WO2021098613A1 - 一种新的小胶质细胞激活方法 - Google Patents

一种新的小胶质细胞激活方法 Download PDF

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WO2021098613A1
WO2021098613A1 PCT/CN2020/128851 CN2020128851W WO2021098613A1 WO 2021098613 A1 WO2021098613 A1 WO 2021098613A1 CN 2020128851 W CN2020128851 W CN 2020128851W WO 2021098613 A1 WO2021098613 A1 WO 2021098613A1
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microglia
mouse
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transgenic mice
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詹阳
吕泽中
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Shenzhen Institute of Advanced Technology of CAS
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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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    • A—HUMAN NECESSITIES
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A01K2217/00—Genetically modified animals
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Definitions

  • the invention relates to the field of biology, in particular to a Cx3cr1CreER-Ai27 genotype double transgenic mouse, a new microglia activation method and application thereof.
  • Microglia are the smallest glial cells in the central nervous system (CNS). They are distributed throughout the central nervous system and account for about 5%-10% of the total number of glial cells. As an immune effector cell resident in the central nervous system, microglia belong to the mononuclear phagocyte family and are widely considered to be the main immune effector of the central nervous system. The morphology of microglia is highly plastic, and its biology The functional status is closely related. In normal brain tissue, microglia are highly branched, with tertiary and quaternary branching structures, and the branches between cells rarely overlap. Branched microglia are often called "resting microglia". Under normal circumstances, highly branched resting microglia provide a highly dynamic and efficient monitoring system for the brain.
  • microglia When inflammation, infection, trauma or other neurological diseases occur in the brain, microglia are quickly activated and gain phagocytosis. Microglia and the neuroinflammation mediated by them play a very important role in the process of central nervous system damage and disease outcome, and are involved in human nervous system disorders such as HIV encephalopathy, Alzheimer's disease, multiple sclerosis, etc. disease.
  • Cre is a site-specific tyrosine recombinase derived from P1 phage, belonging to the lambda integrase superfamily, composed of a pentavalent [Arg-Lys-(His/Lys)-Arg-(His/Trp)] assists in catalyzing the break and reconnection of DNA strands in the process of DNA recombination, and cre inserts introns to form icre.
  • loxP site is a segment with a total length of 34 bp inverted repeat DNA sequence
  • Cre recombinase mediates the recombination between two loxP sites, and the direction and position of the two loxP sites can determine the three results of recombination: deletion, inversion or integration. Since the Cre-loxP system does not require any auxiliary factors, it has been gradually modified and is widely used in eukaryotic cells. It has now played a significant role in the fields of cell development tracking, gene knockout, and gene conditional expression.
  • the chemokine Fractalkine has a CX3C chemokine domain and is named according to the spacing of the N-terminal cysteine. This forms the CX3C family. Unlike other known chemokines, the CX3C module has two isomers. Fractalkine (CX3CL1) is a specific ligand of CX3CR1. It is a transmembrane glycoprotein. Its typical function is to interact with CX3CR1 with high affinity to mediate the blockage of leukocytes in the flow state. The chemokines can be released by the action of proteolytic enzymes. CX3CR1/CX3CX1 signals play different roles in different tissues.
  • the CX3CR1 gene can be expressed in monocytes, dendritic cells (DC), T cell subtypes, and natural killer cells (NK).
  • CX3CL1 can promote the survival of neurons and inhibit the apoptosis of microglia, but in the intact central nervous system, the function of CX3CR1/CX3CL1 signal is still unknown.
  • CX3CR1 can be expressed in peripheral monocytes, NK cells, DC cells, microglia, etc., while in the central nervous system, the CX3CR1 gene is only expressed in microglia.
  • FKN and CX3CR1 are expressed in neurons and microglia, respectively When expressed in plasma cells, the receptor-ligand is essential for the neuron-glia interconnection.
  • microglia Through the communication between cells and the secretion of factors, the resting state of microglia can be maintained under stable conditions.
  • One way is that neurons inhibit the activation of microglia through the CX3CR1/CX3CL1 signaling pathway.
  • CreER Using the CX3CR1 promoter to drive the expression of CreER, through the expression of a large number of CreER, Cre-mediated recombination can be effectively carried out.
  • the CX3CR1 gene is not only expressed in microglia, but also in peripheral myeloid cells. Therefore, when only CX3CR1 knockout mice were used, the function of microglia could not be accurately specified.
  • mice expressed modified hChR2/tdTomato fusion protein after exposure to Cre recombinase.
  • the mouse can be used for optogenetic studies of rapid activation of excitable cells in the body under blue light (450-490 nm) irradiation.
  • the main methods for distinguishing or labeling microglia include morphological observation, characteristic protein immunohistochemical staining, and single-promoter fluorescent protein labeling.
  • these methods have poor specificity and cannot accurately distinguish microglia from the central nervous system.
  • Other cells of the nervous system, and the mouse needs to be killed during the labeling process, and cannot be specifically labeled in a living state.
  • the present invention provides a new microglial cell activation method, which can pass Cx3cr1CreER Knock-in/knock-out mice express a Cre-ERT2 fusion protein and EYFP protein in brain microglia, and then cross with Ai27(RCL-hChR2(H134R)/tdT)-D mice to obtain Cx3cr1CreER-Ai27 genotype double transgenic mice, after intraperitoneal injection of Tamoxifen, an optical fiber is implanted in the target brain area and stimulated with blue light (450-490nm) to specifically activate the microglia in the area under the optical fiber.
  • the microglia activation method of the present invention can specifically activate microglia, accurately distinguish microglia from other cells, and is of great significance in studying the development, behavior, and function of microglia, and is useful in biomedicine.
  • the research field has broad application prospects.
  • an object of the present invention is to provide a new method for activating microglia, which is characterized in that the Cx3cr1CreER-Ai27 gene is obtained by crossing a Cx3cr1CreER transgenic mouse with an Ai27 transgenic mouse.
  • an optical fiber is implanted in the target brain area, and blue light stimulation is used to specifically activate the microglia in the area under the optical fiber.
  • the mouse is a rat or a mouse, and preferably, the mouse is a mouse.
  • the wavelength of the blue light is 450-490 nm
  • the concentration of the intraperitoneal injection of Tamoxifen is 15-25 mg/mL, preferably, the concentration of the intraperitoneal injection of Tamoxifen is 20 mg/mL.
  • Another object of the present invention is to provide a Cx3cr1CreER-Ai27 genotype double transgenic mouse obtained by the above method for activating microglia.
  • the mouse is a rat or a mouse, preferably, the mouse is a mouse.
  • the third objective of the present invention is the application of the above-mentioned method for activating microglia in the study of the development, behavior or function of microglia.
  • the fourth objective of the present invention is the application of the above-mentioned double transgenic mice in the study of the development, behavior or function of microglia.
  • the application can be performed under normal, disease or injury conditions.
  • the present invention provides a new method for activating microglia.
  • This method can specifically mark and activate mouse microglia in vivo, and only microglia express green fluorescent protein, which can accurately distinguish small cells.
  • Glial cells and other nerve cells realize the specific labeling, detection and tracking of microglia in the living state, which can be used to observe the development, behavior or function of microglia in real time. There is no similar method or genetically modified mice. Report.
  • microglia (3)
  • the current research on microglia can also use specific markers for in vitro staining studies.
  • the method of the present invention can be used to achieve specific labeling and activation of microglia in vivo and in vitro.
  • the development, behavior and function of microglia are of great significance and have broad application prospects.
  • FIG. 1 Schematic diagram of transgenic hybridization of the present invention.
  • Cx3cr1-CreER-yfp represents Cx3cr1-CreER transgenic mice
  • ChR2-tdTomato represents Ai27 transgenic mice.
  • Figure 2 is a comparison of specific labeling and activation fluorescence between Cx3cr1-CreER transgenic mice and Cx3cr1-CreER-Ai27 transgenic mice.
  • Figure 3 is a comparison diagram of the fluorescence of activated microglia and non-activated microglia. Different rows represent different magnifications. A and B are activated microglia, C and D are inactivated microglia.
  • Cx3cr1CreER transgenic mice and Ai27 transgenic mice were constructed from Jackson Laboratory in the United States. Rats are 2-3 months old, weigh 20-30g, and are not limited to males or females. The experimental mice were provided with sufficient water and food, and were kept in an environment of 12h darkness and 12h light, and the room temperature was controlled between 20-25°C. Select the normal-growing Cx3cr1CreER mice and the Ai27 gene mice for crossbreeding. After the hybridization, cut and cut the tail tip of the mouse about 4mm. Use an electric iron to stop the scalding of the mouse.
  • the Cx3cr1CreER-Ai27 genotype double transgenic mouse was obtained by crossing Cx3cr1CreER transgenic mice with Ai27 transgenic mice.
  • the Cx3cr1CreER-Ai27 genotype double transgenic mice constructed in Example 1 The transgenic mice were stimulated with 4 mg Tamoxifen (purchased from Sigma) dissolved in 200 ⁇ l corn oil (Sigma), injected subcutaneously or intraperitoneally with Tamoxifen 48 hours apart at two time points. Cx3cr1CreER transgenic mice were used as the control group. The way to deal with it.
  • mice were injected with ethyl carbamate solution into the abdominal cavity. After anesthesia, they were fixed on the dissecting plate, and after cutting the thoracic cavity skin and muscle layers, the heart was exposed, the right atrial appendage was cut and the perfusion needle was inserted Left ventricle, inject 15 mL of 0.15 MPBS, and then inject 15 mL of 4% PFA solution. After cutting off the neck, use forceps to peel off the brain tissue, cerebellum, and part of the brain stem from the skull, and place it in 20 mL of 4% PFA solution , Fix at 4°C for 2 days.
  • microglia can be specifically activated. It can be seen from Figure 2 that, compared with Cx3cr1CreER transgenic mice, Cx3cr1CreER-Ai27 genotype double transgenic mice can specifically distinguish microglia from other cells, the fluorescence intensity is stronger, and the microglia are clearly visible.
  • the optical fiber can be directly implanted in the target brain area, and blue light (450-490nm) stimulation can be used to specifically activate the microglia in the area under the optical fiber.
  • blue light 450-490nm
  • the activated microglia of the Cx3cr1CreER-Ai27 genotype double transgenic mice can be clearly seen, which can specifically distinguish microglia from other cells, while the inactivated microglia cannot Clearly visible fluorescence is detected. It realizes the specific labeling, detection and tracking of microglia in a living state, and can be used to observe the development, behavior or function of microglia in real time.
  • the present invention has successfully constructed a Cx3cr1CreER-Ai27 genotype double transgenic mouse. It also provides a simple and efficient new microglia activation method, which can specifically activate microglia and accurately distinguish microglia from other cells.
  • the transgenic mouse of the present invention is stimulated with blue light, only microglia express green fluorescent protein, which realizes the specific labeling, detection and tracking of microglia in a living state, and can be used for real-time observation of microglia development, Behavior or function.
  • the transgenic mice of the present invention can also accurately distinguish microglia from other cells by the method of in vitro immunohistochemical staining.

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Abstract

提供一种Cx3cr1CreER-Ai27基因型双转鼠以及一种新的小胶质细胞激活方法，通过Cx3cr1CreER小鼠与Ai27基因小鼠进行杂交获得Cx3cr1CreER-Ai27基因型双转鼠，在腹腔注射Tamoxifen后于目标脑区植入光纤，使用蓝光（450-490nm）刺激，即可特异性激活光纤下方区域小胶质细胞。小胶质细胞激活方法可以特异性激活小胶质细胞，准确地区分小胶质细胞与其他细胞，在研究小胶质细胞的发育、行为和功能等方面具有重要意义，操作简便，研究成本和风险降低，具有广阔的应用前景。

Description

一种新的小胶质细胞激活方法 技术领域
本发明涉及生物领域，具体涉及一种Cx3cr1CreER-Ai27基因型双转鼠、一种新的小胶质细胞的激活方法及其应用。
 背景技术
小胶质细胞 (Microglia)是中枢神经系统 (CNS)最小的一种神经胶质细胞，分布于整个中枢神经系统，约占胶质细胞总数的5%-10%。作为常驻中枢神经系统的免疫效应细胞，小胶质细胞隶属于单核吞噬细胞族，被广泛地认为是中枢神经系统的主要免疫效应器，小胶质细胞的形态具有高度可塑性，与其生物学功能状态密切相关。正常脑组织中，小胶质细胞呈高度分枝状，具有三级和四级分枝结构，且细胞间的分枝很少发生重叠。分枝状的小胶质细胞通常被称为“静息小胶质细胞”。正常情况下，高度分枝状静息状态的小胶质细胞为大脑提供了一个高度动态和高效的监测系统。当脑内发生炎症、感染、创伤或其他神经系统疾病时，小胶质细胞迅速被激活并获得吞噬功能。小胶质细胞及其介导的神经炎症在中枢神经系统的损伤及疾病的转归过程中起着非常重要的作用，参与例如HIV脑病，阿尔兹海默病，多发性硬化等人神经系统紊乱疾病。
Cre是一种来源于P1噬菌体的位点特异性络氨酸重组酶，属于λ整合酶超家族，由一个五价物 [Arg-Lys-(His/Lys)-Arg-(His/Trp)] 协助催化DNA重组过程中DNA链的断裂和重连，cre插入内含子后形成icre。loxP位点是一段总长度为34 bp的反向重复DNA序列，Cre重组酶介导两个loxP位点之间的重组，而且，两个loxP位点的方向和位置可以决定重组后的三种结果：删除、翻转或整合。由于Cre-loxP系统不需要任何辅助因子，经逐步改造，被广泛应用于真核细胞，目前已经在细胞发育追踪、基因敲除、基因条件表达等领域发挥了显著作用。
趋化因子Fractalkine具有CX3C趋化因子域，并根据N-末端的半胱氨酸的间距命名，此构成CX3C家族，不同于其他已知的趋化因子，CX3C模块有两种异构体存在。Fractalkine(CX3CL1)是CX3CR1的特异性配体，它是一种跨膜糖蛋白，其典型的功能是与CX3CR1高亲和性的相互作用，从而介导白细胞在流动状态下的阻滞，该可溶性的趋化因子可以被蛋白水解酶作用而释放。CX3CR1/CX3CX1信号在不同组织发挥不同的作用。在循环系统中，CX3CR1基因可在单核细胞、树突状细胞(DC)、T细胞亚型和自然杀伤细胞(NK)中表达。在体外，CX3CL1可促进神经元的存活，抑制小胶质细胞的调亡，但在完整的中枢神经中，CX3CR1/CX3CL1信号的功能还是未知的。CX3CR1可在外周单核细胞、NK细胞、DC细胞、小胶质细胞等中表达，而在中枢神经系统中，CX3CR1基因只在小胶质细胞中表达当FKN和CX3CR1分别在神经元和小胶质细胞中表达时，该受体-配体对神经元-胶质间的相互联系至关重要。通过细胞之间的交流及因子的分泌，可维持小胶质细胞在稳定条件下的静息状态，其中一种方式即为神经元通过CX3CR1/CX3CL1信号通路抑制小胶质细胞的活化。使用CX3CR1启动子驱动CreER的表达，通过大量CreER的表达，可有效进行Cre介导的重组。然而，CX3CR1基因不仅在小胶质细胞中表达，也在外周髓系细胞中表达。因而，当仅仅使用CX3CR1敲除小鼠时，并不能准确指明小胶质细胞的功能。
Ai27小鼠暴露于Cre重组酶后表达改良的hChR2/tdTomato融合蛋白。该小鼠可用于在蓝光(450-490 nm)照射下快速激活体内可兴奋细胞的光遗传学研究。
目前，区分或标记小胶质细胞的主要方法包括形态学观察、特征性蛋白免疫组织化学染色和单启动子荧光蛋白标记，然而这些方法特异性较差，无法准确地区分小胶质细胞与中枢神经系统的其他细胞，且标记过程中需要处死小鼠，无法在活体状态下进行特异性标记。
因而建立有效的离体和在体激活小胶质细胞的方法以及小鼠模型，是现有技术中亟需解决的技术难题。但目前未见类似报道或产品。
 技术问题
基于此，本发明提供了一种新的小胶质细胞激活方法，所述方法通过Cx3cr1CreER knock-in/knock-out小鼠在大脑小胶质细胞中表达一种Cre - ERT2融合蛋白和EYFP蛋白，而后与Ai27(RCL-hChR2(H134R)/tdT)-D基因小鼠进行杂交，获得Cx3cr1CreER-Ai27基因型双转鼠，在腹腔注射Tamoxifen后于目标脑区植入光纤，使用蓝光（450-490nm）刺激，即可特异性激活光纤下方区域小胶质细胞。本发明的小胶质细胞激活方法可以特异性激活小胶质细胞，准确地区分小胶质细胞与其他细胞，在研究小胶质细胞的发育、行为和功能等方面具有重要意义，在生物医药研究领域，有广阔的应用前景。
 技术解决方案
为了解决上述现有技术中存在的问题，本发明的一个目的在于提供一种新的小胶质细胞的激活方法，其特征在于，通过Cx3cr1CreER转基因鼠与Ai27转基因鼠进行杂交，获得Cx3cr1CreER-Ai27基因型双转鼠，在腹腔注射Tamoxifen后于目标脑区植入光纤，使用蓝光刺激，即可特异性激活光纤下方区域小胶质细胞。
优选地，所述鼠为大鼠或小鼠，优选地，所述鼠为小鼠。所述蓝光的波长为450-490nm，所述腹腔注射Tamoxifen的浓度为15-25mg/mL，优选地，所述腹腔注射Tamoxifen的浓度为20mg/mL。
本发明的另一个目的在于提供一种上述小胶质细胞的激活方法获得的Cx3cr1CreER-Ai27基因型双转鼠。所述鼠为大鼠或小鼠，优选地，所述鼠为小鼠。
本发明的第三个目的在于上述小胶质细胞的激活方法在小胶质细胞的发育、行为或功能研究中的应用。
本发明的第四个目的在于上述双转鼠在小胶质细胞的发育、行为或功能研究中的应用。
优选地，所述应用可在正常、疾病或损伤条件下进行。
 有益效果
（1）本发明提供的设计一种新型的激活小胶质细胞方法，该方法可在活体特异性标记激活小鼠小胶质细胞，且仅小胶质细胞表达绿色荧光蛋白，准确的区分小胶质细胞与其他神经细胞，实现了活体状态下对小胶质细胞的特异性标记、检测和追踪，可用于实时观测小胶质细胞发育、行为或功能，目前还没有类似的方法或转基因鼠报道。
（2）目前激活小胶质细胞方法的特异性不够强，操作复杂，成本高，本发明的激活小胶质细胞方法操作简便，成本和风险大大降低。
（3）目前小胶质细胞的研究还可以采用特异性的标记物进行离体染色研究，采用本发明的方法可以实现在体和离体对小胶质细胞的特异性标记和激活，在研究小胶质细胞的发育、行为和功能等方面具有重要意义，具有广阔的应用前景。
 附图说明
图1 本发明转基因杂交示意图。其中Cx3cr1-CreER-yfp代表Cx3cr1-CreER转基因小鼠，ChR2-tdTomato代表Ai27转基因小鼠。
图2 是Cx3cr1-CreER转基因小鼠与Cx3cr1-CreER-Ai27转基因小鼠特异性标记和激活荧光对比图。
图3是激活的小胶质细胞与未激活小胶质细胞荧光对比图。不同行代表不同的放大倍数。Ａ和Ｂ为激活的小胶质细胞，Ｃ和Ｄ为未激活小胶质细胞。
 本发明的实施方式
以下通过具体实施例对本发明作进一步详细说明，以使本领域技术人员能够更好地理解本发明并予以实施，但实施例并不作为本发明的限定。
以下实施例中所使用的实验方法如无特殊说明，均为常规方法。所用的材料、试剂等，如无特殊说明，均可从商业途径得到。其中，
实施例1 Cx3cr1CreER-Ai27基因型双转鼠的构建
Cx3cr1CreER转基因小鼠与Ai27转基因小鼠均构自美国杰克森实验室。鼠龄为2-3月龄，体重20-30g，雌雄不限。实验小鼠水和食物供给充足，饲养于12h黑暗，12h光照的环境中，室温控制在20-25℃之间。挑选生长正常的Cx3cr1CreER小鼠与Ai27基因小鼠进行杂交，杂交后剪取剪取小鼠尾尖端4mm左右，用电烙铁为小鼠烫伤止血，将鼠尾置于1.5mlEppendorf管中，加入500μL鼠尾消化缓冲液和4μL蛋白酶K,60℃水浴锅消化12h。将消化好的组织摇匀后，置于离心机中10000rpm离心6min，弃沉淀。再加入500μL的酚氯仿抽提液，颠倒摇匀后，12000rpm离心10min，吸取上层清液200μL。再加入400μL无水乙醇，轻柔颠倒混匀，DNA呈白色絮状沉淀析出，12000rpm离心5min，弃上清，加入400μL的75%乙醇，12000rpm离心5min，弃上清，待乙醇干透后，加入50μL的ddH 2O溶解DNA。置于4℃进行保存。根据美国杰克森实验室提供的转基因的小鼠的目的基因序列信息设计引物，使用百泰克生物技术公司的PCR反应试剂盒对转基因小鼠进行扩增后测序进行验证，测序工作委托华大基因公司进行。经过测序检测，Cx3cr1CreER-Ai27基因型双转鼠的序列与预期一致，杂交小鼠构建成功。
实施例2小胶质细胞激活验证
参见实施例1的构建方法，通过Cx3cr1CreER转基因小鼠与Ai27转基因小鼠进行杂交，获得Cx3cr1CreER-Ai27基因型双转小鼠，为了诱导Cre重组酶，将实施例1构建的Cx3cr1CreER-Ai27基因型双转小鼠用溶于200μl玉米油（Sigma）的4 mgTamoxifen（购自Sigma）进行刺激，在两个时间点分别隔48小时皮下注射或在腹腔注射Tamoxifen，Cx3cr1CreER转基因小鼠作为对照组，采用同样的处理方式。
实验处理结束后，对小鼠腹腔注射氨基甲酸乙酷溶液，进行麻醉后，将其固定于解剖盘上，剪开胸腔皮肤层和肌肉层后，暴露心脏，剪开右心耳，将灌流针插入左心室，注射0.15 MPBS 15 mL，然后再注射4%PFA溶液15mL，从颈部剪断后，用镊子从颅骨中剥离出大脑组织、小脑、以及部分脑干，置于20 mL 4%PFA溶液中，于4℃固定2天。将脑组织从固定液中取出，以冠状切方向修平小脑，并将修整好的组织粘于振动切片机上，调整刀片位置，切片厚度30μm。采用常规的免疫组化染色后，使用蓝光（450-490nm）刺激，即可特异性激活小胶质细胞。由图2可知，Cx3cr1CreER-Ai27基因型双转小鼠与Cx3cr1CreER转基因小鼠相比，更能特异性地区分小胶质细胞与其他细胞，荧光强度更强，小胶质细胞清晰可见。
另外，Tamoxifen实验处理结束后还可以直接在体于目标脑区植入光纤，使用蓝光（450-490nm）刺激，即可特异性激活光纤下方区域小胶质细胞。由图3可知，蓝光照射后，Cx3cr1CreER-Ai27基因型双转小鼠激活的小胶质细胞清晰可见，能特异性地区分小胶质细胞与其他细胞，而未激活的小胶质细胞未能检测到清晰可见的荧光。实现了活体状态下对小胶质细胞的特异性标记、检测和追踪，可用于实时观测小胶质细胞发育、行为或功能。
由上可见，本发明已成功构建Cx3cr1CreER-Ai27基因型双转小鼠。还提供了一种简单高效的新的小胶质细胞激活方法，可以特异性激活小胶质细胞，准确地区分小胶质细胞与其他细胞。将本发明的转基因小鼠进行蓝光刺激，只有小胶质细胞表达绿色荧光蛋白，实现了活体状态下对小胶质细胞的特异性标记、检测和追踪，可用于实时观测小胶质细胞发育、行为或功能。另外，本发明的转基因小鼠还可以通过离体免疫组化染色的方法准确地区分小胶质细胞与其他细胞。
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围。

Claims (9)

  1. 一种新的小胶质细胞的激活方法，其特征在于，通过Cx3cr1CreER转基因鼠与Ai27转基因鼠进行杂交，获得Cx3cr1CreER-Ai27基因型双转鼠，在腹腔注射Tamoxifen后于目标脑区植入光纤，使用蓝光刺激，即可特异性激活光纤下方区域小胶质细胞。
  2. 根据权利要求1所述的小胶质细胞的激活方法，所述鼠为大鼠或小鼠，优选地，所述鼠为小鼠。
  3. 根据权利要求1或2所述的小胶质细胞的激活方法，所述蓝光的波长为450-490nm。
  4. 根据权利要求1-3任一项所述的小胶质细胞的激活方法，所述腹腔注射Tamoxifen的浓度为15-25mg/mL，优选地，所述腹腔注射Tamoxifen的浓度为20mg/mL。
  5. 权利要求1-4任一项所述小胶质细胞的激活方法获得的Cx3cr1CreER-Ai27基因型双转鼠。
  6. 根据权利要求5所述的Cx3cr1CreER-Ai27基因型双转鼠，所述鼠为大鼠或小鼠，优选地，所述鼠为小鼠。
  7. 权利要求1-4任一项所述小胶质细胞的激活方法在小胶质细胞的发育、行为或功能研究中的应用。
  8. 权利要求5或6所述的双转鼠在小胶质细胞的发育、行为或功能研究中的应用。
  9. 根据权利要求7或8所述的应用，所述应用可在正常、疾病或损伤条件下进行。
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