Congenital pure red blood cell aplastic anemia zebrafish model construction method

Abstract

The invention relates to a construction method of a congenital pure red blood cell aplastic anemia zebrafish model, which comprises the following steps: directionally mutating a bysl gene in a wild type zebrafish embryo to obtain F0 generation zebrafish; mating the F0 generation zebrafish with wild type zebrafish, and screening out positive heterozygote F1 generation zebrafish from the born zebrafish; mating the positive heterozygote F1-generation zebrafish with wild type zebrafish, and screening out positive heterozygote F2-generation zebrafish from the born zebrafish; and hybridizing the male and female positive heterozygote F2-generation zebrafish, and screening positive homozygote zebrafish from the born zebrafish as the congenital pure red blood cell aplastic anemia zebrafish model. The model can simulate the symptoms of the congenital pure red blood cell aplastic anemia, and can be used for mechanism research, drug evaluation and new drug screening of the congenital pure red blood cell aplastic anemia.

Description

Technical Field

[0001] The present application relates to the field of genetic engineering, and in particular to a method for constructing a zebrafish model of congenital pure red blood cell aplasia. Background Art

[0002] Congenital pure red blood cell aplasia, also known as Diamond-Blackfan anemia, or DBA for short, is one of the first batch of rare diseases announced by the National Health Commission in 2018. The main characteristics of patients are congenital red blood cell dysplasia (anemia), congenital malformations, and high incidence of blood tumors. The cause of DBA is the mutation of ribosome-related genes. In 1999, the ribosomal gene RPS19 was first reported to be associated with the onset of DBA. RPS19 is also the gene with the highest mutation frequency in DBA patients, accounting for about 25%. Others include 22 genes such as RPS24, RPS17, and RPL35A. However, there are still about 20%-25% of patients who cannot be diagnosed with mutated genes. Diverse pathogenic genes correspond to diverse pathogenic mechanisms. However, the animal models for congenital pure red blood cell aplasia currently only involve a limited number of genes such as RPS19, RPL11, RPL5, RPS24, RPS7, RPS20, and GATA1, which are not enough to explain the complexity of the pathogenesis of congenital pure red blood cell aplasia. At the same time, it cannot meet the needs of targeted drug screening for different types of congenital pure red blood cell aplasia.

[0003] Therefore, it is necessary to construct a new gene congenital pure red blood cell aplasia animal model. Summary of the invention

[0004] Based on this, one embodiment of the present application provides a method for constructing a zebrafish model of congenital pure red blood cell aplasia.

[0005] The technical solution includes:

[0006] A method for constructing a zebrafish model of congenital pure red blood cell aplasia, comprising:

[0007] Directed mutation of the bysl gene in wild-type zebrafish embryos to obtain F0 generation zebrafish;

[0008] The F0 generation zebrafish are mated with wild-type zebrafish, and positive heterozygous F1 generation zebrafish are screened out from the born zebrafish;

[0009] The positive heterozygous F1 generation zebrafish is mated with wild-type zebrafish, and positive heterozygous F2 generation zebrafish are screened out from the born zebrafish; and,

[0010] The positive heterozygous F2 generation zebrafish are hybridized, and positive homozygous zebrafish are screened out from the born zebrafish as a zebrafish model of congenital pure red blood cell aplasia.

[0011] In one embodiment, directed mutation of the bysl gene in wild-type zebrafish embryos comprises the following steps:

[0012] Prepare sgRNA targeting exon 1 of zebrafish bysl gene, wherein the sequence of the sgRNA is shown in SEQ ID NO.1 and SEQ ID NO.2;

[0013] mixing the sgRNA and Cas9 protein to prepare a Cas9 / sgRNA mixture; and

[0014] The Cas9 / sgRNA mixture was introduced into wild-type zebrafish embryos.

[0015] In one embodiment, the Cas9 / sgRNA mixture is introduced into wild-type zebrafish embryos by microinjection.

[0016] The construction method, the methods used for screening positive heterozygous F1 generation zebrafish, screening positive heterozygous F2 generation zebrafish and screening positive homozygous zebrafish are independently selected from PCR method and Sanger sequencing method.

[0017] In one embodiment, the PCR method uses the detection primer pair shown in SEQ ID NO.6 to SEQ ID NO.7.

[0018] The tissue or organ of the aplastic anemia zebrafish model established by the method for constructing the congenital pure red blood cell aplastic anemia zebrafish model.

[0019] A kit for constructing a congenital pure red blood cell aplasia zebrafish model, comprising a sgRNA targeting exon 1 of the zebrafish bysl gene; the sequence of the sgRNA is shown in SEQ ID NO.1 to SEQ ID NO.2.

[0020] In one embodiment, it also includes one or more of Cas9 protein, PCR reaction buffer and PCR reaction solution.

[0021] In one embodiment, the PCR reaction solution includes Taq DNA polymerase, dNTPs and Mg 2+ One or more of .

[0022] The application of the congenital pure red blood cell aplasia zebrafish model constructed by the construction method in drug screening.

[0023] Compared with the traditional technology, this application has the following beneficial effects:

[0024] The present application constructs a zebrafish model of congenital pure red blood cell aplasia based on bysl gene knockout, which simulates the symptoms of congenital pure red blood cell aplasia and shares a common pathogenic basis with the disease. The model can not only be used for screening and corroboration of new pathogenic genes of congenital pure red blood cell aplasia, but also can be used for mechanism research, drug evaluation and new drug screening of the disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application and to more completely understand the present application and its beneficial effects, the following is a brief introduction to the drawings required for the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present application, and those skilled in the art can obtain other drawings based on these drawings without creative work.

[0026] Figure 1 Prediction results for the gene structure (Figure A), sgRNA target site (Figure A), mutant form (Figure B), and protein translation (Figure C) of bysl.

[0027] Figure 2 Zebrafish homozygous for bysl mutants exhibit hypoplastic head and eyes (Figure B) and cardiac edema (Figure A).

[0028] Figure 3 In situ hybridization experiments were performed using RNA probes for cmyb (Figure A) and hbae1.1 (Figure B) to label hematopoietic stem cells and red blood cells, respectively.

[0029] Figure 4 The qPCR experiment was used to detect the expression results of p53 and its downstream mdm2, p21, ccng1, casp8, and casp10 in bysl homozygous mutants.

[0030] Figure 5 Figure 1 shows abnormal ribosome synthesis in the bysl zebrafish homozygous mutant; Figure A shows the results of transcriptome analysis, Figure B shows the results of qPCR experiment analysis, and Figures C and D show the results of capillary electrophoresis experiment analysis. DETAILED DESCRIPTION

[0031] In order to make the above-mentioned purposes, features and advantages of the present application more obvious and easy to understand, the specific implementation methods of the present application are described in detail below. In the following description, many specific details are set forth to facilitate a full understanding of the present application. However, the present application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present application, so the present application is not limited by the specific embodiments disclosed below.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0033] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0034] The main English explanations involved in this article are as follows:

[0035] CRISPR / Cas9: Clustered Regularly Interspersed Short PalindromicRepeats / Cas, 9 is a system that uses Cas9 protein for gene editing.

[0036] dpf: days post fertilization, hours after fertilization.

[0037] rRNA: ribosomal RNA, ribosomal RNA, is the backbone structure of the ribosome.

[0038] sgRNA: single guide RNA, used in combination with Cas9 protein, can guide the Cas9 protein to locate to its targeted DNA sequence to achieve editing.

[0039] qPCR: Quantitative Real-time PCR, real-time fluorescence quantitative PCR, is a method for real-time monitoring and analysis of PCR amplification products.

[0040] cDNA: complementary DNA, specifically refers to the DNA chain that is complementary to RNA after reverse transcription in vitro.

[0041] The bysl gene is highly conserved in eukaryotes. It has been shown to regulate the processing of 18S ribosomal RNA and participate in the in vivo synthesis of ribosomes. In tumor cells and mice, bysl can regulate cell growth and embryonic development. So far, there has been no report of an animal model that knocks out the bysl gene through the CRISPR / Cas9 system to study the function of the bysl gene in the development of the blood system. Zebrafish is a good model animal for studying the development of the blood system. Its blood system development process is similar to that of humans. In addition, zebrafish embryos develop in vitro, the embryos are transparent, and there are transgenic strains with various blood cell lineages, which makes it easy to use imaging technology to directly observe the growth, differentiation and migration of blood cells. In addition, zebrafish have a large egg-laying volume and low breeding costs, which are suitable for drug screening. Therefore, the zebrafish model is becoming more and more popular in the mechanism research and drug screening of blood system diseases.

[0042] One embodiment of the present application provides a method for constructing a zebrafish model of congenital pure red blood cell aplasia, comprising:

[0043] Directed mutation of the bysl gene in wild-type zebrafish embryos to obtain F0 generation zebrafish;

[0044] The F0 generation zebrafish are mated with wild-type zebrafish, and positive heterozygous F1 generation zebrafish are screened out from the born zebrafish;

[0045] mating the positive heterozygous F1 generation zebrafish with wild-type zebrafish, and screening out positive heterozygous F2 generation zebrafish from the born zebrafish; and

[0046] The positive heterozygous F2 generation zebrafish are hybridized, and positive homozygous zebrafish are screened out from the born zebrafish as a zebrafish model of congenital pure red blood cell aplasia.

[0047] In a specific example, the CRISPR / Cas9 system was used to perform targeted mutations on the bysl gene in wild zebrafish embryos to obtain bysl zebrafish mutants that simulated the symptoms of congenital pure red blood cell aplasia. This method has the advantages of short cycle, stability, low cost and good repeatability.

[0048] Directed mutation of the bysl gene in wild-type zebrafish embryos can reduce the expression of the bysl gene or protein and / or inactivate its function.

[0049] Specifically, the method of mutating the bysl gene in wild-type zebrafish embryos includes the following steps: preparing an sgRNA targeting exon 1 of the zebrafish bysl gene, wherein the sequence of the sgRNA is shown in SEQ ID NO.1 and SEQ ID NO.2; mixing the sgRNA with the Cas9 protein to prepare a Cas9 / sgRNA mixture; and introducing the Cas9 / sgRNA mixture into wild-type zebrafish embryos.

[0050] In one specific example, the Cas9 / sgRNA mixture is introduced into wild-type zebrafish embryos by microinjection.

[0051] In a specific example, PCR and Sanger sequencing methods are used to screen out positive heterozygous F1 generation zebrafish or screen out positive heterozygous F2 generation zebrafish or screen out positive homozygous zebrafish.

[0052] In a specific example, the PCR method uses a detection primer pair as shown in SEQ ID NO.6 to SEQ ID NO.7.

[0053] One embodiment of the present application also provides tissues or organs of a zebrafish model of aplastic anemia established by the above-mentioned method for constructing a zebrafish model of congenital pure red blood cell aplasia.

[0054] One embodiment of the present application also provides a kit for constructing a zebrafish model of congenital pure red blood cell aplasia, comprising an sgRNA targeting exon 1 of the zebrafish bysl gene.

[0055] In a specific example, the sequence of sgRNA is shown as SEQ ID NO.1 and SEQ ID NO.2.

[0056] In a specific example, it also includes one or more of Cas9 protein, PCR reaction buffer and PCR reaction solution.

[0057] In a specific example, the PCR reaction solution includes Taq DNA polymerase, dNTPs and Mg 2+ One or more of .

[0058] The construction method of the present application not only provides a new animal model for the study of the mechanism of congenital pure red blood cell aplasia, helps to reveal the screening and evidence of new pathogenic genes, but also provides a stable, living, reproducible, high-success-rate, and low-cost animal model for drug screening of congenital pure red blood cell aplasia.

[0059] One embodiment of the present application also provides the use of the congenital pure red blood cell aplasia zebrafish model constructed by the above-mentioned construction method in drug screening.

[0060] The embodiments of the present application will be described in detail below in conjunction with examples. It should be understood that these examples are only used to illustrate the present application and are not intended to limit the scope of the present application. The experimental methods for which specific conditions are not specified in the following examples are preferably referred to the guidance provided in the present application, and can also be based on the experimental manual or normal conditions in this area, can also be based on the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0061] In the following specific embodiments, the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified. For temperature and time parameters, acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.

[0062] Example 1

[0063] 1. Model Construction

[0064] 1. Determine the sequence of the sgRNA targeting bysl

[0065] In order to knock out the zebrafish bysl gene, the CRISPRscan website (https: / / www.crisprscan.org / ) was used to predict the sgRNA targeting bysl. In order to improve the efficiency of knocking out bysl, two sgRNAs were selected at the same time and named bysl sgRNA #1 and bysl sgRNA #2. The targeting sequences of these two sgRNAs are sgRNA #1: 5'-GTGTCCTGCGCTCCTCCT-3' (SEQ ID NO.1); sgRNA #2: 5'-GATGAGAAGCTGAGCCGG-3' (SEQ ID NO.2). Both sgRNA targeting sequences are located on exon 1 of the bysl gene and in the coding region of bysl. Subsequently, primers for sgRNA in vitro synthesis were synthesized according to the targeting sequences of sgRNA (all primers used were ordered from Beijing Qingke Biotechnology Co., Ltd.), and the primer sequences are:

[0066] bysl sgRNA #1 fw (SEQ ID NO.3):

[0067] 5'-taatacgactcactataGGGTGTCCTGCGCTCCTCCTgttttagagctagaa-3';

[0068] bysl sgRNA #2 fw (SEQ ID NO.4):

[0069] 5'-taatacgactcactataGGGATGAGAAGCTGAGCCGGgttttagagctagaa-3';

[0070] Tail primer (SEQ ID NO.5):

[0071] 5'-AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC-3'.

[0072] 2. Synthesize the DNA template of bysl sgRNA.

[0073] According to the instructions, add an appropriate amount of distilled water to the obtained sgRNA primers to make the primer concentration 10 mM. Then prepare the PCR system according to the ratio shown in Table 1:

[0074] Table 1

[0075]

[0076] The reaction mixture was then placed in a PCR instrument (Thermo Fisher, Catalog No.: 4375786) and reacted according to the program shown in Table 2:

[0077] Table 2

[0078]

[0079] After the reaction, the product was detected by agarose gel electrophoresis. The product should be a single band of about 117 bp (base pair). The product was then purified using a PCR purification kit (Yisheng Biotechnology (Shanghai) Co., Ltd., Catalog No.: 19106ES50). The product concentration was detected using a NanoDrop micro-spectrophotometer (Thermo Fisher, Catalog No.: ND-2000).

[0080] 3. Synthesize sgRNA

[0081] Transcription was performed using the T7 RNA transcription kit (Nanjing Novogene Biotech Co., Ltd., Catalog No.: TR101-01) using the purified PCR product as a template. The transcription reaction was performed according to the instructions. After transcription, the transcription product was detected by agarose gel electrophoresis, and the product should be a single band. The sgRNA was then purified. First, 1 / 10 volume of ammonium acetate solution (5 M) was added to the transcription system, and water was added to make the total volume 150 μl. Then add an equal volume of phenol-chloroform-isoamyl alcohol mixture (Sigma Aldrich, catalog number: 77617), vortex thoroughly, centrifuge at 12000g for 30 seconds, separate the liquid, aspirate the upper aqueous phase, transfer to a new centrifuge tube, add 1150 μl of chloroform again, vortex thoroughly, centrifuge at 12000g for 30 seconds, separate the liquid, aspirate the upper aqueous phase, transfer to a new centrifuge tube, add 150 μl of isopropanol, mix well, place in a -80℃ refrigerator, and freeze for at least 30 minutes. Then, centrifuge at 12000g for 15 minutes in a centrifuge precooled to 4℃, discard the supernatant, retain the precipitate, add 1 ml of 70% ethanol, centrifuge at 12000g for 15 minutes, discard the supernatant, and retain the precipitate. Then open the lid, place at room temperature, wait for the alcohol to evaporate (about 5 minutes), and add 20 μl of pure water without DNase or RNase. The sgRNA concentration was measured using a NanoDrop micro-spectrophotometer and stored at -80°C.

[0082] 4. Construction of F0 generation bysl mutant zebrafish

[0083] A mixture of Cas9 protein (GenScript Biotech, Cat. No. Z03469) and sgRNA was prepared to a final concentration of 100 ng / μl for Cas9 protein and 50 ng / μl for sgRNA. Subsequently, 2 nl of the mixture was injected into each wild-type embryo (one-cell stage) using a microinjector (Harvard Apparatus, Cat. No. PLI-100A) at the yolk sac of the embryo. After the injection, wait for the embryos to develop to 24 hpf (hours postfertilization), collect 10 embryos, dry them, add 100 μl of NaOH (50 mM), place them in a metal bath at 95°C, incubate for 20 minutes, and then add 10 μl of Tris pH7.5 (10 mM) to neutralize the lysate. Mix well and use it as a DNA template for PCR reaction. The primers used to detect bysl target mutations are bysl fw: 5'-CGCGATTTCACACTCATAATT-3' (SEQ ID NO.6), bysl rv: 5'-AGTCAGTGAGTGCAGCATTCA-3' (SEQID NO.7). After PCR, the PCR product was detected by agarose gel electrophoresis, and the size of the PCR product should be 326 bp. The PCR product was then Sanger sequenced (sequencing service provided by Beijing Qingke Biotechnology Co., Ltd.), and the sequencing primer used was bysl fw. After the bysl gene was successfully knocked out, the sequencing results should show different and chaotic sequences starting from the target site. Finally, the remaining embryos were raised to adulthood.

[0084] 5. Screening of F0 generation bysl mutant zebrafish

[0085] In order to screen out F0 individuals containing the heritable bysl mutant allele, we mated the F0 sexually mature individuals with the wild type and collected the embryos produced. Then, 10 embryos were lysed as DNA templates for PCR, and PCR was performed using bysl fw and bysl rv primers. The PCR products were detected by agarose gel electrophoresis and then Sanger sequencing was performed. If multiple sequences appeared at the target site, it was considered that the parents of these embryos were F0 individuals with bysl mutations. The remaining embryos were then raised to adulthood, which was the F1 generation of bysl mutant zebrafish.

[0086] 6. Screening of F1 generation bysl mutant zebrafish

[0087] To identify the F1 individuals with bysl mutations, we used pointed tweezers to take scales from the sides of the body of different F1 individuals, took 2-3 scales, placed them in a centrifuge tube, added 20 μl NaOH (50 mM), placed them in a metal bath at 95°C, incubated them for 20 minutes, and then added 10 μl Tris pH7.5 (10 mM) to neutralize the lysate. After mixing evenly, it was used as a DNA template for PCR reaction. PCR was performed using bysl fw and bysl rv primers, and the PCR products were detected by agarose gel electrophoresis and then Sanger sequencing. By comparing with the existing bysl gene sequence (the sequence comes from the National Center for Biotechnology Information database, also known as the NCBI database, https: / / www.ncbi.nlm.nih.gov / , Gene ID: 394081), the mutant form of bysl was read out, in which the number of inserted or deleted base pairs is not a multiple of three, which will cause the translation reading frame of mRNA to shift, so it is considered to be an effective knockout form. Among them, a mutant form with a deletion of 58 bp was identified. The heterozygous mutant was then mated with wild-type zebrafish for expansion, and the same method was used to screen and identify the F2 generation bysl mutant zebrafish.

[0088] 7. Morphological and developmental phenotype analysis of bysl homozygous mutants

[0089] In order to analyze the effect of complete knockout of bysl on embryonic development, bysl heterozygous mutants were mated and bysl homozygous mutants were obtained through genotyping. The development of the embryo's head, eyes, heart and other organs was analyzed under a microscope. These organs are commonly affected organs in patients with congenital pure red blood cell aplasia. After analysis, bysl homozygous mutants showed morphological developmental defects such as dysplasia of the head and eyes, and pericardial edema.

[0090] 8. Analysis of hematopoietic stem cells and erythrocytes of bysl homozygous mutants

[0091] Congenital pure red blood cell aplasia is characterized by a decrease in hematopoietic stem cells and anemia, so the hematopoietic stem cells and erythrocytes of the homozygous bysl mutant were analyzed. RNA whole embryo in situ hybridization was used, and RNA probes of cmyb (marking hematopoietic stem cells) and hbae1.1 (marking erythrocytes) were used for labeling, and then photographed under a microscope. The experimental steps of RNA whole embryo in situ hybridization are as follows: First, the RNA probes of cmyb and hbae1.1 were synthesized according to the instructions using the T7 RNA transcription kit and DIG RNA labeling mixture (Roche, catalog number: 11277073910), and then purified according to the instructions and diluted to a concentration of 1 ng / μl with prehybridization solution (50% formamide, 500μg / ml yeast RNA, 50μg / ml heparin, 5× SSC buffer) for use. Then, embryos at 4 dpf (days post fertilization) were collected, fixed overnight at 4°C with 4% paraformaldehyde, and then rinsed 3 times with PBST (PBS buffer containing 0.1% Tween-20), 5 minutes each time, and then proteinase K solution (20 μg / ml, Thermo Fisher, catalog number: EO0491) was added to the embryos, placed in a 37°C metal bath, heated for 35 minutes, and then fixed again with 4% paraformaldehyde at room temperature for 20 minutes, and then rinsed 3 times with PBST, 5 minutes each time. Finally, prehybridization solution was added, incubated at 60°C for 2 hours, and then the prehybridization solution was removed, the probe was added, and incubated at 60°C overnight. On the second day, wash solution 1 (50% formamide, 4× SSC solution, 0.1% Tween-20), wash solution 2 (4× SSC solution, 0.1% Tween-20), and wash solution 3 (0.4× SSC solution, 0.1% Tween-20) were used to incubate at 60°C for 2 times, each time for 30 minutes. Then, embryos were rinsed 3 times at room temperature with maleic acid buffer (MAB, pH 7.5, containing 0.1% Tween-20), and blocking solution (4% BSA solution) was added, and incubated at room temperature for 2 hours. Finally, anti-DIG-AP solution (1:2500 dissolved in 4% BSA solution, Roche, catalog number: 11093274910) was used to incubate embryos overnight at 4°C. Finally, the embryos were rinsed three times with PBST for 30 minutes each time, and incubated with equilibration buffer (25 mM NaOH, 25 mM Tris-HCl pH 9.5, 0.1% Tween-20, 50 mM MgCl2) for 10 minutes. Finally, the embryos were developed in NBT / BCIP colorimetric solution (1:50 diluted in equilibration buffer, Roche, catalog number: 11681451001) until the signal was strong enough. The reaction was terminated with PBST, and the background color was removed with 70% ethanol.

[0092] The results of in situ hybridization experiments showed that the number of hematopoietic stem cells marked by cmyb and red blood cells marked by Hbae1.1 was significantly reduced in bysl homozygous mutants.

[0093] 9. Detection of p53 signaling pathway in bysl homozygous mutants

[0094] One of the characteristics of congenital pure red blood cell aplasia is the activation of the p53 signaling pathway. To this end, qPCR will be used to detect the expression level of the p53 signaling pathway in bysl homozygous mutants. First, 4 dpf bysl homozygous mutant embryos and control group embryos will be collected, and total RNA will be extracted using TRIzol reagent (Thermo Fisher, Catalog No.: 15596026) according to the instructions. Subsequently, a reverse transcription kit (Takara. Catalog No.: RR047Q) was used to synthesize the cDNA library. Finally, qPCR premix (Beijing Quanshijin Biotechnology Co., Ltd., Catalog No. AQ602) was used to prepare the qPCR reaction system according to the instructions, and the qPCR reaction was performed. The real-time fluorescence quantitative PCR instrument used was LightCycler® 480 (Roche, Catalog No.: 05015278001). The primers for detecting the p53 gene and p53 downstream genes, including mdm2, p21, ccng1, casp8, casp10, etc. are shown in Table 3:

[0095] Table 3

[0096]

[0097] The results of qPCR showed that the expression of p53 and its downstream genes was significantly upregulated and the p53 signaling pathway was activated.

[0098] 10. Transcriptome analysis of bysl homozygous mutants

[0099] In order to better verify the similarity between bysl homozygous mutants and congenital pure red blood cell aplasia, 4 dpf bysl homozygous mutants and control group embryos were collected and transcriptome sequencing was performed. Sample preparation, sequencing experiments and data analysis were all completed by Beijing Novogene Technology Co., Ltd. KEGG signaling pathway analysis results showed that the ribosome signaling pathway was significantly upregulated in bysl homozygous mutants. Abnormal ribosome synthesis is also the cause of congenital pure red blood cell aplasia. To further verify this result, qPCR was used to detect the expression of ribosomal genes. The primers used for qPCR are shown in 4.

[0100] Table 4

[0101]

[0102] The results of transcriptome sequencing and qPCR showed that the expression of ribosome processing-related genes was significantly upregulated in the bysl homozygous mutant.

[0103] 11. Detection of bysl homozygous mutant ribosomal RNA

[0104] Ribosomal RNA is the structural skeleton of ribosomes. The content and ratio of ribosomal RNA can reflect whether the biosynthesis of ribosomes is normal. For this reason, when the embryos developed to 4 dpf, the total RNA of bysl homozygous mutants and the control group was extracted. The total RNA samples were then subjected to capillary electrophoresis and quantification using an Agilent 2100 bioanalyzer to obtain the content of 28S and 18S in the samples and calculate the ratio of the two. In the control group, the value of 28S / 18S was about 1.7, while the ratio of bysl homozygous mutants was about 3.2, indicating that the ribosome processing of bysl homozygous mutants was abnormal.

[0105] 2. Result verification:

[0106] 1. Gene structure, sgRNA target site location, mutation form and protein translation prediction of bysl

[0107] like Figure 1 As shown, zebrafish bysl has 7 exons in total, and the sgRNA targets #1 and #2 designed in this application are located on exon 1 ( Figure 1 Middle A). By knocking out bysl through the CRISPR / Cas9 system, the bysl mutant obtained in this application is a deletion of 58 base pairs ( Figure 1 The mutant form of bysl is predicted to encode a truncated polypeptide chain of 79 amino acids, in which the mutation occurs starting from the 52nd amino acid, while the wild-type bysl gene encodes 422 amino acids ( Figure 1 Middle C).

[0108] 2. Bysl zebrafish homozygous mutants show head and eye dysplasia and cardiac edema

[0109] like Figure 2 As shown in the figure, by analyzing the morphology of bysl zebrafish homozygous mutant embryos at 2 dpf, 4 dpf, and 5 dpf, it was found that compared with control embryos, their heads and eyes began to develop poorly from 2 dpf, and at 5 dpf they also showed obvious pericardial edema ( Figure 2 Middle A). In addition, the craniofacial cartilage is also obviously missing ( Figure 2 Middle B).

[0110] 3. Bysl zebrafish homozygous mutants show hematopoietic stem cell reduction and anemia

[0111] like Figure 3As shown, in situ hybridization experiments using RNA probes of cmyb and hbae1.1 to mark hematopoietic stem cells and red blood cells, respectively, revealed that at 4 dpf, the hematopoietic stem cells and red blood cells of the homozygous bysl mutant were significantly reduced. This is consistent with the characteristics of congenital pure red blood cell aplasia ( Figure 3 A and B).

[0112] 4. Activation of p53 signaling pathway in bysl zebrafish homozygous mutants

[0113] like Figure 4 As shown in the figure, qPCR experiments were used to detect the expression of p53 and its downstream mdm2, p21, ccng1, casp8, and casp10 in bysl homozygous mutants. The results showed that the expression of these genes was significantly upregulated in bysl homozygous mutants. This indicates that the expression of p53 and its downstream genes is significantly upregulated, and the p53 signaling pathway is activated. The activation of the p53 signaling pathway is also one of the characteristics of congenital pure red blood cell aplasia.

[0114] 5. Abnormal ribosome synthesis in bysl zebrafish homozygous mutants

[0115] like Figure 5 As shown in Figure 2, transcriptome analysis revealed that the ribosome synthesis signaling pathway was significantly upregulated in the bysl zebrafish homozygous mutant ( Figure 5 Further qPCR experimental results confirmed that genes related to ribosome synthesis, such as wdr43, rpp30, nmd3, utp4, snu13b, eif6, imp4, rrp7a, and csnk2a4, were significantly upregulated ( Figure 5 (B). Subsequent capillary electrophoresis analysis revealed that the 28S / 18S ratio in the bysl homozygous mutant was abnormal ( Figure 5 C and D), while 28S and 18S are the backbone structures of the ribosome, indicating that the ribosome synthesis of the bysl homozygous mutant is disordered, and abnormal ribosome synthesis is the cause of congenital pure red blood cell aplasia.

[0116] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0117] The above-mentioned embodiments only express several implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent of the present application shall be subject to the attached claims, and the description may be used to interpret the content of the claims.

Claims

1. A method for constructing a zebrafish model of congenital pure red blood cell aplasia, characterized in that: include: Directed mutation of the bysl gene in wild-type zebrafish embryos to obtain F0 generation zebrafish; The F0 generation zebrafish are mated with wild-type zebrafish, and positive heterozygous F1 generation zebrafish are screened out from the born zebrafish; The positive heterozygous F1 generation zebrafish is mated with wild-type zebrafish, and positive heterozygous F2 generation zebrafish are screened out from the born zebrafish; as well as, The male and female positive heterozygous F2 generation zebrafish are hybridized, and positive homozygous zebrafish are screened out from the born zebrafish as a zebrafish model of congenital pure red blood cell aplasia.

2. The construction method according to claim 1, characterized in that: Directed mutagenesis of the bysl gene in wild-type zebrafish embryos includes the following steps: Prepare sgRNA targeting exon 1 of zebrafish bysl gene, wherein the sequence of the sgRNA is shown in SEQ ID NO.1 and SEQ ID NO.2; mixing the sgRNA and the Cas9 protein to prepare a Cas9 / sgRNA mixture; and The Cas9 / sgRNA mixture was introduced into wild-type zebrafish embryos.

3. The construction method according to claim 2, characterized in that: The Cas9 / sgRNA mixture was introduced into wild-type zebrafish embryos by microinjection.

4. The construction method according to any one of claims 1 to 3, characterized in that: The methods used for screening positive heterozygous F1 generation zebrafish, screening positive heterozygous F2 generation zebrafish and screening positive homozygous zebrafish are independently selected from PCR method and Sanger sequencing method.

5. The construction method according to claim 4, characterized in that: The PCR method uses the detection primer pair shown in SEQ ID NO.6 to SEQ ID NO.

7.

6. The tissue or organ of the zebrafish model of aplastic anemia established by the method for constructing a zebrafish model of congenital pure red blood cell aplasia according to any one of claims 1 to 5.

7. A kit for constructing a zebrafish model of congenital pure red blood cell aplasia, characterized in that: It includes an sgRNA targeting exon 1 of the zebrafish bysl gene; the sequence of the sgRNA is shown in SEQ ID NO.1 to SEQ ID NO.

2.

8. The kit according to claim 7, characterized in that It also includes one or more of Cas9 protein, PCR reaction buffer and PCR reaction solution.

9. The kit according to claim 8, characterized in that The PCR reaction solution includes Taq DNA polymerase, dNTPs and Mg 2+ One or more of .

10. Use of the congenital pure red blood cell aplasia zebrafish model constructed by the construction method according to any one of claims 1 to 5 in drug screening.

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