Several eif1a genes and uses thereof

Abstract

The application discloses several Eif1a genes and application thereof. The application firstly finds the function of eif1axb gene in cardiovascular development and skeletal development of zebrafish, and finds that folate can inhibit the expression of zebrafish eif1axb and mouse eif1ad7. The application provides a new use of a zebrafish eif1axb, mouse eif1ad7 and human eif1ax modulator in preparation of a drug for treating cardiovascular development disorder and skeletal defect, and provides an animal model for screening of a drug for preventing or treating cardiovascular development disorder and skeletal defect.

Description

Technical Field

[0001] This invention belongs to the field of gene function and application. Specifically, this invention relates to the function and application of a certain Eif1a gene in birth defects. Background Technology

[0002] Eukaryotic translation initiation factors (eIFs) are a class of proteins involved in the initiation of translation in eukaryotes. Among them, eif1a genes are an important member of the translation initiation factor family and play an irreplaceable role in the translation initiation process. eif1a plays a crucial role in the translation initiation scan. Using a hepatitis C virus model, studies have shown that eif1a is indispensable in both the 5' cap-mediated translation initiation scan and the internal ribosome entry site sequence (IRES)-mediated translation initiation scan.

[0003] During embryonic development, the paternal and maternal genomes undergo initial transcription after fertilization, resulting in zygotic gene activation (ZGA). The timing of ZGA varies among different mammals. For a long time, the mainstream understanding of ZGA has remained unchanged: its main function is to provide the RNA and proteins required for preimplantation genetic testing (PGT) and maintain basic embryonic cell functions, such as the expression of housekeeping genes. eif1a is considered a major marker gene for preimplantation genetic activation in mammals. It has been found to be transiently expressed in the two-cell stage of mouse embryos and has been used as a two-cell stage-specific marker. The short duration of ZGA indicates that the embryo needs to rapidly repress the activation of ZGA. Some researchers have shown that extensive transcriptional activation during ZGA can lead to intrinsic DNA damage, which must be corrected for normal development. However, the specific function of genes like eif1a in ZGA remains unreported, and to date, no literature reports the application of eif1a-related genes in the prevention of birth defects. Summary of the Invention

[0004] This invention discovers the uses of the zebrafish eif1axb, mouse eif1ad7, and human eif1ax genes in early embryonic cardiovascular and skeletal development.

[0005] The human gene eif1ax sequence described in this invention is shown in SEQ ID NO.1, the mouse gene eif1ad7 sequence is shown in SEQ ID NO.2, and the zebrafish gene eif1axb sequence is shown in SEQ ID NO.3.

[0006] The first aspect of this invention provides the application of the gene eif1axb in constructing a model of cardiovascular developmental disorders and skeletal abnormalities in zebrafish.

[0007] A second aspect of this invention provides a method for constructing a zebrafish model of cardiovascular developmental disorders and skeletal abnormalities, by knocking out or inhibiting the zebrafish embryo gene eif1axb to obtain the zebrafish model. The method for preparing the zebrafish model described in this invention can employ conventional methods in the art to knock out or inhibit the gene eif1axb in zebrafish embryos. By adjusting the degree of inhibition or knockout, the severity of the model can be controlled.

[0008] A third aspect of this invention provides the application of the zebrafish model of cardiovascular developmental disorders and skeletal abnormalities constructed according to this invention in the screening of therapeutic drugs for cardiovascular developmental disorders and skeletal abnormalities. In a specific example, it is applied in the screening of therapeutic drugs for cardiovascular developmental disorders and skeletal abnormalities caused by gene eif1axb deficiency.

[0009] The fourth aspect of this invention provides the application of the gene Eif1ad7 in constructing mouse models of congenital cardiovascular disease and congenital heart disease.

[0010] The fifth aspect of the present invention provides a method for constructing a mouse model of congenital cardiovascular disease and congenital heart disease, wherein the method is to obtain the model after the mouse embryo has undergone development in the mother mouse following either suppression or overexpression of the gene Eif1ad7.

[0011] The sixth aspect of this invention provides the application of the mouse model of congenital cardiovascular disease and congenital heart disease constructed according to this invention in the screening of therapeutic drugs for congenital cardiovascular disease and congenital heart disease. In one embodiment, it is used in the screening of therapeutic drugs for congenital cardiovascular disease and congenital heart disease caused by gene eif1ad7 deficiency.

[0012] The method for preparing the zebrafish model described in this invention involves inhibiting the expression of the eif1axb gene in zebrafish embryos, knocking down the corresponding eif1axb gene expression using Morpholino technology, or using eif1axb gene siRNA, eif1axb antibody, or other bioengineering techniques that can inhibit eif1axb expression.

[0013] The mouse model described in this invention is obtained by suppressing or overexpressing the Eif1ad7 gene in mouse embryos, followed by embryonic development in the mother mouse. Further, the gene suppression is achieved by regulating the expression of the eif1ad7 gene in mouse embryos using Morpholino technology or antisense RNA technology.

[0014] The seventh aspect of the present invention also provides the application of reagents for detecting the gene eif1ax in the preparation of diagnostic or auxiliary diagnostic reagents for congenital heart disease.

[0015] For example, the zebrafish eif1axb, mouse eif1ad7, and human eif1ax genes are used in the development of gene screening kits. In one embodiment, a diagnostic reagent for screening congenital heart disease can be provided. This reagent targets eif1ax and contains PCR primers designed based on the eif1ax gene and an RNA storage solution. The method of using this diagnostic reagent includes extracting the target DNA, detecting eif1ax expression via real-time PCR, and assessing the risk of congenital heart disease in newborns by comparing the expression levels with those of normal genes.

[0016] The eighth aspect of the present invention also provides the use of the gene eif1ax as a therapeutic target for a drug for treating congenital heart disease.

[0017] The ninth aspect of the present invention also provides the use of folic acid as a regulator of the zebrafish gene eif1axb, the mouse gene eif1ad7, or the human gene eif1ax, mainly as an inhibitor of the zebrafish gene eif1axb, the mouse gene eif1ad7, or the human gene eif1ax.

[0018] Folic acid, rather than the human endogenous 6S-5-methyltetrahydrofolate, has cardiotoxicity; at certain doses, folic acid can induce developmental disorders and malformations of the heart in zebrafish embryos, while 6S-5-methyltetrahydrofolate does not have the aforementioned negative effects. This invention conducted in vitro experiments on mouse blastocysts cultured with FA (folic acid) and MTHF-Ca (calcium 6S-5-methyltetrahydrofolate). Using gene expression profiling, we found that gm5662 was the most significantly differentially expressed mRNA sequence, and this gene is an unknown, unidentified gene. Gm5662 is an mRNA sequence recorded in the mouse genome database. This invention further identifies gm5662 as the mouse eif1ad7 gene, homologous to zebrafish eif1axb and human eif1ax. There is a pseudoautosomal region in the short arms of the X and Y chromosomes. This gene is located in this pseudoautosomal region, indicating that it is likely an ancient and highly conserved gene. This gene may play a key regulatory role in embryonic development. To date, information about this gene is very limited, and the downstream pathways it regulates remain unclear.

[0019] The applicant used a zebrafish model and MO knockdown technology to characterize the biological function of the zebrafish eif1axb gene. By using zebrafish morpholino technology, the expression of the zebrafish homolog Eif1axb gene was targeted and inhibited, and it was determined that the expression of this gene is related to heart development and bone development.

[0020] The applicant discovered through research on the effects of eif1axb gene expression on zebrafish that the zebrafish eif1axb gene is a novel gene target for the treatment of congenital heart disease and skeletal developmental abnormalities. Selective control of eif1axb gene expression can affect the gene network. Therefore, new drugs targeting eif1axb and its homologs, human eif1ax and mouse eif1ad7 genes, could be widely used in the prevention and treatment of birth defects.

[0021] The applicant screened and examined the expression of seven genes previously shown to be closely associated with cardiac defects and / or angiogenesis in 2-dpf and 3-dpf. The applicant found that three of these genes (dll4, efnb2a, and s1pr1) were significantly reduced, while three others (notch1b, ptprb, and s1pr2) were dramatically increased, except for cd146, which remained unchanged. The absence of the Notch ligand dll4 (delta-like 4) in zebrafish leads to defective angiogenesis. Cardiac contraction promotes trabecular formation via epistatic notch1b-efnb2a-nrg1. Previous research has shown that VE-PTP (ptp-rb in zebrafish) is not only a key player in regulating angiogenesis and EC adhesion but also a potential therapeutic target for angiogenesis-dependent diseases.

[0022] This invention also provides a model using zebrafish or mouse gene knockdown or overexpression models for screening drugs to prevent, alleviate, and treat congenital heart disease. The eif1axb gene can also serve as a target gene in gene therapy, allowing for the design and preparation of drugs or biological reagents to prevent, alleviate, and treat congenital heart disease or skeletal development disorders. This provides animal models for drug research on the prevention and treatment of congenital heart disease or skeletal development disorders through genetic engineering. For example, using mouse eif1ad7 as a target gene, a double-stranded siRNA that interferes with eif1ad7 expression can be designed, synthesized chemically, and injected into an animal model to silence the eif1ad7 gene through RNA interference, thus creating a congenital heart disease model in mouse embryos. Gene editing technologies, such as CRISPR / Cas9, can be used. Furthermore, sequences can be inserted into the eif1ad7 gene locus using sgRNA and / or targeting vectors to obtain gene-mutated mouse models. In vitro cell models or animal models with overexpression of eif1a-related genes can also be used for screening to discover molecules that specifically regulate eif1a gene expression, thereby providing new therapeutic molecules for congenital heart disease birth defects.

[0023] This invention studies the biological functions of the zebrafish eif1axb gene and the mouse eif1ad7 gene during embryonic development. By comparing gene mapping methods, it is determined that the human eif1ax gene is homologous to the above genes. This invention provides the application of zebrafish eif1axb, mouse eif1ad7, and human eif1ax as targets in the screening, treatment, and prevention of birth defects, including congenital heart disease, skeletal developmental disorders, and neurodevelopmental disorders.

[0024] This invention provides a method for regulating the expression of zebrafish eif1axb, mouse eif1ad7, and human eif1ax genes to prevent birth defects, including congenital heart disease, skeletal developmental disorders, and neurodevelopmental disorders. The gene regulators include siRNA, antibodies corresponding to the gene expression proteins, and one of the following: gene regulators capable of modulating the expression of zebrafish eif1axb, mouse eif1ad7, and human eif1ax genes using bioengineering techniques.

[0025] A zebrafish embryo model in which the Eif1a gene is either suppressed or overexpressed, and the application of the model in screening drugs for the prevention of birth defects.

[0026] A mouse embryo model in which the Eif1a gene is either suppressed or overexpressed, and the application of the model in screening drugs for the prevention of birth defects.

[0027] The beneficial effects of this invention are:

[0028] This invention is the first to discover the function of the eif1axb gene in the cardiovascular and skeletal development of zebrafish, and to find that folic acid can inhibit the expression of zebrafish eif1axb and mouse eif1ad7. It provides novel uses for zebrafish eif1axb, mouse eif1ad7, and human eif1ax regulators in the preparation of drugs for treating cardiovascular developmental disorders and skeletal defects, and provides animal models for screening drugs for the prevention or treatment of cardiovascular developmental disorders and skeletal defects. Attached Figure Description

[0029] Figure 1 Gm5662 gene identification and analysis, (A&B) GDE (differential gene expression) analysis showed that the expression of gm5662 was most significantly different between FA and MTHF-Ca treated blastocysts; Gm5662 was identified as the eif1ad7 gene in mouse (M. musculus) and eif1axb in zebrafish (D. rerio);

[0030] Figure 2Evolutionary conservation of eif1axb. Amino acid sequences of eif1axb orthologs from three species were compared. The gene is conserved in eukaryotes and shows high homology among humans (EIF1A), zebrafish (eif1axb), and mice (eif1ad7).

[0031] Figure 3 Endogenous zebrafish eif1axb control, FA-treated, or MTHF-Ca-treated embryos were evaluated by qRT-PCR at four embryonic developmental stages (6 hpf, 12 hpf, 24 hpf, and 48 hpf) (n = 30 individual embryos); ***P < 0.001; **P < 0.01; ns, not significant; hpf, hours post-fertilization;

[0032] Figure 4 The efficiency of eif1axb gene knockdown was investigated; (A) The zebrafish eif1axb gene was targeted by a specific morpholino antisense to prevent proper splicing of exon 3 (E3I3-MO). Primers 2F and 5R were used to inquire whether wild-type (non-mutant) transcripts or transcripts with inserted intron 3 were present; (B) RT-PCR of eif1axb transcripts from control-MO and E3I3-MO morpholino-injected embryos 1 day after fertilization confirmed the insertion of intron 3. Injection of 4 ng of eif1axb MO altered the splicing between exon 3 and intron 3, as revealed by the changes in PCR bands between control and eif1axb MO-injected embryos; (C) Quantitative measurement of eif1axb expression levels by qRT-PCR (***P<0.0001). MO-targeted downregulation of eif1axb (injection of 4 ng at the single-cell stage (N=20)) (D) Schematic diagram of the eif1axb-EGFP fluorescent reporter gene, with the eif1axb-ATG-MO target sequence fused to the EGFP box (yellow box) on top; (E) Embryos injected with 50 pg eif1axb-EGFP with standard control morpholine (4 ng) or eif1axb-ATG-MO (4 ng) at 9-hpf and 24-hpf. Embryos injected with eif1axb-GFP plasmid DNA under CMV promoter drive showed green fluorescence. When co-injected with eif1axb-ATG-MO, the green fluorescence was significantly reduced, showing the MO knockdown efficiency. dpf: days post-fertilization.

[0033] Figure 5The absence of the eif1axb phenotype is associated with cardiovascular defects; (AC) General morphology of 3-dpf zebrafish embryos. Compared with control zebrafish, eif1axb knockdown resulted in pericardial edema (BC, red arrow), decreased contractility, and reduced precordial congestion, which are clear signs of heart failure; (D) Time course plot of survival percentage at 3 days in control and eif1axb MO. Panel E shows the percentage of embryos with developmental defects; (F) Quantification of embryonic pericardial area; error bars, indicating ±sem***P<0.001 (n=10; ANOVA); (GO) Representative fluorescent images of Tg(fli1a:EGFP)y1 embryos at 3-dpf; (G&J) Images of the trunk region taken at 3-dpf, with vascular structures visualized by eGFP fluorescence and labeled as ISV and DLAV, showing regular development in embryos injected with control MO; embryos injected with eif1axb-MO showed thinner ISVs (KL, arrow) compared to control MO. In control embryos, parachordal vessels (PAVs) formed normally (J, red arrow). Compared to controls, MO knockdown of eif1axb prevented PAV formation, a precursor to the lymphatic system (KL). The relevant areas are shown at higher magnification. In control embryos, inferior enteric veins (SIVs) developed into smooth basket-like structures on the yolk at 3-dpf (A, E, arrows). Conversely, embryos injected with eif1axb-MO showed reduced ectopic SIV fragments (M, asterisk). (PQ) Quantification of the mean length and diameter of ISVs shows that eif1axb… MO significantly decreased, column, mean; SEM (n=10; ANOVA) ***P<0.001; (R) Quantification of SIV region eif1axb MO significantly decreased, column, mean; bar chart, SEM (n=10; ANOVA; ***P<0.001); DLAV, dorsal longitudinal vessels; ISV, intersegmental vessels; dpf, days post-fertilization;

[0034] Figure 6 eif1axb lacks regulation of cardiovascular signaling pathways; endogenous dll4, notch1b, efnb2a, cd146, ptprb, s1pr1, and s1pr2 were assessed by qRT-PCR in control and eif1axb MO at 2-dpf and 3-dpf (n = 6-10 individual embryos). ***P<0.001; **P<0.01; *P<0.05; ns, not significant; dpf, days post-fertilization.

[0035] Figure 7 The zebrafish model morphology diagram constructed by knocking down eif1axb.

[0036] Figure 8Analysis of intestinal vessels in zebrafish models constructed by knockdown of eif1axb; (A) The intestinal vessels of the control group are normally developed and are smooth basket-like structures; (B) ATG-MO 1ng zebrafish model; (C) ATG-MO 1.5ng zebrafish model; (D) E3I3-MO 1ng zebrafish model; (E) E3I3-MO 1.5ng zebrafish model; (F) Quantitative statistical diagram of the intestinal vessel region of the control group and MO group, SEM (n=10; ANOVA; ***P<0.0001).

[0037] Figure 9 Intersegmental vascular analysis of zebrafish models constructed by eif1axb knockdown; (A) Normal intersegmental vascular development in the control group; (B) Zebrafish model with ATG-MO 1ng; (C) Zebrafish model with ATG-MO 1.5ng; (D) Zebrafish model with E3I3-MO 1ng; (E) Zebrafish model with E3I3-MO 1.5ng; (F) Quantification of average ISV length, SEM (n=10; ANOVA; ***P<0.0001).

[0038] Figure 10 Anatomical and tissue section images of the heart of newborn mice with knockdown of eif1ad7; (A) Anatomical image of the heart of the control group; (B) Anatomical image of the heart of mice after si-RNA intervention; (C) Tissue section image of the heart of the control group; (D) Tissue section image of the heart of mice after si-RNA intervention, and magnified image of the defect area. Detailed Implementation

[0039] The following examples are provided to better understand the present invention, but are not intended to limit the invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0040] In the following examples, FA represents folic acid, and MTHF-Ca represents 6S-5-methyltetrahydrofolate calcium, with molecular weights of 443.40 and 497.52, respectively. 6S-5-methyltetrahydrofolate, not folic acid, is present in the blood circulation.

[0041] Unless otherwise specified, the primers used in the following examples are shown in Table 1.

[0042] Table 1 summarizes the primers used:

[0043]

[0044]

[0045] Example 1: Discovery of the GM5662 gene

[0046] Superovulation induced by exogenous gonadotropin therapy (PMSG / hCG) was used to assess the quality of ovulated oocytes from female mice (C57BL / 6J). In vitro fertilization was then performed. Fertilized oocytes were randomly grouped, with potassium-optimized monosodium oxymethylene (KSOM) medium, KSOM-FA (60 ng / L), and KSOM-MTHF (60 ng / L) as controls. Blastocysts were collected after 24, 48, and 72 hours of culture, observed under a microscope, and preserved in liquid nitrogen.

[0047] Blastocysts from mice were collected 72 hours after in vitro fertilization from three groups: control, FA, and MTHF-Ca. Total RNA was prepared using the RNeasy kit (Qiagen, Germany) according to the manufacturer's instructions. Equal amounts of RNA from all samples were mixed for cDNA library construction to obtain transcriptome data. Normalized cDNA libraries were constructed using 1 μg of total RNA. Sequencing libraries were generated using the NEBNext UltraDirectional RNA Library Prep Kit for Illumina (NEB, USA) and sequenced using paired-end reads on the Illumina HiSeq 2000 platform. All sequencing data were processed and analyzed by Genewiz Bio-pharmTechnology Corporation (Suzhou, China).

[0048] After total RNA extraction and library construction, high-throughput sequencing was performed using the Illumina platform. Sequencing reads were evaluated using Bcl2fastq (V2.17.1.14) to generate FASTQ files. Initial quality control of the FASTQ files was performed using FastQC (V0.10.1) and Cutadapt (V1.9.1) to filter low-quality data, including adapter-introduced contamination and redundant sequences. Clean reads were compared to the mouse genome using Hisat2 (V2.0.1), and gene expression was quantified using Htseq (V0.6.1) based on short reads of fragments per million bases (FPKM). Differentially expressed genes (DEGs) were identified using DESeq2 (V1.6.3) from the Bioconductor package.

[0049] DEG (differentially expressed genes) were identified using DESeq2 (V1.6.3) from the Bioconductor package. Volcano plots were performed in R (V4.0.3) using the ggplot2 package.

[0050] RNA transcriptome analysis of in vitro fertilized and cultured blastocysts showed that Gm5662 in the mouse genome was differentially expressed in fertilized eggs in FA culture medium compared with MTHF-Ca (see [link to data]). Figure 1Homology of Gm5662 among human, zebrafish, and mouse sequences was obtained through DNAMAN (V6.0.3) alignment. Gm5662 is an mRNA sequence recorded in the mouse genome database, but the gene has not yet been identified. Utilizing the advantages of comparative gene mapping, we performed a blast search for homology analysis between Gm5662 (M. musculus) and AAP36772.1 (H. sapiens), finding that human EIF1AX shares 92% homology with Gm5662 in amino acid sequence. Therefore, Gm5662 was identified as the eif1ad7 gene in mouse (M. musculus) and eif1axb in zebrafish (D. rerio). This gene is conserved in eukaryotes and shows high homology among humans (eif1ax), mice (eif1ad7), and zebrafish (eif1axb). Figure 2 During the early development of zebrafish, this gene is orthologous to the human EIF1AX gene. The zebrafish eif1axb gene shares 81.5% homology with the human eif1ax gene, and its protein conservation is 99.3%. Specifically, the human eif1ax gene sequence is shown in SEQ ID NO.1, the mouse eif1ad7 gene sequence is shown in SEQ ID NO.2, and the zebrafish eif1axb gene sequence is shown in SEQ ID NO.3.

[0051] Example 2: Investigating the effect of FA on the expression of the eif1axb gene in zebrafish embryos by RT-PCR.

[0052] In zebrafish, total RNA was extracted from 30 to 50 embryos per group in Trizol (Roche) according to the manufacturer's instructions. RNA was reverse transcribed using the PrimeScript RT kit with gDNA Eraser (Takara). eif1axb gene expression was quantified three times using a Bio-radiQ SYBR Green Supermix (Bio-rad) and detected on a Realplex system (Eppendorf). Relative gene expression quantification was based on the comparative threshold cycling method (2-ΔΔCt). Primers used in the real-time RT-PCR experiments are shown in Table 1. Real-time RT-PCR results showed that the eif1axb gene was downregulated by FA in zebrafish from 12 to 48 hpf, but not by MTHF-Ca ( Figure 3 It was FA, rather than MTHF-Ca, that caused cardiotoxicity, leading to developmental defects in zebrafish embryos. High doses of folic acid also increased the incidence of heart defects in fetal rats; these results have been replicated in two independent laboratories.

[0053] Example 3: Preparation of the zebrafish model

[0054] Using morpholino antisense oligonucleotide (MO) technology, we blocked the expression of eif1axb in embryonic zebrafish and analyzed the effects of knockdown on fish development.

[0055] The zebrafish (Danio rerio, Hamilton, wild type) used in the experiment were adult zebrafish less than 1 year old. The pH of the rearing water was 7±0.2, the temperature was around 28℃, and the light-to-dark ratio was 14h:10h. They were fed brine shrimp eggs twice a day. One female and two male zebrafish were placed in a spawning box the night before, and the eggs were collected the next morning. The eggs were placed in embryo culture medium (prepared with 0.2g / L sea salt) and cultured at 28℃.

[0056] MO sequences were designed targeting the ATG site (ATG-MO) and the exon 3 / intron 3 junction (E3I3-MO) of the zebrafish eif1axb gene, respectively, along with a Standard Control MO. These MOs were designed and purchased by GeneTools (Philomath, OR). The sequences for eif1axb translation blocking and splicing blocking morpholine were 5'-CTCCTTTTCCTTTGTTTTTCGGCAT-3' (ATG-MO) and 5'-CAGCCTGAAGCTCTAAAATGCACCT-3' (E3I3-MO), respectively. The standard control morpholine sequence was 5'-CCTCTTACCTCAGTTACAATTTATA-3' (GeneTools). The MO dosages used for injection were as follows: Control-MO and E3I3-MO, 2 ng per embryo; ATG-MO, 2 ng per embryo. Primers spanning exon 2 (forward primer: 5'-GCGACGTGGTAAGAACGAGAA-3') and exon 5 (reverse primer: 5'-GGCTTTCAGACTCCTAGCCT-3') of eif1axb were used for RT-PCR analysis to confirm the efficacy of E3I3-MO. The primer ef1α sequences used as internal controls were 5'-GGAAATTCGAGACCAGCAAATAC-3' (forward) and 5'-GATACCAGCCTCAAACTCACC-3' (reverse). The CDS region of the eif1axb cDNA, including the eif1axb-ATG-MO target sequence, was cloned into pcDNA3.1-EGFP to test the efficacy of eif1axb morpholino oligonucleotides (MOs).

[0057] Embryos and larvae were analyzed using a Nikon SMZ18 fluorescence microscope, followed by photographs with a digital camera. The levels, brightness, contrast, hue, and saturation of image subsets were adjusted using Adobe Photoshop 7.0 software (Adobe, San Jose, California) to optimize visualization of expression patterns. Quantitative image analysis was performed using image-based morphological analysis (NIS-Elements D4.6) and ImageJ software (NIH, http: / / rsbweb.nih.gov / ij / ). Inverted fluorescence images were used for processing. Positive signals were defined by particle number using ImageJ. Quantification was performed on 10 animals for each treatment, and the total signal for each animal was averaged.

[0058] The MO technique was used to analyze the phenotype of eif1axb knockdown. Figure 4 The effectiveness of eif1axb knockdown was demonstrated, and RT-PCR and real-time quantitative RT-PCR confirmed the knockdown efficiency of eif1axb. Compared with control zebrafish ( Figure 5 Compared to A), knockdown of eif1axb resulted in pericardial edema, decreased contractility, and reduced precordial congestion, which are clear signs of heart failure. Figure 5 (B and C). Increased embryonic defects and decreased survival rates ( Figure 5 D and E). Quantitative analysis of the pericardial region showed a significant increase in the eif1axb mo group (D and E). Figure 5 F). Images of the trunk region taken at 3-dpf, with vascular structures visualized by eGFP fluorescence and labeled as ISVs (intersegmental vessels) and DLAVs (dorsal longitudinal vessels), showing regular development in embryos injected with control MO. Compared to control MO, embryos injected with eif1axb-MO exhibited thinner ISVs (…). Figure 5 K and L, arrows). In controlled embryonic development, parachordal vessels (PAV) form normally ( Figure 5 J). Compared to control, MO knockdown of eif1axb prevented the formation of parachordal vessels (PAVs) in the lymphatic system. The relevant areas are displayed at higher magnification. Figure 5 N&O). Quantification of the average length and diameter of the ISV showed a significant reduction in the eif1axb mo group ( Figure 5 PQ). Therefore, downregulation of the eif1axb gene inhibits early angiogenesis in zebrafish.

[0059] Functions of Eif1a in the development of the heart, trunk, and tail

[0060] Most moderately affected Eif1a (89.1%, n=110) showed significant pericardial cavity enlargement. This also affected embryonic heart rate, particularly at 48 hpf.

[0061] Moderately affected Eif1a (68.9%, n=103) exhibited marked body and / or tail kinking. The defect became apparent at 25 hpf and persisted in older embryos (2–7 days old, data not shown).

[0062] Function of Eif1a in neurodevelopment

[0063] Central nervous system (CNS) development in Eif1a was assessed by examining brain morphology and staining patterns of several neurodifferentiation markers in live embryos. At 25 hpf, different brain regions (e.g., cerebellum, hindbrain, optic tectum) were observed, with the dorsal portion of the posterior wall of the optic tectum and the dorsal cerebellum appearing thinner in Eif1a-MO.

[0064] Example 4 eif1axb deficiency and signaling pathway

[0065] To investigate how eif1axb mediates angiogenesis, we subsequently screened and examined the expression of seven genes previously shown to be closely associated with cardiac defects and / or angiogenesis in 2-dpf and 3-dpf. In zebrafish, total RNA was extracted from 30 to 50 embryos per group in Trizol (Roche) according to the manufacturer's instructions. RNA was reverse transcribed using the PrimeScript RT kit with gDNAEraser (Takara). Relative gene expression quantification was based on the comparative threshold cycling method (2-ΔΔCt). Primers used in the real-time RT-PCR experiments are summarized in Supplementary Table 1.

[0066] We found that three genes (dll4, efnb2a, and s1pr1) were significantly reduced, while three other genes (notch1b, ptprb, and s1pr2) were sharply increased, except for cd146, which remained unchanged. Figure 6 We hypothesize that this is a result of negative feedback regulation to prevent excessive reduction of the vascular system. In this study, we discovered that the zebrafish gene eif1axb controls angiogenesis in vivo by targeting VE-PTP. Eif1axb deficiency may regulate the expression of dll4, ptp-rb, notch1b, and efnb2a through a negative feedback loop. These in vivo results suggest that the eif1axb gene plays an important role in cardiovascular and embryonic development.

[0067] Example 5: Zebrafish Model with Cardiovascular Developmental Defects

[0068] The zebrafish (Danio rerio, Hamilton, wild type) used in the experiment were adult zebrafish less than 1 year old. The pH of the rearing water was 7±0.2, the temperature was around 28℃, and the light-to-dark ratio was 14h:10h. They were fed brine shrimp eggs twice a day. One female and two male zebrafish were placed in a spawning box the night before, and the eggs were collected the next morning. The eggs were placed in embryo culture medium (prepared with 0.2g / L sea salt) and cultured at 28℃.

[0069] Gene Tools, LLC (http: / / www.gene-tools.com / ) designed morpholino (MO). Antisense MO (GeneTools) was microinjected into fertilized single-cell stage embryos according to the standard protocol (ref4). The sequences of eif1axb translation-blocking and splicing-blocking morpholino were 5'-CTCCTTTTCCTTTGTTTTTCGGCAT-3' (ATG-MO) and 5'-CAGCCTGAAGCTCTAAAATGCACCT-3' (E3I3-MO), respectively. The sequence of the standard control morpholino was 5'-CCTCTTACCTCAGTTACAATTTATA-3' (GeneTools). The amounts of MO used for injection were as follows: Control-MO and E3I3-MO, 1 and 1.5 ng per embryo, respectively; ATG-MO, 1 and 1.5 ng per embryo, respectively.

[0070] To assess angiogenesis in zebrafish embryos, embryos were anesthetized with 0.016% MS-222 (tricaine mesylate, Sigma-Aldrich, St. Louis, MO). Zebrafish were then positioned laterally (anterior, left; posterior, right; dorsal, superior) and mounted on concave slides with 3% methylcellulose for observation via fluorescence microscopy. Embryos and larvae were analyzed using a Nikon SMZ18 fluorescence microscope, followed by photographs with a digital camera. The level, brightness, contrast, hue, and saturation of subsets of images were adjusted using Adobe Photoshop 7.0 (Adobe, San Jose, California) to optimize visualization of expression patterns. Quantitative image analysis was performed using image-based morphological analysis (NIS-Elements D4.6) and ImageJ software (NIH, http: / / rsbweb.nih.gov / ij / ). Inverted fluorescence images were used for processing. Positive signals were defined by particle number using ImageJ. Ten animals for each treatment were quantified, and the total signal for each animal was averaged.

[0071] Results showed that zebrafish embryos injected with 1 ng and 1.5 ng of eif1axb-MO reagent experienced slightly delayed development, but all survived within 7 days. Treatment with 1 ng and 1.5 ng of ATG-MO and 1 ng and 1.5 ng of E3I3-MO resulted in slight shortening of body length and pericardial edema. Figure 7 Since the biological function of the eif1axb gene is related to the cardiovascular development of the embryo, we captured fluorescent images of the inferior intestinal vein (SIV) in zebrafish at three days of development and analyzed them. In control embryos, the inferior intestinal vein (SIV) developed into a smooth basket-like structure on the yolk at 3-dpf. Figure 8 A). Zebrafish embryos treated with MO showed ectopic SIV fragments and a reduced number of blood vessels ( Figure 8 BE). Further quantification of the SIV region ( Figure 8 F)SEM (n=10; ANOVA; ***P<0.0001) showed that there is a dose-dependent relationship between interference with MO and vascular development.

[0072] We used eGFP fluorescence visualization to mark ISVs (intersect vessels) and DLAVs (dorsal longitudinal anastomoses). In the control group, the vessels showed regular development. Embryos treated with ATG-MO and E3I3-MO showed thinner ISVs (arrows) or incomplete ISVs and ectopic ISVs (asterisks) (see...). Figure 9 AE). Quantization of the average length of ISVs ( Figure 9 F), SEM (n=10; ANOVA; ***P<0.0001).

[0073] We have created a zebrafish vascular development disorder model. By adjusting the MO dosage, we can achieve mild defects in the intestinal veins and intersegmental vessels of zebrafish embryos, which can be used for the development of related drugs.

[0074] Example 6: Preparation of a mouse model

[0075] Superovulation induced by exogenous gonadotropin therapy (PMSG / hCG) was used to assess the quality of ovulated oocytes from female mice (C57BL / 6J). In vitro fertilization was then performed using potassium-optimized monosodium oxymethylene (KSOM) medium. Blastocysts were collected after 24, 48, and 72 hours of culture, observed under a microscope, and preserved in liquid nitrogen.

[0076] Antisense oligonucleotides (siRNA): 5'-GCCAUAUAAGAGGGAAGCUTT-3', 5'-AGCUUCCCUCUUAUAUGGCTT-3', negative control siRNA 5'-UUCUCCGAACGUGUCACGUTT-3', 5'-ACGUGACACGUUCGGAGAATT-3' were used for research. The above sequences were designed and purchased by Gemma Pharmaceutical Technology Co., Ltd. The siRNA was dissolved in RNase-free buffer and injected at 20 μM into embryos at the one- to four-cell stage using a Narisge MI300 microinjector. After injection, the surviving cells were cultured in KSOM medium and incubated for 3 days. A small hole was made in the oviduct wall between the ampulla and ovary of a pseudopregnant mouse. The tip of a pipette was inserted into the hole, with the pipette opening pointing towards the ampulla, and the embryo was implanted into the pseudopregnant mouse. Female mice were housed separately until birth.

[0077] All newborn mice were observed and their weight was measured on the day of birth. Electrocardiograms were performed on a subset of mice (data not shown). For another subset of newborn mice, when they reached 6 days of age, their thoracic cavities were dissected, and the morphology and pulsation of the heart were observed and photographed under a body field microscope (SMZ168, Motic, Xiamen, Fujian, China). Mouse heart tissue was fixed in 4% paraformaldehyde and embedded in paraffin. H&E-stained tissue sections were examined using a panoramic imaging system IX73 (Olympus, Tokyo, Japan).

[0078] The results showed that the hearts of control mice developed normally, while the hearts of newborn mice were slightly smaller after interfering with eif1ad7 expression (see...). Figure 10 AD), partial myocardial ischemia was observed in newborn mice, and defects were visible in the cardiac wall between the atria and ventricles in 46.5% (n=43), indicating that the model was successfully established.

[0079] The results of the embodiments of the present invention also show that eif1ad7 is involved in the pathogenesis of congenital heart disease in mice, therefore drugs targeting Eif1ad7 have good prospects for treating and preventing birth defects.

Claims

1. Application of morpholine antisense oligonucleotides with nucleotide sequence 5'-CAGCCTGAAGCTCTAAAATGCACCT-3' in constructing a zebrafish cardiovascular developmental disorder model.

2. A method for constructing a cardiovascular developmental disorder zebrafish model, characterized in that, The zebrafish model was obtained by knocking down the eif1axb gene in zebrafish embryos. The method involved knocking down the corresponding eif1axb gene expression using Morpholino technology, which included injecting 1.5 ng to 4 ng of morpholine antisense oligonucleotides that specifically block the eif1axb gene into zebrafish embryo cells. The sequence of the morpholine antisense oligonucleotides was 5'-CAGCCTGAAGCTCTAAAATGCACCT-3'.

3. Application of siRNAs with nucleotide sequences of 5'-GCCAUAUAAGAGGGAAGCUTT-3' and 5'-AGCUUCCCUCUUAUAUGGCTT-3' in constructing a mouse model of congenital heart disease.

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