Method for marking new myelin sheath in central nervous system
By constructing the ENPP6-P2A-CreERT2-T2A-CFP gene fragment and using gene editing technology, combined with tamoxifen-induced activation of Cre recombinase, specific labeling of newly formed myelin in the central nervous system was achieved. This solves the problem that existing technologies cannot distinguish between newly formed and developing myelin in adulthood, and provides a research tool.
Patent Information
- Application Number
- CN202511717816.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-21
- Publication Date
- 2026-02-27
AI Technical Summary
Existing methods have difficulty distinguishing between newly formed myelin sheaths after adulthood and those formed during development, and cannot specifically mark newly formed myelin sheaths at a specific time period.
The ENPP6-P2A-CreERT2-T2A-CFP gene fragment was constructed and ENPP6-CreERT2 mice were obtained through gene editing technology. These mice were then mated with mT/mG reporter mice. The Cre recombinase was activated by tamoxifen, and the oligodendrocytes in the intermediate stage and the myelin sheath structure formed were observed using a fluorescent reporter system.
This study enables specific labeling of newly formed myelin sheaths after adulthood, provides a research tool for manipulating newly formed myelin sheaths at specific time points, and addresses the shortcomings of existing methods.
Smart Images
Figure CN121577592A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of neuroscience, and particularly relates to a method for marking newly formed myelin in the central nervous system. BACKGROUND
[0002] An important component of the central nervous system is white matter, which is composed of a large number of neuronal axons and myelin formed by wrapping of oligodendrocytes (OLs). Myelin ensures the rapid and accurate conduction of axon signals, and also supports the neural axon and the surrounding tissue, and plays a role in insulation and nutrition of the axon. OLs are differentiated from oligodendrocyte precursor cells (OPCs). OPCs have characteristics of stem cells, such as self-renewal, migration, proliferation, etc., and can differentiate into OLs. Studies have shown that myelination mainly occurs during the development stage from birth to adulthood in animals. A large number of myelin is formed in the early stage of the animal and plays a corresponding function. Recent studies have shown that in adult animals, there is still continuous differentiation of OPCs and formation of new myelin, and in some adaptive processes of humans, such as learning new knowledge and skills, the white matter density of the brain changes. More importantly, recent studies have found that inhibition of myelination in adult animals seriously impairs the learning ability of complex motor memory in animals, which shows that newly formed myelin still plays an important role in neural function.
[0003] In adult animals, a large number of myelin already exists in the central nervous system, and conventional staining and marking methods cannot distinguish between newly formed myelin after adulthood and myelin formed during development. For example, the BrdU / EdU incorporation method can only mark a small part of newly formed OLs, and only the cell body can be observed without myelin. The currently available method is to induce OPC-specific gene promoters to drive a fluorescent reporter gene in adult animals, and when these cells differentiate into OLs, the newly formed myelin can be observed. However, this method can mark all newly formed myelin after induction, and cannot specifically mark the newly formed myelin during induction, and it is more difficult to manipulate the newly formed myelin in a specific time period. Therefore, it is particularly important to construct a new animal model capable of marking intermediate-stage oligodendrocytes. SUMMARY
[0004] The present application aims to provide a method for marking newly formed myelin in the central nervous system, and aims to solve the problem that the existing method cannot distinguish between newly formed myelin after adulthood and myelin formed during development. The present application provides a tool for manipulating newly formed myelin in a specific time period.
[0005] To achieve the above objectives, the present invention provides a method for labeling newly formed myelin sheaths in the central nervous system, comprising the following steps: The ENPP6-P2A-CreERT2-T2A-CFP gene fragment was constructed and ENPP6-CreERT2 knock-in mice were obtained using gene editing technology. ENPP6-CreERT2 mice were crossed with mT / mG reporter mice to obtain ENPP6-CreERT;mT / mG double transgenic mice; Tamoxifen-induced treatment was administered to double transgenic mice during the target time period to activate Cre recombinase. Brain tissue was collected after induction, and labeled intermediate-stage oligodendrocytes and the myelin sheath structures they form were observed using a fluorescence reporter system.
[0006] In the section “Tamoxifen induction treatment of double transgenic mice within the target time period to activate Cre recombinase”, the tamoxifen induction treatment is administered by gavage at a dose of 100 mg / kg for 4 consecutive days.
[0007] In the section "Collecting brain tissue after induction and observing labeled intermediate-stage oligodendrocytes and their myelin sheath structures using a fluorescent reporter system", the fluorescent reporter system is the mT / mG system, which expresses the red fluorescent protein Tomato in the unrecombined state and the green fluorescent protein GFP after recombination.
[0008] In the section "Collecting brain tissue after induction and observing labeled intermediate-stage oligodendrocytes and the myelin sheath structures they form using a fluorescence reporter system", the intermediate-stage oligodendrocytes are ENPP6-positive cells.
[0009] The ENPP6 positive cells do not express the oligodendrocyte precursor cell marker PDGFRα, the astrocyte marker GFAP, the microglia marker Iba1, or the neuronal marker NeuN.
[0010] This invention provides a method for labeling newly formed myelin in the central nervous system, comprising the following steps: constructing the ENPP6-P2A-CreERT2-T2A-CFP gene fragment and obtaining ENPP6-CreERT2 knock-in mice using gene editing technology; mating ENPP6-CreERT2 mice with mT / mG reporter mice to obtain ENPP6-CreERT;mT / mG double transgenic mice; inducing the double transgenic mice with tamoxifen during a target time period to activate Cre recombinase; collecting brain tissue after induction and observing the labeled intermediate-stage oligodendrocytes and their formed myelin structures using a fluorescence reporter system. This invention achieves the goal of specifically labeling newly formed myelin during tamoxifen induction. It provides a new research tool for manipulating newly formed myelin during a specific time period. It solves the problem that existing methods label all newly formed myelin after the start of induction and cannot specifically label newly formed myelin during the induction period. Attached Figure Description
[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0012] Figure 1 It is a design strategy diagram.
[0013] Figure 2 This is an immunofluorescence staining co-labeled map. It shows ENPP6 labeling of OLs and myelin sheaths, but not other glial cells and neurons.
[0014] Figure 3 This is a verification diagram of myelin sheath types labeled with ENPP6. It shows that ENPP6 does not label mature myelin sheaths, but rather newly formed myelin sheaths differentiated from OPCs.
[0015] Figure 4 This is a flowchart of a method for labeling newly formed myelin sheaths in the central nervous system provided by the present invention. Detailed Implementation
[0016] Embodiments of the present invention are described in detail below, examples of which are illustrated in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.
[0017] Please see Figures 1 to 4The present invention provides a method for labeling newly formed myelin sheaths in the central nervous system, comprising the following steps: S1 constructed the ENPP6-P2A-CreERT2-T2A-CFP gene fragment and obtained ENPP6-CreERT2 knock-in mice using gene editing technology; Specifically, according to the design scheme, gRNA was designed, constructed, and transcribed in vitro, while a homologous recombination vector (Donorvector) was constructed, and the correctness of the vector sequence was verified by sequencing. Cas9, gRNA, and Donorvector samples were microinjected into mouse zygotes with a C57BL / 6JGpt background; surviving zygotes were then transplanted into pseudopregnant female mice, allowing them to become pregnant and give birth. The F0 generation pups born to the recipient mice were tail-cropped and toe-cropped at 5-7 days for numbering, and genomic DNA was extracted for PCR and sequencing identification to confirm the genotype.
[0018] S2 crossed ENPP6-CreERT2 mice with mT / mG reporter mice to obtain ENPP6-CreERT;mT / mG double transgenic mice; Specifically, after the positive F0 generation mice reach sexual maturity, they are mated with wild-type background mice; the F1 generation mice are tail-cropped and toe-cropped at 5-7 days old, and genomic DNA is extracted for PCR and sequencing identification to confirm the genotype.
[0019] S3 induces double transgenic mice with tamoxifen during the target time period to activate Cre recombinase; The tamoxifen induction treatment consisted of administration of 100 mg / kg by gavage for four consecutive days.
[0020] Specifically, newborn mice were induced with tamoxifen starting one month after birth for four days, and samples were collected at two months of age. Figure 2 B) Immunofluorescence staining showed that it co-labeled with MBP (myelin marker) and CC1 (OL marker), but not with PDGFRα (OPC marker), GFAP (astrocytocyte marker), Iba1 (microglia marker), and Neun (neuronal marker), proving that it labels oligodendrocytes but not oligodendrocyte precursor cells, other glial cells, or neurons. Figure 2 We observed the above phenomenon in multiple mice (A, 2C). Figure 2 D); Since the mice we obtained were Cre / loxP recombinase system mice, after induction with moxifen, the target gene loxp site was specifically knocked out, thereby expressing fluorescence.
[0021] Aged ENPP6-CreERT;MOG-DreERT;mG(rox) / mT(loxp) mice (aged 1 year) were induced with tamoxifen for four days, and samples were collected one month after induction. Figure 3 D,3E) revealed that mGFP and mTomato are not co-labeled ( Figure 3 E,3F) while MOG is a marker of mature myelin, proving that ENPP6 does not mark mature myelin.
[0022] In addition, PDGFRα-CreERT;ENPP6-DreERT;mG(rox) / mT(loxp) mice were used. The first round of tamoxifen induction was performed on day 7 (P7) after birth, lasting for 4 days. The second round of tamoxifen induction was performed on day 17 (P17) after birth, also lasting for 4 days. Samples were collected on day 27 after birth (P27). Figure 3 A), it was observed that 80% of the cells co-labeled with mGFP and mTomato ( Figure 3 B 3C), while PDGFRα is a marker of oligodendrocyte precursor cells OPC, therefore it is believed that ENPP6-positive cells are newly formed myelin differentiated from OPC.
[0023] S4 brain tissue was collected after induction, and labeled intermediate-stage oligodendrocytes and the myelin sheath structures they formed were observed using a fluorescence reporter system.
[0024] The fluorescent reporter system is an mT / mG system that expresses the red fluorescent protein Tomato in the unrecombined state and the green fluorescent protein GFP after recombination.
[0025] The intermediate-stage oligodendrocytes are ENPP6-positive cells. These ENPP6-positive cells do not express the oligodendrocyte precursor cell marker PDGFRα, the astrocyte marker GFAP, the microglia marker Iba1, or the neuronal marker NeuN.
[0026] Specifically, when the mice reach 2 months of age, their brains are collected, and frozen sections are cut into 20μm thick slices, which are then mounted on glass slides and stored at -80°C for later use.
[0027] Brain tissue sections were fixed, perforated, and blocked, then incubated overnight at 4°C with primary antibody. The following day, the samples were washed, incubated with the appropriate host-specific secondary antibody, and counterstained with DAPI. Fluorescence images were captured using a rotating confocal microscope (Olympus, Spin SR10, Shinjuku, Tokyo).
[0028] Verification of laboratory animal genotypes DNA was extracted from newborn mice, and genotyping of offspring was performed using PCR. The primers used in the experiment are as follows: PCR amplification reaction system and conditions The mT / mG system is as follows: The above-disclosed method is merely a preferred embodiment of the present invention for marking newly formed myelin sheaths in the central nervous system. It should not be construed as limiting the scope of the present invention. Those skilled in the art will understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made in accordance with the claims of the present invention are still within the scope of the invention.
Claims
1. A method for labeling newly formed myelin sheaths in the central nervous system, characterized in that, Includes the following steps: The ENPP6-P2A-CreERT2-T2A-CFP gene fragment was constructed and ENPP6-CreERT2 knock-in mice were obtained using gene editing technology. ENPP6-CreERT2 mice were crossed with mT / mG reporter mice to obtain ENPP6-CreERT;mT / mG double transgenic mice; Tamoxifen-induced treatment was administered to double transgenic mice during the target time period to activate Cre recombinase. Brain tissue was collected after induction, and labeled intermediate-stage oligodendrocytes and the myelin sheath structures they form were observed using a fluorescence reporter system.
2. The method for labeling newly formed myelin sheaths in the central nervous system as described in claim 1, characterized in that, In the section "Tamoxifen induction treatment of double transgenic mice during the target time period to activate Cre recombinase", the tamoxifen induction treatment is described as administration by gavage at a dose of 100 mg / kg for 4 consecutive days.
3. The method for labeling newly formed myelin sheaths in the central nervous system as described in claim 1, characterized in that, In the section "Collecting brain tissue after induction and observing labeled intermediate-stage oligodendrocytes and their myelin sheath structures using a fluorescent reporter system", the fluorescent reporter system is the mT / mG system, which expresses the red fluorescent protein Tomato in the unrecombined state and the green fluorescent protein GFP after recombination.
4. The method for labeling newly formed myelin sheaths in the central nervous system as described in claim 1, characterized in that, In the section "Collecting brain tissue after induction and observing labeled intermediate-stage oligodendrocytes and the myelin sheath structures they form using a fluorescence reporter system", the intermediate-stage oligodendrocytes are ENPP6-positive cells.
5. The method for labeling newly formed myelin sheaths in the central nervous system as described in claim 4, characterized in that, The ENPP6 positive cells do not express the oligodendrocyte precursor cell marker PDGFRα, the astrocyte marker GFAP, the microglia marker Iba1, or the neuronal marker NeuN.