CN115992216A - Eif1a genes and application thereof - Google Patents

Abstract

The invention discloses several Eif1a genes and application thereof. The invention discovers the function of the eif1axb gene in the cardiovascular development and skeletal development of the zebra fish for the first time, and discovers that folic acid can inhibit the expression of the zebra fish eif1axb and the mouse eif1ad 7. Provides a new application of zebra fish eif1axb, mouse eif1ad7 and human eif1ax regulator in preparing medicaments for treating cardiovascular development disorder and bone defect, and provides an animal model for screening medicaments for preventing or treating cardiovascular development disorder and bone defect.

Description

Eif1a genes and application thereof

Technical Field

The invention belongs to the field of gene functions and applications, and particularly relates to functions and applications of certain Eif1a genes in birth defects.

Background

Eukaryotic translation initiation factors (eifs) refer to a class of proteins involved in eukaryotic translation initiation, where the eIF1a class of genes is an important member of the translational initiator subfamily and plays an irreplaceable role in initiating translation. Eif1a plays a very important role in translation initiation scanning, and by modeling hepatitis c virus, research finds that Eif1a is an indispensable loop in both the translation initiation scanning process mediated by 5' capping and the translation initiation scanning process mediated by internal ribosome entry site sequence (IRES).

In embryonic development, the parent and maternal genomes are transcribed for the first time after fertilization, and there is a synthon gene activation (ZGA), with ZGA occurring at times that vary from mammal to mammal. The main role of ZGA, which has not been altered for a long time with respect to the mainstream ideas of ZGA, is to provide the RNA and proteins required before embryo cell implantation, maintaining the basic functions of embryo cells, such as the expression of the housekeeper gene. eif1a is considered to be the major marker gene for activation of the genome of the embryo of a mammal prior to implantation, and it was found to be a gene transiently expressed in the two-cell stage of a mouse embryo and has been used as a two-cell stage specific marker. ZGA is of short duration, indicating that embryos need to be rapidly suppressed after activation of the gene, and that a large number of transcriptional activations ZGA may lead to intrinsic DNA damage that must be corrected for proper development. However, the specific function of the eif1a gene in ZGA has not been reported, and so far, no document has been reported about the application of the eif1a related gene in preventing birth defects.

Disclosure of Invention

The invention discovers the application of zebra fish eif1axb, mouse eif1ad7 and human eif1ax genes in early embryo cardiovascular development and skeletal development.

The sequence of the human gene eif1ax is shown as SEQ ID NO.1, the sequence of the mouse gene eif1ad7 is shown as SEQ ID NO.2, and the sequence of the zebra fish gene eif1axb is shown as SEQ ID NO. 3.

The first aspect of the invention provides application of a gene eif1axb in constructing a zebra fish cardiovascular development disorder and bone abnormality model.

The second aspect of the invention provides a method for constructing a zebra fish model with cardiovascular developmental disorder and skeletal abnormality, which is used for knocking out or inhibiting zebra fish embryo genes eif1axb to obtain the zebra fish model. The preparation method of the zebra fish model can adopt a conventional method in the field to knock out or inhibit the gene eif1axb in the zebra fish embryo, and can realize the control of the weight of the model by adjusting the inhibition or knocking-out degree.

The third aspect of the invention provides the use of the constructed cardiovascular developmental disorder and skeletal abnormality zebra fish model of the invention in the screening of drugs for the treatment of cardiovascular developmental disorders and skeletal abnormalities. In a specific example, the use in the screening of a drug for the treatment of cardiovascular developmental disorders and skeletal abnormalities caused by defects in the gene eif1axb.

The fourth aspect of the invention provides application of the gene Eif1ad7 in constructing a model of congenital cardiovascular diseases and congenital heart diseases of mice.

In a fifth aspect, the present invention provides a method for constructing a mouse model of congenital cardiovascular disease and congenital heart disease, wherein the method is that after the expression of gene Eif1ad7 in a mouse embryo is inhibited or gene Eif1ad7 is over-expressed, the embryo is obtained after the development of the embryo in a female mouse.

In a sixth aspect, the present invention provides the use of the congenital cardiovascular disease and congenital heart disease mouse models constructed according to the invention for screening of drugs for the treatment of congenital cardiovascular disease and congenital heart disease. In one embodiment, the use in the screening of drugs for the treatment of congenital cardiovascular diseases and congenital heart diseases caused by defects in the gene eif1ad 7.

The preparation method of the zebra fish model comprises the steps of inhibiting zebra fish embryo eif1axb gene expression, knocking down corresponding eif1axb gene expression by using a Morpholino technology, or adopting siRNA of eif1axb gene, antibody of eif1axb and a method capable of inhibiting eif1axb expression by using other bioengineering technologies.

The construction method of the mouse model is that after Eif1ad7 gene in the mouse embryo is inhibited to express or Eif1ad7 gene is over-expressed, the embryo is obtained after the embryo develops in a female mouse. Further the gene suppression expression was modulating eif1ad7 gene expression in mouse embryos using either Morpholino technology or antisense RNA technology.

The seventh aspect of the invention also provides the application of the reagent for detecting the gene eif1ax in preparing a congenital heart disease diagnosis or auxiliary diagnosis reagent.

For example, the zebra fish eif1axb, the mouse eif1ad7 and the human eif1ax genes are applied to the development of a gene screening kit. In one embodiment, a diagnostic reagent for screening congenital heart disease can be provided, wherein the diagnostic reagent takes eif1ax as a target point, comprises PCR primers designed according to eif1ax genes and RNA storage liquid, the using method of the diagnostic reagent comprises extracting target DNA, detecting eif1ax expression by real-time PCR, and judging the risk of congenital heart disease of a neonate by comparing with normal gene expression level.

The eighth aspect of the invention also provides the use of the gene eif1ax in a therapeutic target for a medicament for treating congenital heart disease.

The ninth aspect of the invention also provides the use of folic acid as a modulator of the zebra fish gene eif1axb or the mouse gene eif1ad7 or the human gene eif1ax, mainly as an inhibitor of the zebra fish gene eif1axb or the mouse gene eif1ad7 or the human gene eif1 ax.

Folic acid, but not 6S-5-methyltetrahydrofolate endogenous to human body, has cardiotoxicity, and folic acid can induce heart development disorder and malformation of zebra fish embryos at a certain dosage, while 6S-5-methyltetrahydrofolate has no negative effects. In vitro experiments were performed on fertilized blasts of mice cultured with FA (folic acid) and MTHF-Ca (6S-5-methyltetrahydrofolate calcium). By using the gene expression profile, we found gm5662 to be the most significantly differentially expressed mRNA sequence, an unidentified unknown gene. Gm5662 is an mRNA sequence recorded in the mouse genome database, and the present invention further determines Gm5662 to be the mouse eif1ad7 gene homologous to zebra fish eif1axb and human eif1 ax. There is a pseudo-autosomal region in the short arm of the X and Y chromosomes. The gene is located in this pseudoautosomal region, indicating that the gene is likely to be an old gene and is highly conserved. The gene may play a key regulatory role in embryo development. So far, information about the gene is very limited, and the downstream pathway of the gene regulation is still unclear.

The applicant adopts a zebra fish model and an MO knockdown technology to characterize the biological function of the zebra fish Eif1axb gene, and determines that the gene expression is related to heart development and bone development through targeting inhibition of the expression of the zebra fish homologous gene Eif1axb gene by using a morpholino technology of the zebra fish.

The applicant discovers through studying the influence of the eif1axb gene expression on the zebra fish that the zebra fish eif1axb gene is a new gene target point for treating congenital heart diseases and skeletal dysplasia, and the selective condition of controlling the eif1axb gene expression can influence a gene network, so that the novel medicine aiming at the eif1axb and homologous human eif1ax of the eif1axb gene target point of mice can be widely applied to the prevention and treatment of birth defects.

Applicants screened and examined the expression of 7 genes that have previously been shown to be closely related to heart defects and/or angiogenesis for 2-dpf and 3-dpf. Applicants found that three of the genes (dll 4, efnb2a and s1pr 1) were significantly reduced, while the other three genes (notch 1b, ptprb and s1pr 2) were dramatically increased, except that cd146 remained unchanged. The absence of Notch ligand dll4 (delta-like 4) in zebra fish leads to defects in vascular sprouting. Systole promotes trabecular formation by notch1b-efnb2a-nrg1 uprightness. Previous studies have shown that VE-PTP (PTP-rb in zebra fish) is not only a key participant in regulating angiogenesis and EC adhesion junctions, but also a potential therapeutic target for angiogenesis-dependent diseases

The invention also provides a model, which is used for screening medicines for preventing, relieving and treating congenital heart diseases by using the zebra fish or mouse gene knockdown or gene overexpression model; the eif1axb gene can also be used as a target gene in gene therapy, and can be used for designing and preparing medicines or biological reagents for preventing, relieving and treating congenital heart diseases or skeletal development disorders, so that animal models required by medicine research for preventing and treating congenital heart diseases or skeletal development disorders can be achieved through genetic engineering technology. For example, a double-stranded siRNA which can interfere with the expression of the eif1ad7 is designed by taking the eif1ad7 of a mouse as a target gene, and after being synthesized by a chemical method, the siRNA is injected into an animal model, and the eif1ad7 gene is silenced by an RNA interference method, so that a congenital heart disease model of the embryo of the mouse is prepared. For example, using gene editing techniques such as CRISPR/Cas9 techniques and, in addition, using sgrnas and/or targeting vectors to insert sequences into the eif1ad7 locus, a mouse model of genetic mutations is obtained. In vitro cell models or animal models of overexpression of the eif1a related genes can be used, and molecules capable of specifically regulating the expression of the eif1a genes can be found through screening, so that novel therapeutic molecules are provided for congenital heart disease birth defects.

The invention provides application of zebra fish Eif1axb, mouse Eif1ad7 and human Eif1ax as targets in screening and treating birth defects, which are congenital heart disease birth defects, skeletal development disorder birth defects and nerve development disorder birth defects, by researching biological functions of the zebra fish Eif1axb genes in the embryo development process and determining homology of human Eif1ax with the genes by comparing a gene mapping method.

Aiming at the functions of zebra fish eif1axb, mouse eif1ad7 and human eif1ax, the application of the regulator of the corresponding genes in preventing birth defects, wherein the birth defects are congenital heart disease birth defects, skeletal development disorder birth defects and neural development disorder birth defects is provided. The gene regulator comprises siRNA, an antibody corresponding to gene expression protein and one of zebra fish eif1axb, mouse eif1ad7 and human eif1ax which can be regulated by utilizing a bioengineering technology.

A zebra fish embryo model, wherein the zebra fish embryo has Eif1a gene inhibited expression or Eif1a gene over-expressed, and the application of the model in screening medicines for preventing birth defects.

A mouse embryo model in which the Eif1a gene is inhibited from expression or the Eif1a gene is overexpressed, the use of the model in screening drugs for preventing birth defects.

The invention has the beneficial effects that:

the invention discovers the function of the eif1axb gene in the cardiovascular development and skeletal development of the zebra fish for the first time, and discovers that folic acid can inhibit the expression of the zebra fish eif1axb and the mouse eif1ad 7. Provides a new application of zebra fish eif1axb, mouse eif1ad7 and human eif1ax regulator in preparing medicaments for treating cardiovascular development disorder and bone defect, and provides an animal model for screening medicaments for preventing or treating cardiovascular development disorder and bone defect.

Drawings

FIG. 1Gm5662 gene discrimination and analysis, (A & B) GDE (differential Gene expression) analysis showed that Gm5662 expression was most significantly different between FA and MTHF-Ca treated blastula; gm5662 was identified as the eif1ad7 gene in mice (M.musculus) and eif1axb in zebra fish (D.rerio);

FIG. 2 evolution conservation of eif1axb. Amino acid sequences of eif1axb orthologs from three species were aligned. The gene is conserved in eukaryotes, with high homology between humans (EIF 1A), zebra fish (EIF 1 axb) and mice (EIF 1ad 7);

FIG. 3 endogenous zebra fish eif1axb control, FA-treated embryos or MTHF-Ca-treated embryos were evaluated by qRT-PCR at four embryo development stages (6 hpf, 12hpf, 24hpf and 48 hpf) (n=30 individual embryos); * P <0.001; * P <0.01; ns, not significant; hpf, hours after fertilization;

FIG. 4eif1axb knockdown efficiency study; (A) The zebra fish eif1axb gene is targeted by a specific morpholino antisense to prevent correct splicing of exon 3 (E3I 3-MO), primers 2F and 5R query for the presence of wild-type (non-mutated) transcripts or transcripts inserted into intron 3; (B) 1 day after fertilization, RT-PCR of eif1axb transcripts of control-MO and E3I3-MO morpholino injected embryos demonstrated the insertion of intron 3, 4ng of eif1axb MO injected altered splicing between exon 3 and intron 3, as revealed by the change in PCR band between control and eif1axb MO injected embryos; (C) Quantitative measurement of eif1axb expression levels measured by qRT-PCR (< 0.0001P), MO targeting down-regulates eif1axb (after injection of 4ng MO at single cell stage (n=20), samples were collected at 1-dpf); (D) Schematic representation of eif1axb-EGFP fluorescent reporter gene, above comprising eif1axb-ATG-MO target sequence fused in-frame with EGFP (yellow box); (E) 50pg eif1axb-EGFP injected with standard control morpholine (4 ng) or eif1axb-ATG-MO (4 ng), embryos were photographed at 9-hpf and 24-hpf, embryos injected with eif1axb-GFP plasmid DNA driven by the CMV promoter showed green fluorescence, which was significantly reduced when co-injected with eif1axb-ATG-MO, showing MO knockdown efficiency, dpf: days after fertilization;

FIG. 5eif1axb phenotype deletions are associated with cardiovascular defects; (A-C) overall morphology of 3-dpf zebra fish embryos. The eif1axb knockdown resulted in pericardial edema (B-C, red arrow), reduced contractility, and reduced precordial congestion, as compared to control zebra fish, an indication of overt heart failure; (D) Time course graph of percent survival of control group versus eif1axb MO for 3 days. Panel E shows the percentage of embryos with developmental defects; (F) quantification of embryo pericardial area; error bars, representing ± s.e.m.. P <0.001 (n=10; analysis of variance); (G-O) representative fluorescent images of Tg (fli 1a: EGFP) y1 embryo at 3-dpf; (G & J) torso area images taken at 3-dpf, vascular structures visualized by eGFP fluorescence and labeled ISV and DLAV, showing regular development of embryos injected with control MO; embryos injected with eif1axb-MO exhibited thinner ISVs (K-L, arrows) compared to control MO, in which parachordal vessels (PAV) formed normally (J, red arrows), and MO knock down eif1axb prevented parachordal vessels (PAV) from forming, a precursor to the lymphatic system (K-L), compared to control MO; the relevant regions are shown at higher magnification, in the control embryo, the hypointestinal venous vessels (SIV) develop into a smooth basket-like structure on the yolk at 3-dpf (A, E, arrow), whereas the eif1axb-MO injected embryo exhibits reduced ectopic SIV fragments (M, asterisks); quantification of (P-Q) ISV average length and diameter showed significant decrease in eif1axb MO, column, average; SEM (n=10; analysis of variance) ×p <0.001; quantification of the (R) SIV region showed a significant decrease in eif1axb MO, columns, average; bar graph, SEM (n=10; analysis of variance; P < 0.001); DLAV, dorsal longitudinal vessel; ISV, internode blood vessel; dpf, days post fertilization;

FIG. 6eif1axb lacks a regulated cardiovascular signal pathway; endogenous dll4, notch1b, efnb2a, cd146, ptprb, s1pr1 and s1pr2 were evaluated 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.

Figure 7 morphology of zebra fish model constructed by eif1axb knockdown.

FIG. 8 intestinal blood vessel analysis of zebra fish model constructed by eif1axb knockdown; (A) The intestinal blood vessel of the control group is normal in development and is of a smooth basket-shaped structure; (B) ATG-MO 1ng zebra fish model; (C) ATG-MO 1.5ng zebra fish model; (D) E3I3-MO 1ng zebra fish model; (E) E3I3-MO 1.5ng zebra fish model; (F) Quantification of vascular areas under the intestines for control and MO groups statistical graphs, SEM (n=10; analysis of variance; P < 0.0001).

FIG. 9 internode vascular analysis of zebra fish model constructed by eif1axb knockdown; (a) normal internode vascular development in the control group; (B) ATG-MO 1ng zebra fish model; (C) ATG-MO 1.5ng zebra fish model; (D) E3I3-MO 1ng zebra fish model; (E) E3I3-MO 1.5ng zebra fish model; (F) ISV mean length was quantified, SEM (n=10; analysis of variance; P < 0.0001).

FIG. 10 is a graph of heart anatomy and tissue section of a mouse knocked down eif1ad7 neonate; (a) a control cardiac anatomic map; (B) mouse heart anatomy following si-RNA intervention; (C) control cardiac tissue section; (D) si-RNA intervention followed by a tissue section of the mouse heart and an enlarged view of the defect site.

Detailed Description

The following examples facilitate a better understanding of the present invention, but are not intended to limit the same. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores.

FA used in the following examples represents folic acid, MTHF-Ca represents 6S-5-methyltetrahydrofolate calcium, molecular weights 443.40 and 497.52, respectively, and 6S-5-methyltetrahydrofolate is present in the blood circulation instead of folic acid.

The primers used in the examples below are shown in Table 1 unless otherwise specified.

Table 1, summarized below using primers:

EXAMPLE 1 discovery of the GM5662 Gene

Exogenous gonadotrophin therapy (PMSG/hCG) induced superovulation was used to assess the quality of ovulated oocytes in female mice (C57 BL/6J). An in vitro fertilization procedure is then performed. Fertilized eggs were randomly grouped and used as controls with potassium only optimized medium (KSOM) medium, KSOM-FA (60 ng/L) and KSOM-MTHF (60 ng/L). After 24, 48 and 72 hours of culture, blastula were collected, observed under a microscope and stored in liquid nitrogen.

Blastocysts of mice 72 hours after in vitro fertilization were collected from three groups of control group, 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. A normalized cDNA library was constructed using 1. Mu.g total RNA. Sequencing libraries were generated using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB, usa) and paired-end read sequencing was performed on Illumina HiSeq 2000 platform. All sequencing was processed and analyzed by Genewiz Bio-pharm Technology Corporation (Suzhou, china).

And extracting total RNA for library construction, and performing high-throughput sequencing by using an Illumina platform. Sequencing reads were evaluated using Bcl2FASTQ (V2.17.1.14) to generate FASTQ files. The initial quality control of the FastQC (V0.10.1) and Cutadapt (V1.9.1) files is performed to filter low quality data, including adapter-introduced contamination and redundant sequences. Clean reads were compared to the mouse genome by Hisat2 (V2.0.1) and gene expression was quantified by Htseq (V0.6.1) based on short reads of fragments per megabase (FPKM). Differentially Expressed Genes (DEGs) were identified using DESeq2 (V1.6.3) in the Bioconductor package.

DEG (differentially expressed gene) was identified using DESeq2 (V1.6.3) in the Bioconductor package. Volcanic mapping was done in R (V4.0.3) using gglot 2 package.

By in vitro fertilization and RNA transcriptome analysis of cultured blasts, the data indicated that fertilized eggs of Gm5662 in the mouse genome were differentially expressed in FA medium compared to MTHF-Ca (see FIGS. 1A, 1B). Homology of Gm5662 between human, zebra fish and mouse sequences was obtained by alignment of DNAMAN (V6.0.3). Gm5662 is the mRNA sequence recorded in the mouse genome database, but the gene has not yet been identified. Using the advantage of comparative gene mapping, we performed a blast search for homology analysis between Gm5662 (M.museuus) and AAP36772.1 (H.sapiens), and found that human EIF1AX has 92% homology with Gm5662 in amino acid sequence. Thus, gm5662 was identified as the eif1ad7 gene in mice (m.musculus) and eif1axb in zebra fish (d.rerio). The gene is conserved in eukaryotes, has high homology between humans (EIF 1 AX), mice (EIF 1ad 7) and zebra fish (EIF 1 axb) (fig. 2), is orthologous to the human EIF1AX gene during early development of zebra fish, has 81.5% homology to the human EIF1AX gene, and is conserved at the protein level at 99.3%. Specifically, the sequence of the human gene eif1ax is shown as SEQ ID NO.1, the sequence of the mouse gene eif1ad7 is shown as SEQ ID NO.2, and the sequence of the zebra fish gene eif1axb is shown as SEQ ID NO. 3.

Example 2 investigation of the Effect of FA on the eif1axb Gene expression of Zebra fish embryos by RT-PCR

In zebra fish, 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 PrimeScript RT kit with gDNA Eraser (Takara). The eif1axb gene expression was quantified three times using Bio-rad iQ SYBR Green Supermix (Bio-rad) and tested on the replex system (Eppendorf). Relative gene expression quantification is based on a comparison threshold cycle method (2- ΔΔct). The primers used in the real-time RT-PCR experiments are shown in Table 1. Real-time RT-PCR results showed that in zebra fish of 12 to 48hpf, the eif1axb gene was down-regulated by FA, but not MTHF-Ca (FIG. 3). I.e., FA, but not MTHF-Ca, causes cardiotoxicity, resulting in a defect in zebra fish embryo development. High doses of folic acid also lead to an increased incidence of heart defects in fetal mice, which have been repeated in two separate laboratories.

Example 3 preparation of zebra fish model

Using morpholino antisense oligonucleotide (MO) technology, we prevented the expression of eif1axb in embryonic zebra fish and analyzed the effect of knockdown on fish development.

The fish used in the experiments of zebra fish (Danio rerio, hamilton, wild type) are adult zebra fish with the age less than 1, the pH value of the breeding water body is 7+/-0.2, the temperature is about 28 ℃, the time ratio of light to darkness is 14h to 10h, and the fish are fed with artemia cysts twice a day. Three zebra fish were placed in spawning boxes one day in the evening, and fish eggs were collected the next morning. The roe is placed in embryo culture solution (prepared from sea salt of 0.2 g/L), and cultured at 28deg.C.

The MO sequence was designed for the ATG site (ATG-MO) of the zebra fish eif1axb gene, and the third exon/third intron junction (E3I 3-MO), with the addition of Standart Control MO. The MO was designed and purchased by Gene Tools (Philomath, OR). The sequences of eif1axb translational and splice-blocking morpholines were 5'-CTCCTTTTCCTTTGTTTTTCGGCAT-3' (ATG-MO) and 5'-CAGCCTGAAGCTCTAAAATGCACCT-3' (E3I 3-MO), respectively. The sequence of the standard control morpholine is 5'-CCTCTTACCTCAGTTACAATTTATA-3' (gene tool). The MO amounts used for injection were as follows: control-MO and E3I3-MO, 2ng per embryo; ATG-MO, 2ng per embryo. Primers spanning eif1axb exon 2 (forward primer: 5'-GCGACGTGGTAAGAACGAGAA-3') and exon 5 (reverse primer: 5'-GGCTTTCAGACTCCTAGCCT-3') were used for RT-PCR analysis to confirm the efficacy of E3I 3-MO. 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 for testing the validity of eif1axb Morpholino Oligonucleotides (MO).

Embryos and larvae were analyzed with a nikon SMZ18 fluorescence microscope and then photographed with a digital camera. The level, brightness, contrast, hue and saturation of the subset of images was adjusted using Adobe Photoshop 7.0 software (Adobe, san Jose, california) to optimize the visualization of the expression pattern. Quantitative image analysis using image-based morphological analysis (NIS-Elements D4.6) and imageJ software (NIH, http:// rsbweb. NIH. Gov/ij /). The fluorescence image is inverted for processing. Positive signals were defined by particle count using ImageJ. The 10 animals per treatment were quantified and the total signal for each animal was averaged.

The phenotype of eif1axb knockdown was analyzed using MO technology. FIG. 4 shows the effectiveness of eif1axb knockdown, and RT-PCR and real-time quantitative RT-PCR confirm knock down efficiency of eif1axb. The eif1axb knockdown resulted in pericardial edema, reduced contractility, and reduced precordial congestion, which is evidence of overt heart failure, as compared to control zebra fish (fig. 5A). Embryo defects increased while survival decreased (fig. 5D and E). Quantification of pericardial area showed a significant increase in eif1axb mo group (FIG. 5F). The torso region images taken at 3-dpf, vascular structures were visualized by eGFP fluorescence and labeled ISV (internode vessels) and DLAV (dorsal longitudinal side vessels), showing regular development of embryos injected with control MO. Embryos injected with eif1axb-MO exhibited thinner ISVs compared to control MO (fig. 5K and L, arrows). In the control embryo, parachordal vessels (PAV) form normally (fig. 5J). MO knockdown of eif1axb prevents the formation of the precursor paravertebral vessels (PAV) of the lymphatic system compared to the control. The relevant area is shown at a higher magnification (fig. 5n & o). Quantification of ISV average length and diameter showed a significant decrease in the eif1axb mo group (FIG. 5P-Q). Thus, down-regulation of the eif1axb gene inhibited early vascular development in zebra fish.

Eif1a function in heart, torso and tail development

Most moderately affected Eif1a (89.1%, n=110) had a significantly enlarged pericardial space. At the same time, the heart rate of the embryo is influenced, and particularly, the heart rate of the embryo at 48hpf is influenced the most.

Moderately affected Eif1a (68.9%, n=103) exhibited significant body and/or tail kinks. Defects became apparent at 25hpf and persisted in older embryos (2-7 days old, data not shown).

Function of Eif1a in neurodevelopment

Central Nervous System (CNS) development of Eif1a was assessed by examining brain morphology and staining patterns of several neural differentiation markers in the biopsies. At 25hpf, with different brain regions (e.g., cerebellum, hindbrain, optic head), the dorsal portion of the optic head posterior wall and the cerebellum dorsal side appear thinner in Eif1 a-MO.

Example 4eif1axb lack of Signal pathway

To investigate how eif1axb mediates angiogenesis, we subsequently screened and examined the expression of 7 genes that previously proved to be closely related to heart defects and/or angiogenesis for 2-dpf and 3-dpf. In zebra fish, 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 PrimeScript RT kit with gDNA Eraser (Takara). Relative gene expression quantification is based on a comparison threshold cycle method (2- ΔΔct). Primers used in the real-time RT-PCR experiments are summarized in supplementary table 1.

We found that three of the genes (dll 4, efnb2a and s1pr 1) were significantly reduced, while the other three genes (notch 1b, ptprb and s1pr 2) were dramatically increased, except that cd146 remained unchanged (FIG. 6). We speculate that this is the result of negative feedback regulation to avoid excessive reduction of the vascular system. In this study we found that the zebra fish gene eif1axb controls angiogenesis in vivo by targeting VE-PTP. Eif1axb deficiency may regulate dll4, ptp-rb, notch1b and efnb2a expression through a negative feedback loop. These in vivo results indicate that the eif1axb gene plays an important role in cardiovascular and embryonic development.

EXAMPLE 5 Zebra fish model for cardiovascular developmental defects

The fish used in the experiments of zebra fish (Danio rerio, hamilton, wild type) are adult zebra fish with the age less than 1, the pH value of the breeding water body is 7+/-0.2, the temperature is about 28 ℃, the time ratio of light to darkness is 14h to 10h, and the fish are fed with artemia cysts twice a day. Three zebra fish were placed in spawning boxes one day in the evening, and fish eggs were collected the next morning. The roe is placed in embryo culture solution (prepared from sea salt of 0.2 g/L), and cultured at 28deg.C.

Gene Tools, LLC (http:// www.gene-tools.com /) designed Morpholino (MO). Antisense MO (GeneTools) were microinjected into fertilized single cell stage embryos according to standard protocols (ref 4). The sequences of eif1axb translational and splice-blocking morpholines were 5'-CTCCTTTTCCTTTGTTTTTCGGCAT-3' (ATG-MO) and 5'-CAGCCTGAAGCTCTAAAATGCACCT-3' (E3I 3-MO), respectively. The sequence of the standard control morpholine is 5'-CCTCTTACCTCAGTTACAATTTATA-3' (gene tool). The amount of MO used for injection is as follows: control-MO and E3I3-MO, 1.5ng per embryo; ATG-MO, 1.5ng per embryo.

To assess vascularization in zebra fish embryos, embryos were anesthetized with 0.016% ms-222 (tricaine mesylate, sigma-Aldrich, st.louis, MO). The zebra fish was then positioned on the sides (front, left; back, right; back, up) and mounted in a recessed slide with 3% methylcellulose for viewing by fluorescence microscopy. Embryos and larvae were analyzed with a nikon SMZ18 fluorescence microscope and then photographed with a digital camera. The level, brightness, contrast, hue and saturation of the subset of images was adjusted using Adobe Photoshop 7.0 software (Adobe, san Jose, california) to optimize the visualization of the expression pattern. Quantitative image analysis using image-based morphological analysis (NIS-Elements D4.6) and imageJ software (NIH, http:// rsbweb. NIH. Gov/ij /). The fluorescence image is inverted for processing. Positive signals were defined by particle count using ImageJ. The 10 animals per treatment were quantified and the total signal for each animal was averaged.

The results showed that the zebra fish embryo injected with 1ng and 1.5ng of eif1axb-MO reagent developed slightly retarded and overall survival was observed within 7 days, and treatment with ATG-MO 1ng, 1.5ng and E3I3-MO 1ng, 1.5ng resulted in a slight length shortening and pericardial edema, see FIG. 7. Since the biological function of the eif1axb gene is related to the cardiovascular development of embryos, we take fluorescent images and count of the three day development of zebra fish subintestinal venous vessels (SIV). The intestinal venous vessels (SIV) developed into a smooth basket-like structure on egg yolk at 3-dpf in the control embryo (FIG. 8A). The MO treated zebra fish embryos showed ectopic SIV fragments and reduced vessel numbers (fig. 8B-E). SIV regions (fig. 8F) SEM (n=10; analysis of variance; × P < 0.0001) were further quantified. It is shown that there is a certain dose relation for interfering MO with vascular development.

We labeled ISV (internode vessels) and DLAV (dorsal longitudinal anastomotic vessels) by eGFP fluorescence visualization, and related vessels were shown to develop regularly in the control group, ATG-MO and E3I3-MO treated embryos exhibited thinner ISV (arrows) or incomplete ISV and ectopic (asterisks) (see FIGS. 9A-E). ISV mean length was quantified (fig. 9F), SEM (n=10; analysis of variance; × P < 0.0001).

The zebra fish vascular development disorder model is prepared, mild defects such as intestinal vein blood vessels, internode blood vessels and the like of zebra fish embryos can be realized through MO dosage adjustment, and the zebra fish embryo can be used for developing related medicines.

Example 6 preparation of mouse model

Exogenous gonadotrophin therapy (PMSG/hCG) induced superovulation was used to assess the quality of ovulated oocytes in female mice (C57 BL/6J). An in vitro fertilization procedure is then performed. Optimized Medium (KSOM) medium was used with potassium alone. After 24, 48 and 72 hours of culture, blastula were collected, observed under a microscope and stored in liquid nitrogen.

Antisense oligonucleotide (siRNA) 5'-GCCAUAUAAGAGGGAAGCUTT-3',5'-AGCUUCCCUCUUAUAUGGCTT-3', negative control siRNA 5'-UUCUCCGAACGUGUCACGUTT-3',5'-ACGUGACACGUUCGGAGAATT-3' was used for the study. The above sequence was designed and purchased by Ji Ma pharmaceutical technologies limited. The siRNA was dissolved in RNase-free buffer, using a Narishige MI300 microinjector and injected into embryos at 20. Mu.M to one to four cell stages. After the injection, the surviving cells were cultured in KSOM medium and transferred to an incubator for 3 days. A small hole is made on the oviduct wall between the ampulla and the ovary of the pseudopregnant mouse, the tip of the liquid-transfering device is inserted into the hole, the opening of the liquid-transfering device points to the ampulla, and the embryo is implanted into the pseudopregnant mouse. Female mice were kept individually until birth.

All neonatal mice were observed and body weight was measured on the day of birth, we measured an electrocardiogram (data not shown) on a portion of the mice and when they grew to 6 days old, dissected the chest of the mice, observed heart morphology and beat under a body field microscope, and photographed (SMZ 168, motic, mansion, fowler, china). The heart tissue of the mice was fixed with 4% paraformaldehyde and paraffin embedded. H & E stained tissue sections were examined with panoramic image acquisition system IX73 (Olympus, tokyo, japan).

The results showed that the heart of the control mice developed normally, and after the eif1ad7 expression was disturbed, the heart of the neonatal mice was slightly smaller (see fig. 10A-D), part of the cardiac ischemia of the neonatal mice, and part of the heart wall between the atrium and ventricle was found to be defective by 46.5% (n=43), indicating that the model preparation was successful.

The result of the embodiment of the invention also shows that the Eif1ad7 participates in the pathogenesis of congenital heart disease of mice, so that the medicine aiming at the target spot of the Eif1ad7 has good prospect of treating and preventing birth defects.

Claims (10)

1. The application of the gene eif1axb in constructing the cardiovascular development disorder and skeletal abnormality model of the zebra fish.

2. A construction method of a zebra fish model with cardiovascular developmental disorder and skeletal abnormality is characterized in that zebra fish embryo genes eif1axb are knocked out or inhibited, and the zebra fish model is obtained.

3. Use of the model constructed according to the method of claim 2 for the screening of drugs for the treatment of cardiovascular developmental disorders and skeletal abnormalities; preferably, the application in the screening of medicaments for treating cardiovascular developmental disorders and skeletal abnormalities caused by defects in the gene eif1axb.

4. The application of the gene Eif1ad7 in constructing a model of congenital cardiovascular diseases and congenital heart diseases of mice.

5. A construction method of a congenital cardiovascular disease and congenital heart disease mouse model is characterized in that the method is that after the gene Eif1ad7 in a mouse embryo is inhibited to express or the gene Eif1ad7 is over-expressed, the embryo is obtained after the embryo develops in a female mouse.

6. Use of the model constructed by the method of claim 5 for screening drugs for congenital cardiovascular diseases and congenital heart diseases; preferably, the application in the screening of drugs for treating congenital cardiovascular diseases and congenital heart diseases caused by the deficiency of the gene eif1ad 7.

7. Application of folic acid as a regulator of zebra fish gene eif1axb or mouse gene eif1ad7 or human gene eif1 ax.

8. Application of folic acid as zebra fish gene eif1axb or mouse gene eif1ad7 or human gene eif1ax inhibitor.

9. Application of a reagent for detecting gene eif1ax in preparation of congenital heart disease diagnosis or auxiliary diagnosis reagent.

10. The application of the gene eif1ax in a therapeutic target of a drug for treating congenital heart disease.

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