A method for constructing a transgenic demyelinating mouse model and its application

Abstract

This invention relates to the field of biotechnology and discloses a method for constructing a transgenic demyelinating mouse model and its application. The method involves introducing the LSL-DTR-IRES-mtdTomato-pA gene fragment into the exon 2 region of the Mapt site in oligodendrocytes of the mouse brain to obtain Mapt-LSL-DTR-IRES-mtdTomato-pA mice. These mice are then crossed with NG2-CreERT tool mice to obtain the NG2-CreERT Mapt-LSL-DTR-IRES-mtdTomato-pA gene knock-in mouse model. This model possesses highly efficient oligodendrocyte lineage tracking capabilities. Tamoxifen induction enables the specific expression of DTR and the fluorescent reporter gene mtdTomato in newly formed oligodendrocytes. Intraperitoneal injection of diphtheria toxin specifically eliminates these new oligodendrocytes, and cell number and function return to normal levels after injection is stopped. This model allows for subjective regulation of newly formed oligodendrocyte function, providing an important tool for studying the biological function of newly formed myelin in the central nervous system, simulating the development of demyelinating diseases, and screening related therapeutic drugs.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for constructing a transgenic demyelinating mouse model and its application. Background Technology

[0002] Myelin is a lipid-rich insulating membrane that surrounds nerve fibers, its core function being to ensure the rapid and low-energy transmission of nerve impulses. In the central nervous system, myelin is formed by the processes of oligodendrocytes wrapping around axons. Oligodendrocytes are generated from oligodendrocyte precursor cells through a series of precise and complex differentiation processes. Notably, oligodendrocytes not only provide the source of myelin formation but also supply essential energy and nutrient support to axons and neurons, playing a crucial role in maintaining axonal integrity, regulating axonal energy metabolism balance, promoting neuronal development, and maintaining neuronal plasticity. Abnormalities in myelin development or impaired regeneration often lead to severe cognitive, learning, and behavioral dysfunctions. Clinically, there are currently no effective treatments for central nervous system demyelinating diseases, such as multiple sclerosis.

[0003] During development, the myelin sheath is rapidly formed, encapsulating key nerve fibers. Its function focuses on "building the framework," and the speed and integrity of myelination directly affect the developmental process. By adulthood, the myelin sheath has largely formed a stable network, and its main function is to maintain the stability of myelination. However, there are still many gaps in research on the function of the myelin sheath in adulthood.

[0004] Therefore, how to construct an animal model that can observe functional changes after the clearing and recovery of newly formed myelin sheath, and thus conduct in-depth research on the biological functions of newly formed myelin sheath in adulthood, has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0005] The purpose of this invention is to provide a method for constructing a transgenic demyelinating mouse model and its application, which solves the problem in the prior art that it is impossible to subjectively regulate the function of newly formed oligodendrocytes after their formation and that it is difficult to conduct in-depth research on the biological function of newly formed myelin in adulthood.

[0006] To achieve the above objectives, the present invention provides a method for constructing a transgenic demyelinating mouse model, comprising the following steps: In mouse oligodendrocyte precursor cells, the LSL-DTR-IRES-mtdTomato-pA gene fragment was introduced into the exon 2 region of the Mapt site to obtain Mapt-LSL-DTR-IRES-mtdTomato-pA mice. The Mapt-LSL-DTR-IRES-mtdTomato-pA mouse was crossed with the NG2-CreERT tool mouse to obtain the NG2-CreERT;Mapt-LSL-DTR-IRES-mtdTomato-pA mouse.

[0007] Specifically, the LSL-DTR-IRES-mtdTomato-pA gene fragment is introduced into the exon 2 region of the Mapt site, including: The Mapt site is the 17762nd site of the Mapt gene.

[0008] Specifically, the insertion of the LSL-DTR-IRES-mtdTomato-pA gene fragment into the exon 2 region of the Mapt site also includes: The Mapt-LSL-DTR-IRES-mtdTomato-pA gene fragment was introduced using gene editing and the Cre-loxP recombinase system.

[0009] The gene editing method is selected from zinc finger nuclease-based gene editing methods, transcription activator-like effector nuclease gene editing methods, or CRISPR / Cas9 gene editing methods.

[0010] The process involves crossing the Mapt-LSL-DTR-IRES-mtdTomato-pA mouse with the NG2-CreERT tool mouse to obtain NG2-CreERT; following the Mapt-LSL-DTR-IRES-mtdTomato-pA mouse, the process further includes: Genotyping of hybrid offspring mice was performed using PCR, including the detection of the Mapt-LSL-DTR-IRES-mtdTomato-pA and NG2-CreERT genes.

[0011] Among them, the NG2-CreERT and Mapt-LSL-DTR-IRES-mtdTomato-pA mice obtained by the method described above for constructing transgenic demyelinating mouse models were used in evaluating brain cell function.

[0012] The brain cells in question are oligodendrocytes of the brain.

[0013] Among them, the NG2-CreERT and Mapt-LSL-DTR-IRES-mtdTomato-pA mice obtained by the method for constructing the transgenic demyelinating mouse model described above are used in screening drugs for the prevention or treatment of neurological diseases.

[0014] This invention discloses a method for constructing a transgenic demyelinating mouse model and its application. Through homologous recombination technology, a tamoxifen-inducible Cre recombinase (CreERT2) is inserted at a specific site into the endogenous regulatory region of the NG2 gene (Cspg4 / Pdgfra), making its expression strictly driven by the NG2 promoter. Using gene editing and the Cre-loxP recombinase system, the LSL-DTR-IRES-mtdTomato-pA gene fragment is knocked into the second exon region of the Mapt (17762) site in mouse oligodendrocyte precursor cells in a highly efficient manner. The two genotype mice are then crossed to construct the target genotype. The animal models constructed in this invention have the following characteristics: the NG2-CreERT and Mapt-LSL-DTR-IRES-mtdTomato-pA gene knock-in mouse models exhibit highly efficient oligodendrocyte lineage tracking capabilities; intraperitoneal injection of diphtheria toxin (DT) specifically eliminates newly formed oligodendrocytes; after stopping DT injection, the number and function of oligodendrocytes can still recover to normal levels; simultaneously, compared with the commonly used PLP-CreERT and ROSA26-eGFP-DTA models in the neurological field, this model has the advantage of allowing for subjective regulation of newly formed oligodendrocytes after their functional formation. Using the animal models constructed in this invention, it is possible to study the biological function of newly formed myelin in the central nervous system, gain a deeper understanding of the functional changes in the loss and recovery of newly formed myelin, simulate the occurrence and development of related human diseases, reveal the pathogenesis of related diseases, provide new ideas for the prevention and treatment of clinical demyelinating diseases, and use gene-edited animal models to screen for effective drugs or treatments for these diseases, providing new options for clinical treatment. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0016] Figure 1 This is a schematic diagram of the gene knock-in and hybridization process of the target mouse of this invention.

[0017] Figure 2 This is a diagram showing the design and identification results of the transgenic mouse of this invention.

[0018] Figure 3This is a diagram showing the results of gene knock-in verification in this invention. Histology represents histological verification, CC1+ cells represent mature oligodendrocytes, MBP specifically labels myelin, PDGFRα+ cells represent oligodendrocyte precursor cells, VE represents the intraperitoneal injection saline group, DT represents the intraperitoneal injection DT group, fold change of CC1+ and mTOMATO+ cells in cortex represents the fold change in the number of CC1 (specifically labeled oligodendrocytes) and mTOMATO (fluorescent reporter gene) double-positive cells in the cerebral cortex, and fold change of CC1+ and mTOMATO+ cells in CC represents the number of CC1 (specifically labeled oligodendrocytes) and mTOMATO (fluorescent reporter gene) double-positive cells in the corpus callosum region.

[0019] Figure 4 This is a flowchart illustrating the steps of constructing the transgenic demyelinating mouse model of the present invention. Detailed Implementation

[0020] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0021] Please refer to Figures 1 to 4 This invention provides a method for constructing a transgenic demyelinating mouse model, comprising the following steps: S101: In mouse oligodendrocyte precursor cells, the LSL-DTR-IRES-mtdTomato-pA gene fragment was introduced into the exon 2 region of the Mapt site to obtain Mapt-LSL-DTR-IRES-mtdTomato-pA mice. S102: Cross the Mapt-LSL-DTR-IRES-mtdTomato-pA mouse with the NG2-CreERT tool mouse to obtain the NG2-CreERT;Mapt-LSL-DTR-IRES-mtdTomato-pA mouse.

[0022] Specifically, (I) laboratory animal breeding and laboratory animal genotype verification: In the following experiments, all mice were housed in individually ventilated cages (IVC) and were allowed to choose their feeding method under standard 12-hour light / dark cycles. Mapt-LSL-DTR-IRES-mtdTomato-pA mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd., Cat#T050171) were crossed with NG2-CreERT tool mice (commercially available). Genotyping of the offspring was performed using PCR. NG2-CreERT tool mice were used as a specific induction method for recombinant mice with oligodendrocyte precursor cells. Figure 2 A), the specific steps are as follows: To obtain transgenic mice with conditional regulation of newly formed oligodendrocytes, the LSL-DTR-IRES-mtdTomato-pA gene fragment was efficiently introduced into the exon 2 region of the Mapt (17762) site in mouse brain oligodendrocyte precursor cells, resulting in Mapt-LSL-DTR-IRES-mtdTomato-pA mice (i.e., target mice 1, as shown in the image). Figure 1 (As shown); Mapt-LSL-DTR-IRES-mtdTomato-pA mice were crossed with NG2-CreERT tool mice to obtain NG2-CreERT; Mapt-LSL-DTR-IRES-mtdTomato-pA mice (i.e., target mice 2, such as...) Figure 1 (as shown) The specific steps for genotyping (example) are as follows: The PCR amplification reaction system for the gene Mapt-LSL-DTR-IRES-mtdTomato-pA is: 2*Taq The PCR reaction mixture consisted of 12.5 μL MasterMix (PCR reaction premix), 1 μL upstream primer (10 pmol / μL), 1 μL downstream primer (10 pmol / μL), 1 μL template (approximately 100 ng / μL), and 9.5 μL ddH2O. The PCR amplification conditions were as follows: pre-denaturation at 95℃ for 5 min, followed by cycles of 30 s at 98℃, 30 s at 65℃ (-0.5℃ / cycle), and 45 s at 72℃, repeated 20 times. Then, a cycle of 30 s at 98℃, 30 s at 55℃, and 45 s at 72℃ was performed, repeated 20 times. Finally, the mixture was incubated at 72℃ for 5 min and stored at 10℃. Gel electrophoresis was then performed, and band size was used for identification. Primer sequences are shown in Table 1. The gel electrophoresis results are as follows. Figure 2 As shown in B, two mice were heterozygous for Mapt-LSL-DTR-IRES-mtdTomato-pA (6# and 7#), one mouse was homozygous for Mapt-LSL-DTR-IRES-mtdTomato-pA (8#), and one mouse was wild-type (5#).

[0023] The PCR amplification reaction system for the NG2-CreERT gene was as follows: 10 μL of 2*Taq Master Mix (i.e., PCR reaction premix), 1 μL of upstream primer (10 pmol / μL), 1 μL of downstream primer (10 pmol / μL), 2 μL of template (approximately 100 ng / μL), and 6 μL of ddH2O. The PCR amplification conditions were as follows: pre-denaturation at 94℃ for 2 min, followed by cycles of 30 s at 94℃, 30 s at 65℃ (-0.5℃ / cycle), and 20 s at 68℃, repeated 10 times. Then, cycles of 30 s at 94℃, 30 s at 60℃, and 20 s at 72℃ were repeated 30 times. Finally, the gene was treated at 72℃ for 2 min and stored at 16℃. Gel electrophoresis was then performed, and the bands were identified based on their size. Primer sequences are shown in Table 1. The gel electrophoresis results are as follows: Figure 2 As shown in B, one mouse was a homozygous NG2-CreERT mouse (1#), and three mice were heterozygous NG2-CreERT mice (2#, 3#, 4#).

[0024] Table 1 Primer Information (II) Verification of DTR expression effect: Since the obtained mice are from the Cre / loxP recombinase system, these mice can specifically knock out the STOP gene after induction with moxifen, expressing the target gene DTR gene sequence and the fluorescent reporter gene mtdTomato gene sequence. Therefore, this invention uses the Cre / loxP-induced recombination method to verify the gene expression results. The specific steps are as follows: Two target mice were administered tamoxifen (30 mg / ml) by gavage at a dose of 50 mg / kg at 1 month of age (i.e., 1 month) for four consecutive days. The mice were then allowed to develop to 7 months of age before their brains were collected. Figure 3 A) The mouse brain was cut into 10μm or 20μm thick sections by freezing, mounted on glass slides and stored at -80°C for later use; Since the mice obtained can specifically knock out the STOP gene and express the target gene DTR gene sequence and the fluorescent reporter gene mtdTomato gene sequence after induction with DT, this invention uses Cre / loxP-induced recombination and intraperitoneal injection of DT to verify the killing effect of DT. The specific steps are as follows: Two-month-old mice were administered tamoxifen (30 mg / ml) by gavage at a dose of 50 mg / kg for four consecutive days starting at 1 month of age. When the mice reached 2 months of age, they were intraperitoneally injected with DT for 3 days. Eight days later, the mice's brains were collected. Figure 3B) The mouse brain was cut into 10μm or 20μm thick sections by freezing, mounted on glass slides and stored at -80°C for later use; Immunofluorescence staining and image acquisition: Table 2 Antibody Information Methods: Brain tissue sections were fixed, perforated, and blocked. They were then incubated overnight at 4°C with three primary antibodies (see Table 2), one for each antibody. The next day, the samples were washed and incubated with the corresponding host-specific secondary antibodies, followed by counterstaining with DAPI. Fluorescence images were captured using a rotating confocal microscope (Olympus, Spin SR10, Shinjuku, Tokyo). Results are shown below. Figure 3 As shown in Figure A.

[0025] like Figure 3 As shown in Figure A, significant co-labeling of mtdTomato was detected in CC1-positive oligodendrocytes of NG2-CreERT;Mapt-LSL-DTR-IRES-mtdTomato-pA mice. Figure 3 A), significant co-labeling of mtdTomato was detected in the MBP-positive myelin of NG2-CreERT;Mapt-LSL-DTR-IRES-mtdTomato-pA mice (A). Figure 3 A) No co-labeling was found in PDGFRα-positive oligodendrocyte precursor cells of NG2-CreERT;Mapt-LSL-DTR-IRES-mtdTomato-pA mice ( Figure 3 A). This result indicates that DTR is successfully expressed in mature oligodendrocytes, but not in oligodendrocyte precursor cells.

[0026] like Figure 3 As shown in Figure B, immunofluorescence results indicated that, compared to the littermate control group VE, the DT group mice showed a significant reduction in the area of ​​CC1-positive myelin sheaths in the cerebral cortex and corpus callosum, the number of mtdTomato-positive oligodendrocytes, and the area of ​​mtdTomato-positive myelin sheaths. Figure 3 B). The above results indicate that Mapt-LSL-DTR-IRES-mtdTomato-pA can be efficiently and specifically expressed in TMF-induced oligodendrocytes, and intraperitoneal injection of DT can efficiently kill TMF-induced oligodendrocytes. In other words, this invention successfully constructed NG2-CreERT;Mapt-LSL-DTR-IRES-mtdTomato-pA mice.

[0027] The present invention also provides an NG2-CreERT;Mapt-LSL-DTR-IRES-mtdTomato-pA mouse obtained according to the above construction method.

[0028] This mouse model can be used to evaluate brain cell function, particularly the function of oligodendrocytes in the brain. Furthermore, this mouse model can be used to screen drugs for the prevention or treatment of neurological diseases.

[0029] The NG2-CreERT and Mapt-LSL-DTR-IRES-mtdTomato-pA gene knock-in mouse models exhibit highly efficient oligodendrocyte lineage tracking capabilities. Intraperitoneal injection of diphtheria toxin (DT) specifically eliminates newly formed oligodendrocytes. After discontinuation of DT injection, the number and function of oligodendrocytes can recover to normal levels. Furthermore, compared to the commonly used PLP-CreERT and ROSA26-eGFP-DTA models in the neurology field, this model has the advantage of allowing for subjective regulation after the formation of oligodendrocyte function. Using the animal models constructed in this invention, the biological functions of newly formed myelin can be studied, providing a deeper understanding of the functional changes in the loss and recovery of newly formed myelin, simulating the occurrence and development of related human diseases, revealing the pathogenesis of related diseases, providing new ideas for the prevention and treatment of clinical demyelinating diseases, and using gene-edited animal models to screen for effective drugs or treatments for these diseases, providing new options for clinical treatment.

[0030] The above-disclosed embodiments are merely one or more preferred embodiments of this application and should not be construed as limiting the scope of this application. Those skilled in the art can understand that all or part of the processes for implementing the above embodiments and equivalent changes made in accordance with the claims of this application still fall within the scope of this application.

Claims

1. A method for constructing a transgenic demyelinating mouse model, characterized in that, Includes the following steps: In mouse oligodendrocyte precursor cells, the LSL-DTR-IRES-mtdTomato-pA gene fragment was introduced into the exon 2 region of the Mapt site to obtain Mapt-LSL-DTR-IRES-mtdTomato-pA mice. The Mapt-LSL-DTR-IRES-mtdTomato-pA mouse was crossed with the NG2-CreERT tool mouse to obtain the NG2-CreERT;Mapt-LSL-DTR-IRES-mtdTomato-pA mouse.

2. The method for constructing a transgenic demyelinating mouse model as described in claim 1, characterized in that, In mouse oligodendrocyte precursor cells, the LSL-DTR-IRES-mtdTomato-pA gene fragment was introduced into exon 2 of the Mapt site, specifically including: The Mapt site is the 17762nd site of the Mapt gene.

3. The method for constructing a transgenic demyelinating mouse model as described in claim 2, characterized in that, The LSL-DTR-IRES-mtdTomato-pA gene fragment was introduced into the exon 2 region of the Mapt site, and specifically included: The Mapt-LSL-DTR-IRES-mtdTomato-pA gene fragment was introduced using gene editing and the Cre-loxP recombinase system.

4. The method for constructing a transgenic demyelinating mouse model as described in claim 3, characterized in that, The gene editing method is selected from zinc finger nuclease-based gene editing methods, transcription activator-like effector nuclease gene editing methods, or CRISPR / Cas9 gene editing methods.

5. The method for constructing a transgenic demyelinating mouse model as described in claim 1, characterized in that, The Mapt-LSL-DTR-IRES-mtdTomato-pA mouse was crossed with the NG2-CreERT tool mouse to obtain NG2-CreERT; after the Mapt-LSL-DTR-IRES-mtdTomato-pA mouse, the following was also included: Genotyping of hybrid offspring mice was performed using PCR, including the detection of the Mapt-LSL-DTR-IRES-mtdTomato-pA and NG2-CreERT genes.

6. The application of NG2-CreERT;Mapt-LSL-DTR-IRES-mtdTomato-pA mice obtained by the method for constructing the transgenic demyelinating mouse model as described in any one of claims 1-5 in evaluating brain cell function.

7. The application as described in claim 6, characterized in that, The brain cells in question are oligodendrocytes of the brain.

8. The use of NG2-CreERT;Mapt-LSL-DTR-IRES-mtdTomato-pA mice obtained by the method for constructing the transgenic demyelinating mouse model as described in any one of claims 1-5 in screening drugs for the prevention or treatment of neurological diseases.

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