CN110564771B - Preparation method of cerebral calcification disease model - Google Patents

Abstract

The invention relates to and discloses a preparation method of a cerebral calcification disease experimental model. The method of the invention takes MYORG gene as a target point, and the MYORG gene is deleted, reduced in expression or inactivated. The experimental model obtained by the method is an effective and typical model of the cerebral calcification disease, can be used for researching the cerebral calcification disease and can be used for screening and testing specific medicines.

Description

Preparation method of cerebral calcification disease model

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a novel cell or animal model for treating cerebral calcification disease and a preparation method thereof.

Background

Cerebral calcification disease is a disease in which calcium salt deposition occurs in the brain, which affects brain substance metabolism and normal functions, and induces various sensory, motor, cognitive and emotional disorders. The brain calcification disease is widely dangerous, the incidence rate of the brain calcification disease is 1% in young people, the detection rate of the brain calcification disease is increased to 20% in old people, and the detection rate of the brain calcification disease is up to 45% in a few diseases such as Down syndrome patients. The clinical symptoms are various and mainly manifested as dyskinesia, mental disorder, cognitive disorder, epileptic seizure, dizziness, headache and the like. The mild one has no obvious clinical symptoms, while the severe one can have personality and behavior changes, which finally results in psychosis or dementia. However, the current clinical response strategy mainly adopts symptomatic treatment and can not radically cure, and no effective medicine can remove or reduce abnormal calcium deposition at present, mainly because the basic mechanism of the occurrence of the brain calcification is not known at present. Among them, Primary familial calcification of the brain (PFBC) is a genetic disease of the nervous system characterized by symmetrical calcification of the basal ganglia or other brain sites. The development of genomics in recent years is benefited, genetic pathogenic factors of the cerebral calcification disease are discovered in succession, and the understanding of human beings on the disease is gradually expanded. Since this disease was described systemically by Fahr scholars in 1930, it is also called Fahr disease. The disease is more than the disease after middle-aged, the disease course progresses slowly, and no obvious sex difference exists. There may be significant heterogeneity in its clinical presentation between different families or patients within the same family. Craniocerebral CT is the main means for diagnosing the disease, and can be expressed as symmetrical massive calcifications at globus pallidus, caudate nucleus, dentate nucleus, white brain matter, thalamus, cortex and other parts (figure 1), and the calcifications can be connected into large sheets along with the progress of the disease. The pathology of the disease is found to be mainly deposition of calcium salt particles (taking hydroxyapatite as a main component) in tissues around terminal arterioles, veins and capillaries.

PFBC is sporadic or familial in onset, has high genetic heterogeneity, and has more autosomal dominant genetic studies. Starting in the 90 s of the 20 th century, researchers around the world performed extensive genetic studies on PFBC, until 2012, by professor of liu statics university, china science and technology university, wuhan, cloned the first causative gene SLC20a2 of PFBC. Thereafter, pathogenic genes mainly including PDGFRB, PDGFB, XPR1 and the like were successively discovered and verified. The SLC20A2 gene encodes sodium-phosphorus cotransporter (PiT2) protein, which is a main transporter molecule responsible for extracellular phosphorus uptake expressed in brain. The protein encoded by the XPR1 gene is involved in the excretion of phosphorus from cells. The PDGFRB gene encodes a platelet-derived growth factor receptor, belonging to the type III receptor tyrosine kinase family; the PDGFB gene encodes a platelet-derived growth factor, and the combination of the platelet-derived growth factor and the PDGFB gene activates downstream signaling pathways, participates in the development of human pericephalic cells (percyte), and maintains the integrity of the blood brain barrier.

In conclusion, the analysis of the pathogenic gene and pathogenesis of PFBC at the present stage is helpful for developing the understanding of the pathogenesis of the cerebral calcification disease. According to the research rules of other diseases, any disease can be divided into hereditary and non-hereditary factors, and the identification of hereditary factors and the research on related molecular cellular mechanisms can reveal the internal cause of the disease, which is the most critical breakthrough for the elucidation of the pathological molecular signaling network driving the disease. At present, the cognition of the cerebral calcification disease is still in an early stage, a new pathogenic gene is discovered, the pathological mechanism of the cerebral calcification disease is cleared, a novel animal model is developed, and a new treatment means is developed on the basis of the pathogenic gene, so that the cerebral calcification disease is a current urgent task.

Disclosure of Invention

The invention aims to provide a preparation method of an experimental model of cerebral calcification disease. On the basis, a specific experimental model and pathological feature description thereof are provided.

In a first aspect of the invention, there is provided a method of preparing a model of cerebral calcification disease, said method comprising deleting, reducing expression or inactivating the MYORG gene in the genome of the model.

In a preferred embodiment, the method for deleting, reducing expression or inactivating the MYORG gene in the genome of the model includes (but is not limited to): gene knockout, gene editing, RNA interference, epigenetic suppression, blocking or inhibiting the activity of a MYORG protein or a homologous protein thereof with an antibody against the MYORG protein or a homologous protein thereof.

In another preferred embodiment, the gene knockout includes (but is not limited to): stem cell targeting technology based on homologous recombination, gene editing technology, chemical molecule mutagenesis genome mutation, transposon site-directed inactivation and the like; such gene editing includes (but is not limited to): CRISPR/cas9, spf1, TALEN, Zinc Finger Nuclease (ZFN), Base editing.

In another preferred embodiment, the MYORG gene is deleted, reduced in expression or inactivated in the genome of the model using gene editing techniques; preferably, the base deletion or insertion is caused by gene editing, resulting in premature termination of the protein coding frame

In another preferred embodiment, the gene editing techniques target the MYORG gene, thereby causing corresponding changes in the amino acid sequence, including (but not limited to): p.w75, p.w443, p.m35v, p.r441g, p.s232l, p.q203, p.r261l, p.116_117insLAFR and p.365_366 delFD.

In another preferred example, a stop codon is introduced in the MYORG gene after amino acid 63 of its encoded polypeptide to terminate translation prematurely; preferably, sgRNA of the nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 are used for gene editing.

In another preferred example, the MYORG gene in the genome of the model is deleted, expression is reduced or inactivated while a reporter gene is introduced; preferably, the reporter gene replaces the original MYORG gene for expression; more preferably, the reporter gene is introduced after the MYORG gene promoter.

In another preferred embodiment, the model is an animal model or a cell model.

In another preferred embodiment, the animal is a mammal including, but not limited to, a rodent, a non-human primate; preferably, the rodent comprises a mouse (mouse) or a rat (rat); the non-human primate comprises a macaque or a marmoset; the cell includes a eukaryotic cell.

In another preferred embodiment, the cerebral calcification disease is primary familial cerebral calcification disease.

In another preferred embodiment, said deleting, reducing expression or inactivating the MYORG gene in the genome of the model comprises: simulating MYORG gene pathogenic mutation of human brain calcification patients; preferably, the mutations comprise mutations corresponding to positions of the human MYORG genes c.225g > a, c.1328g > a, c.103a > G, c.1321c > G, c.695c > T, c.607c > T, c.782_783GC > TT, c.348_349insCTGGCC TTCCGC and c.1092_1097 delCTTCGA.

In another aspect of the present invention, there is provided a use of the model of cerebral calcification disease obtained by any one of the above-mentioned methods for preparing a model for indicating and analyzing the onset stage of cerebral calcification disease, or for screening a candidate drug or treatment for treating cerebral calcification disease; or as a model for studying brain calcification.

In another aspect of the present invention, there is provided the above kit for preparing a model of cerebral calcification disease, said kit comprising: an agent that deletes, reduces expression, or inactivates the MYORG gene in the genome of the model.

In a preferred embodiment, the agent that deletes, reduces expression or inactivates the MYORG gene in the genome of the model includes, but is not limited to: gene knockout agents, gene editing agents, RNA interference agents, epigenetic suppressive agents.

In another preferred embodiment, the agent that causes deletion, decreased expression or inactivation of the MYORG gene in the model genome comprises CRIPSR/cas9 gene editing agent.

In another preferred embodiment, said cas9 gene editing agent produces a genomic fragment deletion in the MYORG gene, thereby causing premature translation termination of the protein encoded by the MYORG gene after amino acid number 63; more preferably, the sgRNA is a nucleotide sequence shown in SEQ ID NO. 1 and SEQ ID NO. 2; or the cas9 gene editing reagent introduces a reporter gene after the promoter of the MYORG gene and causes the MYORG gene to be deleted.

In another preferred embodiment, the gene editing reagent further comprises: CRISPR/Cas9mRNA or protein, or an agent, such as a plasmid, that forms CRISPR/Cas9mRNA or protein within a cell.

In another aspect of the present invention, there is provided a method for screening a candidate drug or a therapeutic regimen for treating a cerebral calcification disease using the model of the cerebral calcification disease obtained by any one of the methods described above, comprising: administering a candidate substance or treatment regimen to said test model, and observing the test model for the presence of remission or improvement (either statistically significant or significant) of the disease, and if so, for the potential drug or treatment of the underlying brain calcification disease.

In a preferred embodiment, the screening method is for the purpose of screening for a drug, and is not diagnostic or therapeutic.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, PFBC patient brain calcification CT image of Myorg gene inactivation mutation.

FIG. 2, analysis of Myorg gene expression in mouse brain.

FIG. 3, schematic of Myorg knock kout mouse construction.

FIG. 4, schematic representation of the preparation of Myorg-GFP mice.

Figure 5, Myorg knockout mouse calcification plot.

Detailed Description

The inventor of the invention discloses a method for establishing a cerebral calcification disease experimental model and application thereof through intensive research. The method of the invention takes Myorg gene as a target spot to delete, reduce expression or inactivate the Myorg gene. The experimental model obtained by the method is an effective and typical model of the cerebral calcification disease, can be used for researching the cerebral calcification disease, and can be used for screening and testing specific medicines, gene therapy or other treatment schemes.

MYORG gene

The inventor prepares a cerebral calcification disease experimental model by knocking out and inactivating mutation of a target gene by locking a MYORG gene as a new pathogenic gene of the cerebral calcification disease through genetic research of human cerebral calcification patient families and repeated research and demonstration. The experimental model comprises an animal model or a cell model and the like.

In humans (Homo Sapiens), the MYORG gene is globally called a myogenesis modulating glycosidase (pure), its NCBI gene ID is 57462, also known as KIAA1161 or NET37, is located on chromosome 9p13.3, its cDNA sequence (open reading frame) has NCBI number NM-020702.4 (see also SEQ ID NO:9 of the present invention), and protein sequence number NP-065753.2; in mice (Mus Musculus), the Myorg gene, also known as AI464131, has an NCBI gene ID of 329828, located on chromosome 4; 4A5, the cDNA sequence number is NM-001085515.2, the protein sequence number is NP-001078984.1. In other species, this gene is now commonly named Myorg, and homologous genes from different species are available via the NCBI website HomoloGene database under number 19853. The human MYORG gene encodes a 714 amino acid protein (716 amino acids in the mouse MYORG protein) that is a member of the glycosyl hydrolase 31 family. MYORG encoding protein has a certain function in muscle cell development, so that the MYORG encoding protein is named as myocyte generation regulation type glycosidase.

In the existing research, the MYORG gene is found to be involved in regulating the development of muscle cells, but both researches only adopt in vitro cultured cells, and whether the MYORG gene plays a key regulation role in the development of the muscle cells still needs research data of an animal model. Prior to the research work of the applicant team, there was no evidence or report of any correlation with human brain calcification. The inventor discovers that MYORG gene is an important autosomal recessive inheritance PFBC pathogenic gene for the first time, and applies the MYORG gene to preparation of a cerebral calcification disease experimental model for the first time, so that the MYORG gene is taken as a research platform, the pathological mechanism of the cerebral calcification disease can be researched, a novel biomarker and a novel diagnostic index can be researched and discovered in the middle stage, and the MYORG gene can be used for screening and testing a new candidate drug, gene therapy or other treatment strategies in the later stage.

Expression analysis revealed that MYORG was specifically expressed in murine brains in S100 positive astrocytes. The cauda poda of astrocytes, together with perivascular cells, form a neurovascular unit, and this is precisely the site of most craniocerebral pathological calcium deposition; however, it remains to be investigated how MYORG loss leads to brain calcification, which is one of the values of the model described in the present invention. Unlike the 4 disease-causing genes discovered in the past, the MYORG mutation accounts for more than half of the PFBC families inherited in a stealth manner, is a newly disclosed recessive disease-causing gene, and suggests that a brand-new and important gene and cell type participate in the disease-causing process of PFBC. For genetic diseases, the development of animal models depends on the discovery and validation of pathogenic genes and specific pathogenic mutations. Different from other current animal models related to pathogenic genes, the animal model mainly shows brain calcification, and other models have problems in aspects of brain development disorder, brain metabolism, birth death and the like, so the animal model has irreplaceable effect in research and development of new treatment means. The invention provides a powerful tool for exploring PFBC pathogenesis and developing novel treatment strategies.

It is understood that the term "MYORG" also includes variants of the naturally occurring MYORG gene, homologous genes, specific versions of which can be obtained via a database query from NCBI's gene database, Ensemble gene browser database, UCSC gene database, and uniport. Representative examples include: nucleotide sequence encoding a MYORG protein identical to the wild type due to codon degeneracy, nucleotide sequence encoding a conservatively variant polypeptide of a wild type MYORG protein.

Preparation of Experimental model

The invention provides a method for preparing a brain calcification disease experimental model. The method comprises the following steps: the MYORG gene in the genome is deleted, obviously reduced or inactivated.

According to the new discovery of the invention, the idea of preparing the experimental model can be applied to preparing cell models and animal models. The cell model includes mammalian cell models or other eukaryotic cell models, such as human cells or mouse cells. Such animals include, but are not limited to: non-human primates (including stone crab monkeys, rhesus monkeys, marmosets, etc.), rodents (including rats, mice, hamsters, etc.) and the like, in which the MYORG gene is highly conserved, i.e., the presence of the MYORG gene, a variant thereof, or a homologous gene thereof, occurs in different animals. Preferably, the animals are rodents and non-human primates, including but not limited to: rat, mouse, macaque, marmoset. It will be appreciated that the core of the protection of the present invention lies in the model design and implementation strategy of the association of a loss-of-function mutation in the MYORG gene leading to cerebral calcification, including but not limited to the protocol described in the subsequent examples of the invention.

As used herein, the term "animal with brain calcification disease" or "animal model with brain calcification disease" refers to an animal that produces one or more conditions selected from the group consisting of: symmetric calcification occurring in basal ganglia, thalamus, hindbrain, cortex and other brain regions, different degrees of somatomotor disorders, psychotic disorders, cognitive disorders, different degrees and forms of epilepsy, dizziness, headache, etc. The intracerebral calcification can be detected by imaging techniques such as craniocerebral CT, MRI and the like, and can also be analyzed by brain tissue section and calcium salt deposition staining.

As used herein, the term "MYORG gene expression deletion, reduction or inactivation" refers to a homozygous deletion, reduction or inactivation of the MYORG gene. However, it cannot be excluded that experimental models of MYORG gene expression deletion, reduction or inactivation, based on heterozygosity, add other gene manipulation methods to achieve a scheme similar to homozygous knockout and triggering intracerebral calcification. Most preferably, the model is a homozygous experimental model.

As used herein, the expression "lack, reduction of expression of a MYORG gene" refers to a modified animal (whole body or partial tissue) or cell that does not express a MYORG gene, or that has a significantly reduced amount of expression, e.g., to less than 30% of the wild-type, as compared to the wild-type animal or cell; preferably to less than 20% of wild type; more preferably to less than 10% or 5% of wild type. Also, by "MYORG gene inactivation" is meant that the engineered gene expresses only deletion-type products without complete function, such as protein fragments lacking a critical functional domain (C-terminal, N-terminal or middle segment).

As used herein, the term "deletion, reduction or inactivation of MYORG gene expression" includes deletion, reduction or inactivation of MYORG gene expression, either systemically, or in a particular cell type, or in a particular tissue range. In particular, equivalent mutations in model animals generated by mimicking MYORG gene-causing mutations in human PFBC patients (c.225g > a, c.1328g > a, c.103a > G, c.1321c > G, c.695c > T, c.607c > T, c.782_783GC > TT, c.348_349insCTGGCCTTCCGC and c.1092_1097delC TTCGA) result in loss, reduction or inactivation of expression of the MYORG gene in the model animals.

Various methods may be used to delete, reduce or inactivate "MYORG gene expression" including: performing gene knockout through stem cell targeting (homologous recombination, transposon site-directed inactivation and the like); or by gene editing techniques (including CRISPR/cas9 (or spf1), TALENs, zinc finger nucleases, Base editing techniques, etc.); or by RNAi or epigenetic suppression, etc.; or treatment with an anti-MYORG antibody renders the MYORG protein inactive. It will be appreciated that a variety of methods known in the art to down-regulate expression or inactivation of a gene or polypeptide are available.

As a preferred mode of the invention, the MYORG gene is knocked out by means of gene editing, resulting in the MYORG gene not expressing a fully functional protein product. Preferably, the CRISPR/Cas9 system is used for gene editing. By selecting an appropriate sgRNA target site, high gene editing efficiency is brought about.

In a preferred embodiment of the present invention, a preferred target site is designed and provided. Introducing the sgRNA or a nucleic acid capable of forming the sgRNA, Cas9mRNA or a nucleic acid capable of forming the Cas9mRNA into a fertilized egg of an animal (e.g., a mouse) to obtain a gene-edited animal (e.g., a mouse). Cas9mRNA and sgRNA can be obtained through in vitro transcription, nucleic acid capable of forming the sgRNA can be used as a nucleic acid construct or an expression vector, or the nucleic acid capable of forming the Cas9mRNA can be used as a nucleic acid construct or an expression vector, and the expression vectors are introduced into cells, so that active sgRNA and Cas9mRNA are formed in the cells. The mode of introduction is preferably microinjection into fertilized eggs, and electroporation or lipofection into cells, but does not exclude other effective introduction methods.

As an alternative of the invention, the Cre and loxp methods are applied to selectively knock out, reduce or inactivate the MYORG gene in the genome of the animal or cell.

The experimental model constructed by the invention can be used for screening and testing specific drugs, can be used as a powerful tool for scientific research and new drug evaluation, and has good stability.

The invention also provides a method for screening candidate drugs or therapeutic agents for treating the cerebral calcification disease by using the experimental model, which comprises the steps of administering a candidate substance to the experimental model, observing whether the experimental model has remission or improvement of the disease, and if so, determining that the candidate substance is a potential drug for the cerebral calcification disease.

In the present invention, a candidate drug or therapeutic agent refers to a substance known to have a certain pharmacological activity or being tested, which may have a certain pharmacological activity, and includes, but is not limited to, nucleic acids, proteins, saccharides, chemically synthesized small or large molecular compounds, cells, natural product extracts, and complex components thereof. The candidate drug or therapeutic agent may be administered orally, intravenously, intraperitoneally, subcutaneously, intragastrically, electroporated, intraspinal, or by direct intracerebral injection.

It should be noted that the research on PFBC, a kind of brain calcification, not only promotes the understanding of hereditary brain calcification disease, but also promotes the research on the pathogenesis of global brain calcification disease. In contrast to diabetes, type one diabetes is hereditary and type two diabetes is mainly non-hereditary. The research on the type I diabetes provides great help for the pathogenesis and the treatment method of the type II diabetes. In addition, the first type diabetes and the second type diabetes are greatly overlapped in medication, and insulin is not only suitable for the first type but also suitable for the second type. Therefore, for the cerebral calcification disease, the cerebral calcification disease model contained in the invention is not only suitable for PFBC, but also a valuable tool for the pathological research and the development of treatment schemes of the non-PFBC cerebral calcification disease.

Reagent kit

Based on the method, the invention also provides a kit for preparing the experimental model of the cerebral calcification disease, wherein the kit comprises: an agent that deletes, reduces or inactivates MYORG gene expression in the genome.

The kit can also comprise an instruction manual for implementing the method for preparing the animal model with the cerebral calcification disease, so that the application of the method is convenient for a person skilled in the art.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 expression analysis of MYORG Gene

A6-8 week C57BL/6N mouse (20-30g) was anesthetized and then treated with PBS,taking brain after 4% paraformaldehyde perfusion, fixing in 4% paraformaldehyde, dehydrating with 30% sucrose, and cutting into brain slices with thickness of 40um (RNase-Free is required). Applications of

2.5HD Assay-BROWN kit (Advanced Cell Diagnostics, Hayward, Calif.) and a designed and synthesized MYORG Probe (Probe C1Cat.300031) were used for in situ acrobatic analysis of MYORG genes in brain slices.

As a result of experiments, it was found that MYORG has wide expression in the brain cortex (Ctx), hippocampus (Tha), thalamus (Hip), striatum (Str), and cerebellum (Crb), and has expression in brain regions with more calcification, such as cerebellum, thalamus, and striatum (FIGS. 2a, 2b, and 2 c).

In the cerebellum, the Purkinje Cell Layer (PCL) has high hybridization signal intensity, and according to the core and distribution characteristics, it is suggested that it is likely to be a special morphological type of astrocyte, i.e., Bergman Glia (FIG. 2 b). To further analyze the expression pattern of MYORG in cells, fluorescence in situ hybridization was used (

The analysis of neuronal Cell types was performed by the Fluorescent Reagent Kit v2, Advanced Cell Diagnostics, Hayward, CA) and immunohistochemistry (S100. beta. (1:300, ab868, abcam)) co-labeling method. The results showed that, in the brain, MYORG was specifically expressed in S100b positive astrocytes (fig. 2e-2g) and also in part of the ventricular or paraventricular choroid plexus (Choriod plexus) cells, which may be cells of the same lineage as astrocytes.

Example 2 establishment of MYORG cas9 knockout mice

Synthesizing a gene editing technology mediated by CRISPR/cas9, wherein the gene editing technology is characterized in that the sequence of the sgRNA is as follows:

sgRNA1：5’-CTGCTGTGCTCCGCGGTACT-3’(SEQ ID NO:1)；

sgRNA2：5’-TTCTCCATCCGTAACCAGAA-3’(SEQ ID NO:2)；

mRNA transcribed from two sgRNAs and mRNA of Cas9 were introduced into fertilized egg cells of C57BL/6J mouse by microinjection at a concentration of 100 ng/. mu.l, respectively.

Then, embryos meeting the embryo development quality standard are transplanted into a surrogate recipient mouse for pregnancy, and the mouse is born 20 days later.

10 days after birth, the tails of 100 newborn mice are respectively collected, genomic DNA of the mice is extracted, an editing target segment is amplified through PCR by identifying primers, Sanger sequencing is carried out by adopting one primer, and the genotype of the mice is determined according to a sequencing result. The PCR primer sequences were as follows:

Myorg-gF1：5’-CCGACATCGACCTGGTAG-3’(SEQ ID NO:7)

Myorg-gR1：5’-TGGAAGGGCACTGAATCA-3’(SEQ ID NO:8)

the genotypes of the different founder mice were analyzed and homozygous mice were selected for which a fragment of the coding region of the MYORG gene (SEQ ID NO:4) was deleted, resulting in premature termination of translation of the MYORG protein (SEQ ID NO: 5). Among them, a part of mice in which translation was terminated early in amino acid No. 63 (MYORG-P63, SEQ ID NO:6) was concentrated was used for breeding mice for expansion. A schematic diagram of gene editing is shown in FIG. 3.

Example 3 preparation of MYORG-GFP mice

Cleavage was generated in the MYORG gene by CRISPR/Cas9 technology using one sgRNA sequence. The sgRNA sequence is as follows:

5’-TTGGCAGGTGA AAAATTGTC-3’(SEQ ID NO:3)

through a cell endogenous homologous recombination system, a targeting sequence of gene synthesis introduced by microinjection is integrated into a MYORG gene target position, and then a MYORG-GFP mouse is generated. The mRNA transcription of sgRNA, Cas9, microinjection of mRNA and donor targeting sequences, embryo transfer and surrogate pregnancy, rat tail genomic DNA extraction and genotyping methods and procedures required for mice were substantially similar to those of example 1.

The target positions of the introduced MYORG gene are as follows: the GFP coding sequence was inserted just after the ATG start codon (SEQ ID NO:4) of the MYORG gene, resulting in, on the one hand, knockout of the mouse MYORG gene, and, on the other hand, placing GFP expression under the control of the endogenous MYORG gene promoter, so that GFP expression can reflect the cellular expression specificity of MYORG. A schematic of gene editing is shown in FIG. 4 (wherein WPRE is a viral-derived post-translational regulatory element that enhances expression levels of the pre-expression cassette gene).

In example 1, the first demonstration that MYORG is specifically expressed in S100 b-positive glial cells, GFP mice could be used to label S100 b-positive glial cells. The heterozygote mouse has another copy of MYORG gene, and the brain development and the anatomical structure, the motor function, the cognitive function, the emotion function and the like of the heterozygote mouse are all normal, so the heterozygote mouse is a very good mouse strain marked with S100b positive glial cells. The heterozygous mice are paired to obtain homozygote Myorg-GFP mice, the mice not only realize the knockout of Myorg genes, but also mark cells originally expressing the genes, and the mark can be used for subsequent mechanism research and can also be used for the effectiveness evaluation of candidate drugs or treatment scheme test experiments at the cell level.

Example 4 histochemical analysis of MYORG knockdown murine brain calcified deposits

MYORG cas9 knockout mice (20-30g) of different ages, constructed as described in example 2 above, were anesthetized, perfused with PBS and 4% paraformaldehyde, brains were harvested, postfixed in 4% paraformaldehyde, paraffin-embedded, and dissected into 4um thick brain slices, which were individually stained with Alizarin Red (Alizarin Red), Von Kossa, HE, etc. to analyze calcified lesions. Furthermore, the elemental composition of the calcific focus deposit can be analyzed by combining a scanning electron microscope and X-ray energy spectrum analysis. And (3) placing the paraffin section which is positive in tissue staining and is not sealed in a gold plating machine, and spraying gold for ten minutes to enable the sample to have conductivity. Under a stereoscopic microscope, a staining positive focus is found, and after the conductive adhesive is connected, an energy spectrometer is used for analyzing element components and comparing the element components with adjacent normal tissues.

The results are shown in FIG. 5. Symmetric calcium salt deposition was found in brains of 8 and 9 month old MYROG knockdown mice, HE staining showed a change in tissue, alizarin red and Von Kossa staining indicated indeed calcium salt deposition. As a control, no such deposited plaques were observed in wild-type mice of the same age.

All documents mentioned in this application are incorporated by reference in this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

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gatgccggct tccgcgtcac actttgggtg catccgtttg tcaactataa ctcgtcgagc 1200

ttcggcgaag gtgtggagcg cgagctgttc gtgcgcgagc ccacgggccg gctgcccgcg 1260

ctggtgcgct ggtggaacgg tattggcgca gtgctggact tcacgcaccc agaggcccga 1320

gagtggttcc agggacacct acggcgcctg cgcttgcgct acaacgtgac ctcctttaag 1380

ttcgacgcgg gtgaggtcag ctacctgccc agggacttca gcacctacag gcctctgtct 1440

gaccccagtg tgtggagcag gcggtacacc gagatggcgg agcccttctt ctcgctagcc 1500

gaggtccgcg tggggtacca gtcacagaac atctcctgct tcttccgtct agtggaccgc 1560

gactcggtat ggggctacga cctggggctg cgctctctta tccctgcggt gctcaccgtc 1620

agtatgctgg gctatccgtt catcttgccc gatatgatag gtggcaacgc ggtgccggag 1680

cgcacagccg gccgccaaga tgggccgggg ccagagcgcg agctctacgt gcgctggctg 1740

gaggtggccg ccttcatgcc tgccatgcag ttttcaatcc ctccttggca gtacgacgca 1800

gaagtggtag ccatcgcaca caaattcgct gctctgcgcg cctccctcgt ggcgccactg 1860

ctgctagagc ttgctggtga gatcaccgac actggcgacc ccatcgtgcg ccccctgtgg 1920

tggatcgcac cgggggatga aacggcccat cgcattgact cacagtttct catcggggac 1980

acgctgttgg tggcgccggt gctagagcct ggtaagcaag agcgtgatgt ctacctgcct 2040

gccggcaaat ggcgaagcta caagggtgaa ctttttgaca agacgccggt gctgctcacg 2100

gattaccctg tcgacctgga tgaggtcgcc tatttcacct gggcttcctg a 2151

<210> 5

<211> 716

<212> PRT

<213> mouse (Mus musculus)

<400> 5

Met Ser Gln Asn Leu Gln Glu Thr Ser Gln Ala Tyr Pro Arg His Arg

1 5 10 15

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

20 25 30

Thr Met Tyr Thr Phe Leu Pro Asp Asn Phe Ser Pro Ala Lys Pro Lys

35 40 45

Pro Thr Lys Glu Leu Arg Pro Leu Leu Cys Ser Ala Val Leu Gly Leu

50 55 60

Leu Leu Val Leu Ala Ala Val Val Ala Trp Cys Tyr Tyr Ser Ala Ser

65 70 75 80

Leu Arg Lys Ala Glu Arg Leu Arg Ala Glu Leu Leu Asp Leu Asn Arg

85 90 95

Gly Gly Phe Ser Ile Arg Asn Gln Lys Gly Glu Gln Val Phe Arg Leu

100 105 110

Ala Phe Arg Ser Gly Ala Leu Asp Leu Asp Ser Cys Ser Arg Asp Gly

115 120 125

Ala Leu Leu Gly Cys Ser Arg Ala Ala Asp Gly Arg Pro Leu His Phe

130 135 140

Phe Ile Gln Thr Val Arg Pro Lys Asp Thr Val Met Cys Tyr Arg Val

145 150 155 160

Arg Trp Glu Glu Ala Val Pro Gly Arg Ala Val Glu His Ala Met Phe

165 170 175

Leu Gly Asp Ala Ala Ala His Trp Tyr Gly Gly Ala Glu Met Arg Thr

180 185 190

Gln His Trp Pro Ile Arg Leu Asp Gly Gln Gln Glu Pro Gln Pro Phe

195 200 205

Val Thr Ser Asp Val Tyr Ser Ser Asp Ala Ala Phe Gly Gly Ile Leu

210 215 220

Glu Arg Tyr Trp Leu Ser Ser Arg Ala Ala Ala Ile Lys Val Asn Asp

225 230 235 240

Ser Val Pro Phe His Leu Gly Trp Asn Ser Thr Glu Arg Ser Met Arg

245 250 255

Leu Gln Ala Arg Tyr His Asp Thr Ser Tyr Lys Pro Pro Ala Gly Arg

260 265 270

Thr Ala Ala Pro Glu Leu Ser Tyr Arg Val Cys Val Gly Ser Asp Val

275 280 285

Thr Ser Ile His Lys Tyr Met Val Arg Arg Tyr Phe Asn Lys Pro Ser

290 295 300

Arg Val Pro Ala Ser Glu Ala Phe Arg Asp Pro Ile Trp Ser Thr Trp

305 310 315 320

Ala Leu His Gly Arg Ala Val Asp Gln Asn Lys Val Leu Gln Phe Ala

325 330 335

Gln Gln Ile Arg Gln His Arg Phe Asn Ser Ser His Leu Glu Ile Asp

340 345 350

Asp Met Tyr Thr Pro Ala Tyr Gly Asp Phe Asn Phe Asp Glu Gly Lys

355 360 365

Phe Pro Asn Ala Ser Asp Met Phe Arg Arg Leu Arg Asp Ala Gly Phe

370 375 380

Arg Val Thr Leu Trp Val His Pro Phe Val Asn Tyr Asn Ser Ser Ser

385 390 395 400

Phe Gly Glu Gly Val Glu Arg Glu Leu Phe Val Arg Glu Pro Thr Gly

405 410 415

Arg Leu Pro Ala Leu Val Arg Trp Trp Asn Gly Ile Gly Ala Val Leu

420 425 430

Asp Phe Thr His Pro Glu Ala Arg Glu Trp Phe Gln Gly His Leu Arg

435 440 445

Arg Leu Arg Leu Arg Tyr Asn Val Thr Ser Phe Lys Phe Asp Ala Gly

450 455 460

Glu Val Ser Tyr Leu Pro Arg Asp Phe Ser Thr Tyr Arg Pro Leu Ser

465 470 475 480

Asp Pro Ser Val Trp Ser Arg Arg Tyr Thr Glu Met Ala Glu Pro Phe

485 490 495

Phe Ser Leu Ala Glu Val Arg Val Gly Tyr Gln Ser Gln Asn Ile Ser

500 505 510

Cys Phe Phe Arg Leu Val Asp Arg Asp Ser Val Trp Gly Tyr Asp Leu

515 520 525

Gly Leu Arg Ser Leu Ile Pro Ala Val Leu Thr Val Ser Met Leu Gly

530 535 540

Tyr Pro Phe Ile Leu Pro Asp Met Ile Gly Gly Asn Ala Val Pro Glu

545 550 555 560

Arg Thr Ala Gly Arg Gln Asp Gly Pro Gly Pro Glu Arg Glu Leu Tyr

565 570 575

Val Arg Trp Leu Glu Val Ala Ala Phe Met Pro Ala Met Gln Phe Ser

580 585 590

Ile Pro Pro Trp Gln Tyr Asp Ala Glu Val Val Ala Ile Ala His Lys

595 600 605

Phe Ala Ala Leu Arg Ala Ser Leu Val Ala Pro Leu Leu Leu Glu Leu

610 615 620

Ala Gly Glu Ile Thr Asp Thr Gly Asp Pro Ile Val Arg Pro Leu Trp

625 630 635 640

Trp Ile Ala Pro Gly Asp Glu Thr Ala His Arg Ile Asp Ser Gln Phe

645 650 655

Leu Ile Gly Asp Thr Leu Leu Val Ala Pro Val Leu Glu Pro Gly Lys

660 665 670

Gln Glu Arg Asp Val Tyr Leu Pro Ala Gly Lys Trp Arg Ser Tyr Lys

675 680 685

Gly Glu Leu Phe Asp Lys Thr Pro Val Leu Leu Thr Asp Tyr Pro Val

690 695 700

Asp Leu Asp Glu Val Ala Tyr Phe Thr Trp Ala Ser

705 710 715

<210> 6

<211> 63

<212> PRT

<213> mouse (Mus musculus)

<400> 6

Met Ser Gln Asn Leu Gln Glu Thr Ser Gln Ala Tyr Pro Arg His Arg

1 5 10 15

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

20 25 30

Thr Met Tyr Thr Phe Leu Pro Asp Asn Phe Ser Pro Ala Lys Pro Lys

35 40 45

Pro Thr Lys Glu Leu Arg Pro Leu Leu Cys Ser Ala Val Glu Gly

50 55 60

<210> 7

<211> 18

<212> DNA

<213> primers (Primer)

<400> 7

ccgacatcga cctggtag 18

<210> 8

<211> 18

<212> DNA

<213> primers (Primer)

<400> 8

tggaagggca ctgaatca 18

<210> 9

<211> 2145

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 9

atgctccaga accctcagga gaagagccag gcctaccccc gccgccgccg gcctggctgc 60

tacgcatacc gtcagaaccc cgaggccatc gcagccgcag ctatgtacac cttcctgccc 120

gacaacttct cacctgccaa gcccaagcct tccaaagacc tgaagccgct gctgggctcc 180

gcggttctgg ggctgctgct tgtgctggcc gcggtggtgg cctggtgcta ctacagcgtc 240

tccctacgca aggcggagcg acttcgcgcg gagctgctgg acctgaaagc tggcggcttc 300

tccatccgca atcagaaggg agagcaggtc ttccgcctgg ccttccgctc cggcgcgctg 360

gaccttgact cctgcagccg cgatggcgcc ctgctgggct gctcgctcac ggccgacggg 420

ctgccgctgc acttcttcat ccagactgtg cggcccaagg acacggtcat gtgctaccgc 480

gtgcgctggg aggaggcagc gccgggccgg gccgtggagc acgccatgtt cttgggcgac 540

gcggcggccc actggtatgg tggcgccgag atgaggacgc aacactggcc catccgcctg 600

gatggccagc aggagcccca gccgttcgtc accagcgatg tctactcctc cgacgccgcg 660

tttgggggca tcctcgagcg ctactggcta tcttcgcgcg cggccgccat caaagtcaat 720

gactcagtgc ccttccacct gggctggaac agcacggagc gctcgctgcg gcttcaggcg 780

cgctaccacg acacgcccta caagccaccc gccggccgcg ccgcagcgcc agagctgagc 840

taccgagtgt gcgtgggctc agacgtcacc tccatccaca agtacatggt gcgtcgctac 900

ttcaacaagc cgtcaagggt gccagcaccc gaggccttcc gagaccccat ttggtccaca 960

tgggcgctgt acgggcgcgc cgtggaccag gacaaggtgc tgcgttttgc ccaacagatc 1020

cgcctgcacc acttcaacag cagccacctg gaaatcgacg acatgtacac acctgcttat 1080

ggcgacttcg acttcgatga ggtcaaattc cccaacgcca gcgacatgtt ccgccgcctg 1140

cgcgacgccg gcttccgcgt cacgctctgg gtgcaccctt ttgtcaacta caactcgtcg 1200

cgcttcggcg agggcgtgga gcgcgagctg ttcgtgcgcg aacccacggg ccggttacct 1260

gcgctggtgc gctggtggaa cggcatcggc gcggtgctag acttcacgca cccaaaggcc 1320

cgcgactggt tccagggaca cctgcggcgg ctgcgctctc gctactccgt ggcttccttc 1380

aagttcgacg cgggcgaggt cagctacctg ccgcgggact tcagcaccta ccggccgctg 1440

ccggacccca gcgtctggag ccggcgctac actgagatgg cgctgccctt cttctcgctg 1500

gcggaggtgc gcgtaggcta ccagtcacag aacatctcct gcttcttccg cctggtggat 1560

cgcgactctg tgtggggcta cgacctgggg ttgcgctcac tcatccccgc ggtgctcacc 1620

gtcagcatgc tgggctaccc attcatccta cccgatatgg tgggcggcaa cgccgtgccc 1680

cagcggacag ccggcggcga tgtgcccgag cgcgagctct acattcgctg gctggaagtg 1740

gccgccttta tgccggccat gcagttctct atcccgccct ggcgctacga cgcggaagtg 1800

gtggccatcg cgcagaagtt cgccgccctg cgggcctcgc ttgtggcacc gctgttgctt 1860

gagctggcgg gcgaggtcac cgacacgggt gaccctatcg tgcgccccct ttggtggatt 1920

gcgcccggcg acgagacagc tcaccgtatc gactcgcagt tccttattgg ggacacgctg 1980

cttgtggccc cggtgctgga gccaggcaag caggagcgcg acgtctattt gcccgccggc 2040

aagtggcgca gctacaaggg tgagcttttc gacaagacgc cggtgctgct caccgattac 2100

ccggtcgacc tggatgagat cgcctacttt acctgggcgt cctga 2145

Claims (15)

1. A method of making an animal model of brain calcification disease, said method comprising deleting or inactivating the MYORG gene in the genome of the model.

2. The method of claim 1, wherein the method of deleting or inactivating a MYORG gene in the genome of the model comprises: gene knock-out, gene editing, RNA interference, epigenetic suppression, blocking or inhibiting the activity of a MYORG protein with an antibody against a MYORG protein.

3. The method of claim 2, wherein the knockout comprises: stem cell targeting technology based on homologous recombination, gene editing technology, chemical molecule mutagenesis genome mutation, transposon site-directed inactivation and the like; the gene editing comprises the following steps: CRISPR/cas9, spf1, TALEN, zinc finger nuclease, Base editing.

4. The method of claim 3, wherein the MYORG gene in the genome of the model is deleted or inactivated using gene editing techniques.

5. The method of claim 4, wherein the base deletion or insertion is generated by gene editing, resulting in premature termination of the protein coding cassette.

6. The method of claim 5, wherein the translation is terminated prematurely by introducing a stop codon in the MYORG gene corresponding to amino acid 63 of the polypeptide encoded thereby.

7. The method of claim 6, wherein sgRNA of the nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2 are used for gene editing.

8. The method of claim 1, wherein the MYORG gene in the genome of the model is deleted or inactivated simultaneously with the introduction of a reporter gene; the reporter gene replaces the original MYORG gene for expression; the reporter gene was introduced after the MYORG gene promoter.

9. The method of claim 1, wherein the animal is a mouse.

10. The method of claim 9, wherein the mouse is a mouse or a rat.

11. Use of a model of cerebral calcification disease obtained by the method of any one of claims 1 to 10 as a candidate drug for screening drugs for the treatment of cerebral calcification disease.

12. A method for screening a drug candidate for treating a cerebral calcification disease using the model of the cerebral calcification disease obtained by the method as defined in any one of claims 1 to 10, comprising: administering the candidate drug to the test model, observing whether the test model has remission or improvement of the disease, and if so, determining that the candidate drug is a potential drug of the potential cerebral calcification disease; the screening method is aimed at screening drugs, and is non-diagnostic or therapeutic.

13. The method of claim 12, wherein the alleviation or improvement is a statistically significant or significant alleviation or improvement.

14. The method of claim 12, wherein the drug candidate is selected from the group consisting of nucleic acids, proteins, carbohydrates, chemically synthesized small or large molecular compounds, cells, natural product extracts, and combinations thereof.

15. The method of claim 12, wherein the drug candidate is administered orally, intravenously, intraperitoneally, subcutaneously, intragastrically, electroporated, intraspinal, or directly intracerebrally.

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