Construction of transgenic mouse capable of photoactivating astrocytes and related application of transgenic mouse

Abstract

The invention discloses construction and related application of a transgenic mouse capable of photoactivating astrocytes, and relates to the field of animal models. The construction method comprises the steps that photosensitive protein is conditionally expressed in astrocytes of a target animal, so that the animal model capable of photoactivating the astrocytes is obtained, and the model can be applied to research of astrocyte dysfunction disease mechanisms and development of related drugs. It is verified for the first time that after photosensitive protein is conditionally expressed in astrocytes, ASTs in the hippocampus CA1 region are activated through light, reactive hyperplasia of the ASTs can be stimulated, and the function of a glial network can be improved; the ASTs are specifically controlled through a photogenetic method, anxiety behaviors caused by early-stage stress such as maternal and infant isolation can be relieved, and a way is provided for medicine development combined with photoactivation treatment.

Description

Technical Field

[0001] The present invention relates to the field of animal models, and in particular to the construction of transgenic mice capable of photoactivating astrocytes and related applications thereof. Background Art

[0002] Astrocytes (ASTs) are the most numerous and complex type of glial cells in the brain. They can participate in the regulation of emotional behaviors such as anxiety by connecting proteins on their surfaces, affecting synapse formation, and regulating neuronal activity. Therefore, it is expected that anxiety-like behaviors can be treated by manipulating the activity of ASTs. A large number of studies have focused on the connection between neuronal function and mental disorders caused by chronic stress. Recent studies have found that abnormal glial cells also play an important role in this. Previous studies have shown that abnormal development of ASTs plays an important role in early stress-induced mental disorders.

[0003] Early chronic stress is a key risk factor for anxiety disorders. Early stress refers to the psychological and physical trauma caused by external pressure and stimulation in early life that exceeds one's ability to withstand. It will have long-term effects on brain development, memory formation, etc. Individuals will show susceptibility to anxiety mental illness in adulthood and may even develop suicidal tendencies. Therefore, it is of great clinical significance to understand the biological mechanism of early chronic stress and find treatments to improve anxiety caused by early chronic stress.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide the construction of transgenic mice capable of light-activating astrocytes and related applications thereof.

[0006] The present invention is achieved in that:

[0007] In a first aspect, an embodiment of the present invention provides a method for constructing an animal model of astrocytes that can be photoactivated, comprising: conditionally expressing a photosensitive protein in astrocytes of a target animal.

[0008] In a second aspect, embodiments of the present invention provide use of an animal model constructed by the construction method described in the preceding embodiments in developing or screening drugs for preventing, treating or assisting in treating diseases associated with abnormal astrocyte function.

[0009] The present invention has the following beneficial effects:

[0010] The present invention provides a method for constructing an animal model. By conditionally expressing a photosensitive protein in astrocytes of a target animal, an animal model of astrocytes that can be photoactivated can be obtained, which can be applied to studying the disease mechanism of abnormal astrocyte function and related drug development.

[0011] The present invention verifies for the first time that after conditional expression of photosensitive proteins in astrocytes, light activation of ASTs in the CA1 region of the hippocampus can stimulate reactive proliferation of ASTs and improve glial network function; specific manipulation of ASTs by optogenetic methods can alleviate anxiety behaviors caused by early stress such as maternal-infant separation, providing a path for the development of drugs used in combination with light activation therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0013] Figure 1 A is a schematic diagram of the construction process of the animal model; B is a schematic diagram of the Cre gene carried by the Cre mouse and the LSL-photosensitive protein gene carried by the tool mouse;

[0014] Figure 2 YFP + The proportion of cells with ASTs in the cortex and hippocampus;

[0015] Figure 3 The results are for the co-labeling of Rhodopsin with neurons, microglia, and oligodendrocytes;

[0016] Figure 4 The effect of light-activated ASTs in Example 4 on the number and activation state of ASTs in maternal-infant isolated mice;

[0017] Figure 5 The effect of light-activated ASTs in Example 4 on the number of ASTs in maternal-infant isolated mice;

[0018] Figure 6 The effect of light-activated ASTs on the expression of CX30 and CX43 in maternal-infant isolated mice in Example 5;

[0019] Figure 7 This is the result of the recovery of glial network function in maternal-infant isolated mice after high-intensity fluorescence quenching by light-activated ASTs in Example 5;

[0020] Figure 8 The effect of light-activated ASTs on the central grid movement time of maternal-infant isolated mice in the open field experiment of Example 6;

[0021] Fig. 9The effect of light-activated ASTs on the time it takes maternal-infant isolated mice to enter the open arms in the elevated plus maze experiment of Example 6;

[0022] Fig.10 The effect of light-activated ASTs on the ratio of exploration time to total time of new objects by maternal-infant separated mice in the new object recognition test in Example 6;

[0023] Fig.11 This is the cliff avoidance result in Example 6;

[0024] Fig.12 This is the result of the tail suspension experiment in Example 6. DETAILED DESCRIPTION

[0025] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0026] On the one hand, an embodiment of the present invention provides a method for constructing an animal model of astrocytes that can be photoactivated, comprising: conditionally expressing a photosensitive protein in astrocytes of a target animal.

[0027] In some embodiments, the construction method uses Cre-Loxp technology to conditionally express a light-sensitive protein in astrocytes of a target animal.

[0028] In some embodiments, the target animal comprises a mouse or a rat.

[0029] In some embodiments, the construction method comprises:

[0030] Obtaining Cre mice that specifically express Cre recombinase in astrocytes and tool mice that carry LoxP-Stop-LoxP-target genes; the target genes include genes for light-sensitive proteins;

[0031] The Cre mice were mated with the tool mice to obtain double-positive heterozygous mice.

[0032] In some embodiments, the Cre mouse comprises ALDH1L1 CreERT Transgenic mice or ALDH1L1 CreERT2 Transgenic mice. Specifically, the Chinese name of ALDH1L1 is aldehyde dehydrogenase 1 family member L1.

[0033] The embodiment of the present invention is based on the Cre-loxP recombinase system, selects ALDH1L1 as the ASTs-specific driver, and obtains an animal model by mating Cre mice that specifically express Cre recombinase in ASTs with tool mice carrying the LoxP-Stop-LoxP-photosensitive protein gene. Tamoxifen can be used to induce the specific expression of photosensitive protein in ASTs, avoiding tissue damage caused by traditional virus injection methods.

[0034] In some embodiments, the light-sensitive protein includes any one or more of ReaChR, ChR2, ChrimsonR, CheRiff, ChETA, hChR2(C128S / D156A), C1V1(t / t), oChIEF(E163A / T199C), hChR2(E123T / T159C) and hChR2(H134R).

[0035] In some embodiments, the photosensitive protein is ReaChR. ReaChR is a red-shifted photosensitive protein with an excitation wavelength of 590-630 nm, higher tissue penetration and light transmission efficiency, and is more suitable for brain stimulation than ChR.

[0036] In some embodiments, the animal model is a disease model associated with abnormal astrocyte function.

[0037] In some embodiments, the disease associated with abnormal astrocyte function includes anxiety or early stress.

[0038] In some embodiments, the early stress includes early stress caused by mother-infant separation.

[0039] In some embodiments, when the animal model is a disease model of anxiety or early stress, the construction method further comprises: separating the double-positive heterozygous mice or their double-positive offspring mice from their mothers after birth to obtain a disease model. The double positive refers to the simultaneous expression of Cre and LoxP-Stop-LoxP-target genes.

[0040] In some embodiments, the mother-infant separation time is: 3 to 7 hours per day during the period from day 1 to 3 to day 13 to 15 after the mouse is born. Optionally, the day 1 to 3 can be day 1, 2 or 3; the day 13 to 15 can be day 13, 14 or 15; the 3 to 7 hours can be any one of 3, 4, 5, 6 and 7 hours or a range between any two of them.

[0041] In addition, an embodiment of the present invention also provides the use of an animal model constructed by the construction method described in any of the aforementioned embodiments in the development or screening of drugs for preventing, treating or assisting in the treatment of diseases associated with abnormal astrocyte function.

[0042] In some embodiments, the disease associated with abnormal astrocyte function includes anxiety or early stress.

[0043] In some embodiments, the drug includes: a drug used in combination with light-activated therapy.

[0044] As used herein, "treating" includes preventing or alleviating a condition, reducing the rate at which a condition develops or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition, reducing or stopping symptoms associated with a condition, producing complete or partial reversal of a condition, curing a condition, or a combination of the above.

[0045] For cancer, "treatment" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumors or malignant cells, or some combination of the above. For tumors, "treatment" includes eliminating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying the development of the tumor, or some combination of the above.

[0046] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0047] Example 1: Construction of ASTs-specific light-activated mouse ALDH1L1 CreERT :R26 LSL-ReaChR-mCit

[0048] ALDH1L1 CreERT Mice and R26 LSL-ReaChR-mCit Mice (source: Jackson Lab) were mated, and the offspring were genotyped and ALDH1L1 CreERT :R26 LSL-ReaChR-mCit Heterozygous mice, the construction process diagram can be found in Figure 1 .

[0049] Example 2: ALDH1L1 CreERT :R26 LSL-ReaChR-mCit Mouse model validation

[0050] ALDH1L1 CreERT :R26 LSL-ReaChR-mCitThe expression of the fluorescent protein YFP can be started after continuous induction of mice with tamoxifen at a dose of 100 mg / kg per day for 4 days. The mice were anesthetized by intraperitoneal injection of 20% urethane solution. After the mice were anesthetized successfully, they were fixed on a foam board, the chest cavity of the mice was opened to completely expose the heart, and a suitable perfusion needle was inserted into the left ventricle along the apex of the heart. After cutting the right atrial appendage, physiological saline and 4% paraformaldehyde solution were used for perfusion in sequence. The perfusion was stopped after the whole body of the mouse was stiff. After the brain tissue of the mouse was peeled off, fixed and dehydrated, the brain tissue was cut into 20μm slices using a freezing slicer. AST was labeled with ALDH1L1 antibody, and the expression of YFP+ cells in AST in the hippocampus and cortex regions was observed under a microscope. Rhodopsin was used to label ReaChR photosensitive protein, and neurons (NeuN + ), microglia (Iba1 + ), oligodendrocytes (PDGFRα + ) to observe the co-labeling of Rhodopsin with neurons, microglia, and oligodendrocytes in the hippocampus and cortex, and to verify the expression of ALDH1L1 CreERT :R26 LSL-ReaChR-mCit Specificity of double-transgenic mice.

[0051] Depend on Figure 2 and Figure 3 It can be seen that in the mouse cortex and hippocampus, YFP+ cells accounted for 67% of ASTs, and Rhodopsin + NeuN + Cells, Iba1 + Cells co-labeled with PDGFRα + Cell co-labeling proves that ALDH1L1 constructed in the present embodiment CreERT :R26 LSL-ReaChR-mCit Mice have better effectiveness and specificity.

[0052] Example 3: Construction of an early stress model caused by maternal-infant isolation that can be used for light activation

[0053] Take ALDH1L1 CreERT :R26 LSL-ReaChR-mCit Double-transduced newborn mice (P2, the second day after birth) were placed in a new cage and isolated from their parents for 5 hours every day from P2 to P14. After the isolation, the mice were restored to their original living conditions to obtain an early stress model (same as "mother-infant isolation mice"). After induced isolation of mice with tamoxifen at a dose of 100 mg / kg per day for 4 consecutive days, optical fibers were implanted in the CA1 region of the mouse hippocampus at P26.

[0054] Example 4: Effect of light-activated ASTs on ASTs in mice isolated from their infants

[0055] Learning from light-activated ALDH1L1 CreERT :R26 LSL-ReaChR-mCit For maternal-infant isolation mice, the mice were perfused and brain tissues were peeled off according to the method of Example 2. After tissue sections, ASTs were labeled with ALDH1L1 antibody and GFAP antibody, respectively, and the effect of optogenetic activation on the number and activation state of ASTs in the CA1 region of the hippocampus of maternal-infant isolation mice was observed under a microscope.

[0056] Depend on Figure 4-5 It can be seen that after light stimulation of the CA1 region of the hippocampus of isolated mice, ALDH1L1 + The increase in the number and branches of ASTs demonstrated that light stimulation could induce reactive proliferation of ASTs in the CA1 region of the hippocampus of maternal-infant isolated mice.

[0057] Example 5: Effects of light-activated ASTs on glial network function in maternal-infant isolated mice

[0058] The brain slices of Example 4 were taken, and the CX30 and CX43 of the tissues were respectively labeled, and the expression of the important structure of the gap junction protein in the glial network of the isolated mouse brain after light-activated ASTs was observed under a microscope.

[0059] Prepare artificial cerebrospinal fluid and slicing fluid, pre-cool the slicing fluid and pour it into the groove of the oscillating slicer, and continue to inject mixed gas into the slicing fluid. CreERT :R26 LSL-ReaChR-mCit For the maternal-infant isolation model mice, the mice were perfused and the brain tissue was peeled off according to the method of Example 2, and the brain slices were quickly transferred to the slice solution for immersion to prepare brain slices with a thickness of 300 μm, and then transferred to artificial cerebrospinal fluid filled with mixed gas and incubated at 37°C for 1 hour, and then transferred to room temperature for incubation for 1 hour to prepare acute ex vivo brain slices. The ex vivo brain slices were stained with SR101, immersed in artificial cerebrospinal fluid injected with mixed gas, photographed and observed by laser confocal microscopy, and the improvement of the molecular transmission function of the mouse glial network by light-activated ASTs was evaluated by fluorescence quenching recovery experiment.

[0060] Depend on Figure 6 It can be seen that after light stimulation isolated the CA1 region of the mouse hippocampus, the expression of CX30 increased by 26.0% and the expression of CX43 increased by 18.7%. Figure 7 The results showed that after high-intensity fluorescence quenching, the speed and degree of recovery of glial network function in isolated mouse brain slices with light-specific stimulation were faster. The above results prove that light activation of ASTs can improve the glial network function of isolated mice.

[0061] Example 6: Effects of light-activated ASTs on anxiety behavior in mice isolated from their mothers

[0062] Tamoxifen-induced ALDH1L1 CreERT:R26 LSL-ReaChR-mCit In the maternal-infant isolation mice, the mice with implanted optical fibers were set as the experimental group, and the mice without implanted optical fibers were set as the control group. The two groups of mice were given pulsed light activation for 12 consecutive days starting from P28, with a wavelength of 593nm and an activation paradigm of 10mW, 20Hz, 100ms light on, 1s light off. The mice were behaviorally tested at P40: the effects of light-activated ASTs on the anxiety behavior of maternal-infant isolation mice were tested by open field test and elevated plus maze test; the effects of light-activated ASTs on the depression and impulsive behavior of maternal-infant isolation mice were evaluated by novel object recognition test, cliff avoidance test and tail suspension test.

[0063] Depend on Figure 8-Figure 9 It can be seen that after light stimulation, the central motor test of isolated mice increased significantly compared with the control group, and the ratio of time in the open arm to the total time increased, proving that light activation of ASTs can improve the anxiety-like behavior of isolated mice. Fig.10 It can be seen that the ratio of the exploration time of the new object to the total time of the mice before and after light stimulation did not change significantly, and the cliff avoidance ( Fig.11 ) and tail suspension test ( Fig.12 ) There was no significant difference in the statistical results. Figure 10 to Figure 12 The results demonstrated that light activation did not aggravate the depressive and impulsive-like behaviors of isolated mice.

[0064] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for constructing an animal model capable of photoactivating astrocytes, characterized in that: It includes: Conditionally express light-sensitive proteins in astrocytes of target animals.

2. The construction method according to claim 1, characterized in that: The construction method uses Cre-Loxp technology to conditionally express light-sensitive proteins in astrocytes of target animals; Optionally, the target animal comprises a mouse or a rat.

3. The construction method according to claim 2, characterized in that: The construction method comprises: Obtaining Cre mice that specifically express Cre recombinase in astrocytes and tool mice that carry LoxP-Stop-LoxP-target genes; the target genes include genes for light-sensitive proteins; The Cre mice were mated with the tool mice to obtain double-positive heterozygous mice.

4. The construction method according to claim 3, characterized in that: The Cre mice include ALDH1L1 CreERT Transgenic mice or ALDH1L1 CreERT2 Transgenic mice.

5. The construction method according to claim 1, characterized in that: The photosensitive protein includes any one or more of ReaChR, ChR2, ChrimsonR, CheRiff, ChETA, hChR2, C1V1, oChIEF, hChR2, and hChR2; Optionally, the photosensitive protein is ReaChR.

6. The construction method according to claim 3, characterized in that: The animal model is a disease model associated with abnormal astrocyte function; Optionally, the diseases associated with abnormal astrocyte function include: anxiety or early stress; Optionally, the early stress includes early stress caused by mother-child separation.

7. The construction method according to claim 6, characterized in that: When the animal model is a disease model of anxiety or early stress, the construction method further comprises: separating the double-positive heterozygous mice or their double-positive offspring mice from their mothers after birth to obtain a disease model.

8. The construction method according to claim 7, characterized in that: The mother-infant separation time is: within the period from the 1st to the 3rd day to the 13th to the 15th day after the mouse is born, the separation is 3 to 7 hours every day.

9. Use of the animal model constructed by the construction method according to any one of claims 1 to 8 in developing or screening drugs for preventing, treating or assisting in treating diseases associated with abnormal astrocyte function.

10. The use according to claim 9, characterized in that: The diseases associated with abnormal astrocyte function include: anxiety or early stress; Optionally, the drug includes: a drug used in combination with light-activated therapy.

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