of lines and single gene mutationscmyb ^+/- Strain;

s2: single gene mutantikzf1 ^+/- Strains and single gene mutationscmyb ^+/- Mating the strain to obtain F1 generation zebra fish; the F1 generation zebra fish comprises heterozygotes with double gene mutationsikzf1 ^+/- cmyb ^+/- Strain;

S3：ikzf1 ^+/- cmyb ^+/- the strain is selfed to obtain F2 generation zebra fish; screening from said F2 zebra fishikzf1 ^-/- cmyb ^-/- Obtaining a granulocytopenia animal model;

wherein, + represents a wild type allele, -represents a mutant allele; mutant alleles were obtained from wild type alleles after gene knockout.

2. The method for constructing an animal model for agranulocytosis as claimed in claim 1, wherein,ikzf1 ^-/- cmyb ^-/- the strain isikzf1Gene and genecmybZebra fish lines in which all alleles of the gene are knocked out; the saidikzf1The cDNA sequence of the wild allele of the gene is shown as SEQ ID NO.1The saidcmybThe cDNA sequence of the wild type allele of the gene is shown as SEQ ID NO. 2.

3. The method for constructing an animal model for agranulocytosis according to claim 2, wherein the following steps are performedikzf1The cDNA sequence of the mutant allele of the gene is shown as SEQ ID NO.3, whichcmybThe cDNA sequence of the mutant allele of the gene is shown as SEQ ID NO. 4.

4.ikzf1Gene and genecmybThe application of the gene in constructing the zebra fish model of the granulocytopenia is characterized in thatikzf1All alleles of a gene and thecmybAll alleles of the gene were knocked out.

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