Research method of DNA (Deoxyribose Nucleic Acid) methylated transferase regulation mechanism of oligodendrocyte myelin sheath abnormality
By constructing a gene-edited mouse model and detecting the DNA methylation status of oligodendrocytes, we have solved the research challenges in existing technologies, achieved a deeper understanding of the regulatory mechanism of DNMTs, and provided treatment options for demyelinating diseases.
Patent Information
- Application Number
- CN202511063909.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-31
- Publication Date
- 2025-12-02
AI Technical Summary
Existing technologies for studying the regulation mechanism of DNA methyltransferases in oligodendrocyte myelin abnormalities suffer from problems such as high lethality in animal models, off-target effects, and low myelination efficiency in cell models, making it difficult to accurately reproduce the spatiotemporal specificity of DNMT regulation.
Gene knockout and overexpression mouse models were constructed using gene editing technology. Oligodendrocytes were isolated and cultured, and high-throughput sequencing and bioinformatics analysis were combined to detect methyltransferase activity and DNA methylation levels, as well as the expression of myelin marker MBP.
This study provides a more in-depth and precise research method that integrates molecular biology and cell biology techniques to systematically reveal the regulatory mechanisms of DNMTs in myelination, providing new therapeutic targets for demyelinating diseases.
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Figure CN121046468A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of detection and research technology, specifically involving a research method for the regulation mechanism of DNA methyltransferase in oligodendrocyte myelin abnormalities. Background Technology
[0002] Oligodendrocytes (OLs) are the sole source of myelination in the central nervous system (CNS), and their myelination capacity (i.e., the ability to generate multiple layers of dense myelin sheaths to encapsulate axons) is epigenetically regulated, particularly by DNA methylation modification. Current research primarily relies on animal models such as conditional knockout mice and cell models derived from primary oligodendrocyte precursor cell (OPC) cultures.
[0003] In animal models, systemic DNMT knockout is highly lethal, while conditional knockout may have off-target effects (such as Cre enzyme leakage into neurons or astrocytes). 5-aza toxicity may interfere with methylation in other cell types, leading to non-specific phenotypes. In cell models, primary OPCs easily lose epigenetic memory in vitro and lack neuron-glial cell interactions; although brain organoids simulate a 3D environment, their myelination efficiency is low (<30%), and the spatiotemporal specificity of DNMT regulation is difficult to reproduce.
[0004] Revealing how DNMTs inhibit myelin gene transcription through methylation modification can fill the knowledge gap in the "epigenetic-myelination" mechanism and provide new therapeutic targets for demyelinating diseases (such as multiple sclerosis and premature infant brain white matter injury). Summary of the Invention
[0005] In view of this, the purpose of this invention is to provide a research method for the regulation mechanism of DNA methyltransferase in oligodendrocyte myelin abnormalities, so as to solve the above-mentioned problems.
[0006] To achieve the above objectives, the present invention provides the following technical solution:
[0007] This invention provides a method for studying the regulatory mechanism of DNA methyltransferases in oligodendrocyte myelin abnormalities, comprising the following steps:
[0008] S1. Gene knockout mice and gene overexpression mice were constructed using gene editing technology, and normal mice were set up as a control group.
[0009] S2. Isolate and culture oligodendrocytes;
[0010] S3. Detect methyltransferase activity, DNA methylation level, and oligodendrocyte myelin indices.
[0011] Furthermore, in step S1, the gene knockout mouse is constructed as follows:
[0012] sgRNA targeting the methyltransferase gene was designed and mixed with Cas9 protein to form a ribonucleoprotein complex. The ribonucleoprotein complex was then injected into the pronucleus of mouse zygotes using microinjection technology. The injected zygotes were then transplanted into the oviduct of surrogate mother mice. After the mice developed and gave birth, the genotypes of the newborn mice were identified. Mice with successful knockout of the target gene were screened using PCR amplification and sequencing technology.
[0013] Gene overexpression mice were constructed by microinjecting the expression vector into the pronucleus of mouse zygotes. The injected zygotes were then transplanted into the oviducts of surrogate mother mice. After the mice developed and gave birth, the genotypes of the newborn mice were identified. Mice with successful target gene knockout were screened using PCR amplification and sequencing technology.
[0014] Furthermore, in step S2, the method for isolating and culturing oligodendrocytes is as follows:
[0015] Mouse brain tissue was placed in PBS buffer, and the meninges and blood vessels were removed. The brain tissue was then cut into small pieces and incubated with a digestive solution containing 0.25% trypsin and 0.02% DTA.
[0016] Digestion was stopped by adding DMEM medium containing 10% fetal bovine serum. After pipetting, a single-cell suspension was prepared. The single-cell suspension was spread on Percoll density gradient centrifuge medium. After centrifugation, oligodendrocytes located in specific density layers were collected. The cells were resuspended in DMEM medium containing 10% fetal bovine serum and seeded into culture flasks coated with poly-L-lysine. The flasks were then incubated at 37°C in a 5% CO2 incubator.
[0017] Furthermore, in step S3, the method for detecting methyltransferase activity is as follows:
[0018] Cultured oligodendrocytes were added to a reaction buffer containing a radioactive methyl donor and incubated at 37°C. The reaction was then terminated by adding acid, and the reaction product was centrifuged.
[0019] The supernatant was separated and analyzed by HPLC to detect the content of radiolabeled methylation products, and the activity of methyltransferase was calculated based on the content of methylation products.
[0020] Furthermore, in step S3, the method for detecting DNA methylation level is as follows:
[0021] Genomic DNA was extracted from oligodendrocytes and fragmented. The DNA fragments were then repaired at the ends, A-tailed, and methylated adapters were added. The adapter DNA fragments were then subjected to bisulfite treatment to convert unmethylated cytosine to uracil, while the methylated cytosine remained unchanged.
[0022] The processed DNA was amplified by PCR to construct a sequencing library. The sequencing library was then subjected to high-throughput sequencing to obtain sequencing data. The sequencing data was then compared and analyzed to determine the methylation status of each site in the genome and to calculate the overall DNA methylation level.
[0023] Furthermore, in step S3, the oligodendrocyte myelin indices are detected by myelin basic protein expression, and the method is as follows:
[0024] Cultured oligodendrocytes were seeded into well plates pre-placed with coverslips. After the cells adhered and grew, they were fixed with paraformaldehyde, washed with PBS buffer, and then incubated with PBS solution containing Triton X-100 at room temperature.
[0025] The cells were then rinsed with PBS buffer, and goat serum blocking solution was added. After blocking at room temperature, the blocking solution was discarded, and MBP primary antibody was added. The cells were incubated overnight at 4°C. The cells were then rinsed with PBS buffer again, and fluorescently labeled secondary antibody was added. The cells were incubated at room temperature in the dark. The cells were rinsed with PBS buffer again, and DAPI staining solution was added. The cells were stained at room temperature in the dark. Finally, the coverslips were mounted with anti-fluorescence quenching mounting medium, and the fluorescence intensity was observed and photographed under a fluorescence microscope.
[0026] The beneficial effects of this invention are as follows:
[0027] 1. This invention focuses on a novel research method for investigating the regulatory mechanism of DNA methyltransferases in oligodendrocyte myelin sheath abnormalities. Using mice as an experimental model, it aims to analyze this regulatory mechanism more deeply and precisely. The method of this invention integrates advanced molecular biology techniques, cell biology techniques, and bioinformatics analysis methods, providing a comprehensive and systematic solution for related research.
[0028] Other advantages, objectives, and features of the invention will be set forth in the following description and will be apparent to those skilled in the art in some respects, or may be learned by practice of the invention. The objectives and other advantages of the invention can be realized and obtained through the following description. Attached Figure Description
[0029] To make the purpose, technical solution, and beneficial effects of the invention clearer, the following figures are provided for illustration:
[0030] Figure 1 This is a diagram illustrating the genotype identification and verification of the gene knockout mice of this invention. Detailed Implementation
[0031] This invention provides a method for studying the regulatory mechanism of DNA methyltransferases in oligodendrocyte myelin abnormalities.
[0032] Example 1
[0033] S1. Mouse model construction
[0034] Constructing gene knockout mice: sgRNA targeting the methyltransferase gene was designed and mixed with Cas9 protein to form a ribonucleoprotein complex. The ribonucleoprotein complex was then injected into the pronucleus of C57BL / 6 mouse zygotes using microinjection. The injected zygotes were then transplanted into the oviducts of surrogate mother mice, and after development and delivery, the newborn mice were genotyped. Mice with successful target gene knockout were screened using PCR amplification and sequencing techniques.
[0035] Constructing gene-overexpressing mice: An expression vector containing the target methyltransferase gene was constructed, with a strong promoter to drive efficient gene expression. The expression vector was injected into the pronucleus of C57BL / 6 mouse zygotes using microinjection. The injected zygotes were then transplanted into the oviducts of surrogate mother mice, and after development and delivery, the newborn mice were genotyped. Mice with successful target gene knockout were screened using PCR amplification and sequencing techniques.
[0036] S2. Isolation and culture of oligodendrocytes
[0037] Mouse brain tissue was placed in PBS buffer, and the meninges and blood vessels were removed. The brain tissue was minced and then digested with a solution containing 0.25% trypsin and 0.02% EDTA. The mixture was incubated at 37°C for 15-20 minutes. Then, DMEM medium containing 10% fetal bovine serum was added to stop the digestion. The cells were gently pipetted to prepare a single-cell suspension. The single-cell suspension was spread on Percoll density gradient centrifuge medium and centrifuged at 2000 rpm for 20 minutes. Oligodendrocytes located in specific density layers were collected, resuspended in DMEM medium containing 10% fetal bovine serum, and seeded into culture flasks pre-coated with poly-L-lysine. The flasks were then incubated at 37°C in a 5% CO2 incubator.
[0038] S3. Detection of DNA methyltransferase activity
[0039] Take cultured oligodendrocytes and add... 3The reaction buffer of HS-syladenosylmethionine was incubated at 37°C to allow DNA methyltransferase to catalyze the methyl transfer reaction. After the reaction was completed, acid was added to terminate the reaction, and the reaction product was centrifuged. The supernatant was then collected and analyzed by HPLC to detect the content of radiolabeled methylation products. The activity of methyltransferase was calculated based on the content of methylation products.
[0040] Detecting DNA methylation levels
[0041] Genomic DNA was extracted from oligodendrocytes and fragmented using ultrasound. The DNA fragments underwent end repair, A-tailing, and methylation adapter ligation. The ligated DNA fragments were then subjected to bisulfite treatment to convert unmethylated cytosine (C) to uracil (U), while methylated cytosine remained unchanged. The treated DNA was then amplified by PCR to construct a sequencing library. The sequencing library was then subjected to high-throughput sequencing to obtain sequencing data. Bioinformatics analysis software was used to compare and analyze the sequencing data to determine the methylation status of each site in the genome and calculate the overall DNA methylation level.
[0042] Detection of myelin basic protein (MBP) expression
[0043] Cultured oligodendrocytes were seeded into 24-well plates with coverslips. After cell adhesion and growth, the cells were fixed with 4% paraformaldehyde for 15-20 minutes, then washed three times with PBS buffer. PBS solution containing 0.3% Triton X-100 was added, and the cells were incubated at room temperature for 15 minutes to increase cell membrane permeability. The cells were then washed three more times with PBS buffer, followed by the addition of goat serum blocking solution and blocking at room temperature for 30 minutes. The blocking solution was discarded, and MBP primary antibody was added and incubated overnight at 4°C. The cells were washed three times with PBS buffer, and fluorescently labeled secondary antibody was added and incubated at room temperature in the dark for 1 hour. The cells were washed three more times with PBS buffer, and DAPI staining solution was added and stained at room temperature in the dark for 5 minutes. The coverslips were then mounted with anti-fluorescence quenching mounting medium, and the cells were observed and photographed under a fluorescence microscope to analyze the fluorescence intensity of MBP.
[0044] In this invention, the gene knockout principle is based on the CRISPR / Cas9 system, which originates from the acquired immune defense mechanism of bacteria. In this system, the Cas9 protein can accurately recognize and bind to the target DNA sequence under the guidance of sgRNA, and then cut the double-stranded DNA, causing DNA double-strand breaks (DSBs). During the process of repairing DSBs, cells are prone to errors such as base insertion or deletion, which leads to the loss of gene function and achieves the purpose of gene knockout.
[0045] In gene overexpression, a constructed expression vector carrying the target gene is introduced into a fertilized egg. The strong promoter in the vector can initiate the transcription process of the target gene, thereby enabling the target gene to be expressed in large quantities in mice, thus achieving gene overexpression.
[0046] When isolating and culturing oligodendrocytes, trypsin and EDTA can disrupt intercellular connections and proteins on the cell membrane surface, thereby separating the cells from the tissue block. Percoll density gradient centrifugation can separate oligodendrocytes from other cells based on differences in cell density. Poly-L-lysine can promote the adherent growth of oligodendrocytes on the surface of the culture flask.
[0047] When detecting DNA methyltransferase activity, DNA methyltransferases can transfer methyl groups from a methyl donor to a specific substrate molecule. Using a radioisotope-labeled methyl donor, if the methyltransferase is active, it will transfer the radioactive methyl group to the substrate during the reaction, generating a radiolabeled methylated product. The reaction product can be separated by HPLC, and the radioactivity intensity can be detected, thus indirectly reflecting the activity of the methyltransferase.
[0048] When detecting DNA methylation levels, bisulfite can deaminate unmethylated cytosine into uracil. In the subsequent PCR amplification process, uracil is amplified into thymine (T). Methylated cytosine, due to the protection of the methyl group, is not modified by bisulfite and remains cytosine after PCR amplification. By comparing the DNA sequences before and after bisulfite treatment through high-throughput sequencing and bioinformatics analysis, the methylation status of each site in the genome can be determined, and the overall DNA methylation level can be calculated.
[0049] When detecting myelin basic protein (MBP) expression, MBP is one of the main structural proteins of myelin and has high specificity. The primary antibody can specifically recognize and bind to MBP, while the fluorescently labeled secondary antibody can bind to the primary antibody. By detecting the fluorescence intensity under a fluorescence microscope, the expression level of MBP can be directly reflected, and the formation of myelin can be inferred. DAPI staining solution can stain the cell nucleus and be used to locate cells.
[0050] like Figure 1 As shown, genotyping of gene knockout mice using PCR amplification and sequencing techniques revealed that wild-type mice exhibited bands of a specific length in the PCR amplification gel electrophoresis results, while gene knockout mice showed significantly shortened or absent PCR amplification bands due to the excision of the target gene fragment.
[0051] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.
Claims
1. A method for studying the regulatory mechanism of DNA methyltransferases in oligodendrocyte myelin abnormalities, characterized by: Includes the following steps, S1. Gene knockout mice and gene overexpression mice were constructed using gene editing technology, and normal mice were set up as a control group. S2. Isolate and culture oligodendrocytes; S3. Detect methyltransferase activity, DNA methylation level, and oligodendrocyte myelin indices.
2. The method for studying the DNA methyltransferase regulatory mechanism of oligodendrocyte myelin abnormalities according to claim 1, characterized in that: In step S1, the gene knockout mouse is constructed as follows: sgRNA targeting the methyltransferase gene was designed and mixed with Cas9 protein to form a ribonucleoprotein complex. The ribonucleoprotein complex was then injected into the pronucleus of mouse zygotes using microinjection technology. The injected zygotes were then transplanted into the oviduct of surrogate mother mice. After the mice developed and gave birth, the genotypes of the newborn mice were identified. Mice with successful knockout of the target gene were screened using PCR amplification and sequencing technology. Gene overexpression mice were constructed by microinjecting the expression vector into the pronucleus of mouse zygotes. The injected zygotes were then transplanted into the oviducts of surrogate mother mice. After the mice developed and gave birth, the genotypes of the newborn mice were identified. Mice with successful target gene knockout were screened using PCR amplification and sequencing technology.
3. The method for studying the DNA methyltransferase regulatory mechanism of oligodendrocyte myelin abnormalities according to claim 1, characterized in that: In step S2, the method for isolating and culturing oligodendrocytes is as follows: Mouse brain tissue was placed in PBS buffer, and the meninges and blood vessels were removed. The brain tissue was then cut into small pieces and incubated with a digestive solution containing 0.25% trypsin and 0.02% DTA. Digestion was stopped by adding DMEM medium containing 10% fetal bovine serum. After pipetting, a single-cell suspension was prepared. The single-cell suspension was spread on Percoll density gradient centrifuge medium. After centrifugation, oligodendrocytes located in specific density layers were collected. The cells were resuspended in DMEM medium containing 10% fetal bovine serum and seeded into culture flasks coated with poly-L-lysine. The flasks were then incubated at 37°C in a 5% CO2 incubator.
4. The method for studying the DNA methyltransferase regulatory mechanism of oligodendrocyte myelin abnormalities according to claim 1, characterized in that: In step S3, the method for detecting methyltransferase activity is as follows: Cultured oligodendrocytes were added to a reaction buffer containing a radioactive methyl donor and incubated at 37°C. The reaction was then terminated by adding acid, and the reaction product was centrifuged. The supernatant was separated and analyzed by HPLC to detect the content of radiolabeled methylation products, and the activity of methyltransferase was calculated based on the content of methylation products.
5. The method for studying the DNA methyltransferase regulation mechanism of oligodendrocyte myelin abnormalities according to claim 1, characterized in that: In step S3, the method for detecting DNA methylation level is as follows: Genomic DNA was extracted from oligodendrocytes and fragmented. The DNA fragments were then repaired at the ends, A-tailed, and methylated adapters were added. The adapter DNA fragments were then subjected to bisulfite treatment to convert unmethylated cytosine to uracil, while the methylated cytosine remained unchanged. The processed DNA was amplified by PCR to construct a sequencing library. The sequencing library was then subjected to high-throughput sequencing to obtain sequencing data. The sequencing data was then compared and analyzed to determine the methylation status of each site in the genome and to calculate the overall DNA methylation level.
6. The method for studying the DNA methyltransferase regulatory mechanism of oligodendrocyte myelin abnormalities according to claim 1, characterized in that: In step S3, the oligodendrocyte myelin sheath marker is the detection of myelin basic protein expression, which is performed as follows: Cultured oligodendrocytes were seeded into well plates pre-placed with coverslips. After the cells adhered and grew, they were fixed with paraformaldehyde, washed with PBS buffer, and then incubated with PBS solution containing Triton X-100 at room temperature. The cells were then rinsed with PBS buffer, and goat serum blocking solution was added. After blocking at room temperature, the blocking solution was discarded, and MBP primary antibody was added. The cells were incubated overnight at 4°C. The cells were then rinsed with PBS buffer again, and fluorescently labeled secondary antibody was added. The cells were incubated at room temperature in the dark. The cells were rinsed with PBS buffer again, and DAPI staining solution was added. The cells were stained at room temperature in the dark. Finally, the coverslips were mounted with anti-fluorescence quenching mounting medium, and the fluorescence intensity was observed and photographed under a fluorescence microscope.