WO2021098613A1 - Novel microglial cell activation method - Google Patents
Novel microglial cell activation method Download PDFInfo
- Publication number
- 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
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- microglia
- mouse
- cells
- transgenic mice
- double transgenic
- Prior art date
Links
- 238000000034 method Methods 0.000 title claims abstract description 31
- 230000002025 microglial effect Effects 0.000 title abstract description 8
- 230000020411 cell activation Effects 0.000 title abstract description 4
- 238000011830 transgenic mouse model Methods 0.000 claims abstract description 35
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 claims abstract description 22
- 229960001603 tamoxifen Drugs 0.000 claims abstract description 11
- 238000011161 development Methods 0.000 claims abstract description 10
- 239000013307 optical fiber Substances 0.000 claims abstract description 10
- 210000004556 brain Anatomy 0.000 claims abstract description 8
- 239000007928 intraperitoneal injection Substances 0.000 claims abstract description 8
- 238000011160 research Methods 0.000 claims abstract description 4
- 210000000274 microglia Anatomy 0.000 claims description 67
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 24
- 241000699660 Mus musculus Species 0.000 claims description 23
- 230000003213 activating effect Effects 0.000 claims description 13
- 201000010099 disease Diseases 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- 230000000638 stimulation Effects 0.000 claims description 4
- 230000006378 damage Effects 0.000 claims description 3
- 208000014674 injury Diseases 0.000 claims description 3
- 208000027418 Wounds and injury Diseases 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 210000004027 cell Anatomy 0.000 description 10
- 108090000835 CX3C Chemokine Receptor 1 Proteins 0.000 description 9
- 102100039196 CX3C chemokine receptor 1 Human genes 0.000 description 9
- 210000003169 central nervous system Anatomy 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- 230000004913 activation Effects 0.000 description 8
- 238000002372 labelling Methods 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 102100020997 Fractalkine Human genes 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 210000001642 activated microglia Anatomy 0.000 description 4
- 210000002569 neuron Anatomy 0.000 description 4
- 101150062345 CX3CR1 gene Proteins 0.000 description 3
- 108010051219 Cre recombinase Proteins 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- 101000854520 Homo sapiens Fractalkine Proteins 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 210000005013 brain tissue Anatomy 0.000 description 3
- 210000004443 dendritic cell Anatomy 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000011532 immunohistochemical staining Methods 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 210000000822 natural killer cell Anatomy 0.000 description 3
- 210000004498 neuroglial cell Anatomy 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 108010078239 Chemokine CX3CL1 Proteins 0.000 description 2
- 108010012236 Chemokines Proteins 0.000 description 2
- 102000019034 Chemokines Human genes 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- 208000012902 Nervous system disease Diseases 0.000 description 2
- 210000001638 cerebellum Anatomy 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 238000009396 hybridization Methods 0.000 description 2
- 210000004713 immature microglia Anatomy 0.000 description 2
- 239000012642 immune effector Substances 0.000 description 2
- 229940121354 immunomodulator Drugs 0.000 description 2
- 238000011813 knockout mouse model Methods 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000014644 Brain disease Diseases 0.000 description 1
- 102000011815 CX3C chemokine domains Human genes 0.000 description 1
- 108050002276 CX3C chemokine domains Proteins 0.000 description 1
- 238000012270 DNA recombination Methods 0.000 description 1
- 208000032274 Encephalopathy Diseases 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- JOYRKODLDBILNP-UHFFFAOYSA-N Ethyl urethane Chemical compound CCOC(N)=O JOYRKODLDBILNP-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 208000036110 Neuroinflammatory disease Diseases 0.000 description 1
- 208000025966 Neurological disease Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 206010057249 Phagocytosis Diseases 0.000 description 1
- 108010091086 Recombinases Proteins 0.000 description 1
- 102000018120 Recombinases Human genes 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000133 brain stem Anatomy 0.000 description 1
- 230000008568 cell cell communication Effects 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000002026 chloroform extract Substances 0.000 description 1
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000002285 corn oil Substances 0.000 description 1
- 235000005687 corn oil Nutrition 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000834 fixative Substances 0.000 description 1
- 108091006047 fluorescent proteins Proteins 0.000 description 1
- 102000034287 fluorescent proteins Human genes 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000007862 fractalkine signaling Effects 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 210000005240 left ventricle Anatomy 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000002864 mononuclear phagocyte Anatomy 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 201000006417 multiple sclerosis Diseases 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000003959 neuroinflammation Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000008782 phagocytosis Effects 0.000 description 1
- 210000004180 plasmocyte Anatomy 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 210000005247 right atrial appendage Anatomy 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- 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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
-
- 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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- 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
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0278—Knock-in vertebrates, e.g. humanised vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
-
- 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
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
-
- 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
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- 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
- A01K2217/00—Genetically modified animals
- A01K2217/15—Animals comprising multiple alterations of the genome, by transgenesis or homologous recombination, e.g. obtained by cross-breeding
-
- 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
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- 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
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0312—Animal model for Alzheimer's disease
-
- 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
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
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.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Animal Behavior & Ethology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Developmental Biology & Embryology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Neurology (AREA)
- Microbiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Public Health (AREA)
- Neurosurgery (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Ophthalmology & Optometry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Provided is a Cx3cr1CreER-Ai27 genotype double transgenic mouse, and a novel microglial cell activation method. The Cx3cr1CreER-Ai27 genotype double transgenic mouse is obtained by crossing a Cx3cr1CreER mouse with an Ai27 gene mouse. After intraperitoneal injection of Tamoxifen, a target brain area is implanted with an optical fiber and stimulated with blue light (450-490 nm), to specifically activate microglial cells in the area implanted with the optical fiber. The microglial cell activation method can specifically activate microglial cells and accurately distinguish the microglial cells from other cells. The method is of great significance in studying the development, behavior, and function of microglial cells, is easy to operate, reduces research costs and risks, and has broad application prospects.
Description
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。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.
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。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. 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。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.
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。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. In the circulatory system, the CX3CR1 gene can be expressed in monocytes, dendritic cells (DC), T cell subtypes, and natural killer cells (NK). In vitro, 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. When 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. 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. 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. However, 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.
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。Ai27 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.
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。At present, the main methods for distinguishing or labeling microglia include morphological observation, characteristic protein immunohistochemical staining, and single-promoter fluorescent protein labeling. However, 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.
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。Therefore, the establishment of effective methods and mouse models for activating microglia in vitro and in vivo is a technical problem that needs to be solved urgently in the prior art. But there are no similar reports or products.
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。Based on this, 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.
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。In order to solve the above-mentioned problems in the prior art, 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 In type double-transformed mice, after intraperitoneal injection of Tamoxifen, 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.
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。Preferably, 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.
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。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.
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。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.
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。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.
优选地，所述应用可在正常、疾病或损伤条件下进行。Preferably, the application can be performed under normal, disease or injury conditions.
（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。(1) 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.
（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。(2) The specificity of the current method for activating microglia is not strong enough, the operation is complicated, and the cost is high. The method for activating microglia of the present invention is easy to operate, and the cost and risk are greatly reduced.
（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。(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.
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。Figure 1 Schematic diagram of transgenic hybridization of the present invention. Among them, Cx3cr1-CreER-yfp represents Cx3cr1-CreER transgenic mice, and ChR2-tdTomato represents Ai27 transgenic mice.
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。Figure 2 is a comparison of specific labeling and activation fluorescence between Cx3cr1-CreER transgenic mice and Cx3cr1-CreER-Ai27 transgenic mice.
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。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.
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。Hereinafter, the present invention will be further described in detail through specific examples, so that those skilled in the art can better understand and implement the present invention, but the examples are not intended to limit the present invention.
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，Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. The materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial sources. among them,
实施例1 Cx3cr1CreER-Ai27基因型双转鼠的构建Example 1 Construction of double transgenic mice of Cx3cr1CreER-Ai27 genotype
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。
Both 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℃. 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. Place the tail in a 1.5ml Eppendorf tube and add 500μL of the mouse The tail digestion buffer and 4μL proteinase K were digested in a 60℃ water bath for 12h. After shaking the digested tissues, place them in a centrifuge at 10,000 rpm for 6 min, and discard the precipitate. Then add 500 μL of phenol chloroform extract, invert and shake well, centrifuge at 12000 rpm for 10 min, and draw 200 μL of the supernatant. Then add 400μL of absolute ethanol, gently invert and mix, the DNA is white flocculent precipitate, centrifuge at 12000rpm for 5min, discard the supernatant, add 400μL of 75% ethanol, centrifuge at 12000rpm for 5min, discard the supernatant, after the ethanol is dry, add 50μL of ddH 2 O dissolves DNA. Store at 4°C. Design primers based on the target gene sequence information of transgenic mice provided by the Jackson Laboratory of the United States, and use Biotech's PCR reaction kit to perform amplification and sequencing of transgenic mice for verification. The sequencing work was entrusted to BGI get on. After sequencing, the sequence of the Cx3cr1CreER-Ai27 genotype double transmouse mouse was consistent with expectations, and the hybrid mouse was successfully constructed.
实施例2小胶质细胞激活验证Example 2 Verification of Microglia Activation
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。Referring to the construction method of Example 1, the Cx3cr1CreER-Ai27 genotype double transgenic mouse was obtained by crossing Cx3cr1CreER transgenic mice with Ai27 transgenic mice. In order to induce Cre recombinase, 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 μl 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.
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。After the experimental treatment, the 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℃ for 2 days. The brain tissue was taken out of the fixative solution, the cerebellum was trimmed in the coronal direction, and the trimmed tissue was glued to the vibrating microtome, the blade position was adjusted, and the slice thickness was 30 μm. After using conventional immunohistochemical staining and stimulation with blue light (450-490nm), 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.
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。In addition, after the Tamoxifen experimental treatment is completed, 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. It can be seen from Figure 3 that after blue light irradiation, 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.
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。It can be seen from the above that 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. When 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. In addition, the transgenic mice of the present invention can also accurately distinguish microglia from other cells by the method of in vitro immunohistochemical staining.
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。The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. range.
Claims (9)
- 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。 A new method for activating microglia, which is characterized by hybridizing Cx3cr1CreER transgenic mice with Ai27 transgenic mice to obtain Cx3cr1CreER-Ai27 genotype double transgenic mice. After intraperitoneal injection of Tamoxifen, optical fibers are implanted in the target brain area. Using blue light stimulation can specifically activate the microglia in the area under the optical fiber.
- 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。 The method for activating microglia according to claim 1, wherein the mouse is a rat or a mouse, preferably, the mouse is a mouse.
- 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。 The method for activating microglia according to claim 1 or 2, wherein the wavelength of the blue light is 450-490 nm.
- 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。 The method for activating microglia according to any one of claims 1-3, wherein 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.
- 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。 The Cx3cr1CreER-Ai27 genotype double transgenic mouse obtained by the method for activating microglia of any one of claims 1-4.
- 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。 The Cx3cr1CreER-Ai27 genotype double transgenic mouse according to claim 5, wherein the mouse is a rat or a mouse, preferably, the mouse is a mouse.
- 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。 Application of the method for activating microglia according to any one of claims 1 to 4 in the development, behavior or function research of microglia.
- 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。 The use of the double transgenic mouse of claim 5 or 6 in the study of the development, behavior or function of microglia.
- 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。The application according to claim 7 or 8, which can be performed under normal, disease or injury conditions.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911155688.XA CN112825816A (en) | 2019-11-22 | 2019-11-22 | Novel microglial cell activation method |
| CN201911155688.X | 2019-11-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2021098613A1 true WO2021098613A1 (en) | 2021-05-27 |
Family
ID=75921834
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/CN2020/128851 WO2021098613A1 (en) | 2019-11-22 | 2020-11-13 | Novel microglial cell activation method |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN112825816A (en) |
| WO (1) | WO2021098613A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104846014A (en) * | 2015-02-28 | 2015-08-19 | 中国科学院深圳先进技术研究院 | Specificity excitory astrocyte composition and application of composition thereof for improving schizophrenia abnormal behavior |
| CN109641030A (en) * | 2016-06-13 | 2019-04-16 | 优特埃合伙有限公司 | For adjusting the method and composition of Opiate withdrawal symptoms |
-
2019
- 2019-11-22 CN CN201911155688.XA patent/CN112825816A/en active Pending
-
2020
- 2020-11-13 WO PCT/CN2020/128851 patent/WO2021098613A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104846014A (en) * | 2015-02-28 | 2015-08-19 | 中国科学院深圳先进技术研究院 | Specificity excitory astrocyte composition and application of composition thereof for improving schizophrenia abnormal behavior |
| CN109641030A (en) * | 2016-06-13 | 2019-04-16 | 优特埃合伙有限公司 | For adjusting the method and composition of Opiate withdrawal symptoms |
Non-Patent Citations (6)
| Title |
|---|
| LINDA MADISEN, TIANYI MAO, HENNER KOCH, JIA-MIN ZHUO, ANTAL BERENYI, SHIGEYOSHI FUJISAWA, YUN-WEI A HSU, ALFREDO J GARCIA, XUAN GU: "A toolbox of Cre-dependent optogenetic transgenic mice for light-induced activation and silencing", NATURE NEUROSCIENCE, NATURE AMERICA , INC, US, vol. 15, no. 5, US, pages 793 - 802, XP055400277, ISSN: 1097-6256, DOI: 10.1038/nn.3078 * |
| PARKHURST CHRISTOPHER�N.; YANG GUANG; NINAN IPE; SAVAS JEFFREY�N.; YATES JOHN�R.; LAFAILLE JUAN�J.; HEMPSTEAD BARBARA�L.; LITTMAN : "Microglia Promote Learning-Dependent Synapse Formation through Brain-Derived Neurotrophic Factor", CELL, ELSEVIER, AMSTERDAM NL, vol. 155, no. 7, 19 December 2013 (2013-12-19), Amsterdam NL, pages 1596 - 1609, XP028806602, ISSN: 0092-8674, DOI: 10.1016/j.cell.2013.11.030 * |
| TING JONATHAN T.; FENG GUOPING: "Development of transgenic animals for optogenetic manipulation of mammalian nervous system function: Progress and prospects for behavioral neuroscience", BEHAVIOURAL BRAIN RESEARCH., ELSEVIER, AMSTERDAM., NL, vol. 255, 6 March 2013 (2013-03-06), NL, pages 3 - 18, XP028740249, ISSN: 0166-4328, DOI: 10.1016/j.bbr.2013.02.037 * |
| YAWO HIROMU, ASANO TOSHIFUMI, SAKAI SEIICHIRO, ISHIZUKA TORU: "Optogenetic manipulation of neural and non-neural functions", DEVELOPMENT GROWTH AND DIFFERENTIATION., RICHMOND, VIC. : BLACKWELL PUBL. ASIA, US, vol. 55, no. 4, 1 May 2013 (2013-05-01), US, pages 474 - 490, XP055814088, ISSN: 0012-1592, DOI: 10.1111/dgd.12053 * |
| ZHANG, B. ET AL.: "The specificity and role of microglia in epieptogenesis in mouse models of tuberous sclerosis complex", EPILEPSIA, vol. 59, no. 9, 5 August 2018 (2018-08-05), XP055814084 * |
| ZHAO XIAO-FENG, ALAM MAHABUB MARAJ, LIAO YUAN, HUANG TINGTING, MATHUR RAMKUMAR, ZHU XINJUN, HUANG YUNFEI: "Targeting Microglia Using Cx3cr1-Cre Lines: Revisiting the Specificity", ENEURO, vol. 6, no. 4, 1 July 2019 (2019-07-01), pages ENEURO.0114 - 19.2019, XP055814080, DOI: 10.1523/ENEURO.0114-19.2019 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112825816A (en) | 2021-05-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Inoue et al. | Development of new inbred transgenic strains of rats with LacZ or GFP | |
| JP2022031487A (en) | Genetically modified cells, tissues and organs for treating disease | |
| Petrich et al. | Temporal activation of c‐Jun N‐terminal kinase in adult transgenic heart via cre‐loxP‐mediated DNA recombination | |
| Lü et al. | Both the transplantation of somatic cell nuclear transfer-and fertilization-derived mouse embryonic stem cells with temperature-responsive chitosan hydrogel improve myocardial performance in infarcted rat hearts | |
| JPH1057054A (en) | Unrestricted donor cell | |
| Liu et al. | Versatile cell ablation tools and their applications to study loss of cell functions | |
| Yeo et al. | Development of a Pde6b gene knockout rat model for studies of degenerative retinal diseases | |
| Krug et al. | Generation of a transparent killifish line through multiplex CRISPR/Cas9mediated gene inactivation | |
| CN114747542B (en) | Construction method and application of DSC2 gene knockout mouse model | |
| Kramer et al. | Generation of a DAT-P2A-Flpo mouse line for intersectional genetic targeting of dopamine neuron subpopulations | |
| CN106620721B (en) | Application of monocyte chemotactic protein-1 induced protein1 (Mcpip 1) in liver ischemia-reperfusion injury | |
| TWI263784B (en) | Encapsulated cell indicator system | |
| JP2009544362A (en) | Use of mitotically inactivated stem cells for the repair of damaged tissue | |
| WO2013063076A1 (en) | Compositions for and methods of modulating complications, risks and issues with xenotransplantation | |
| WO2021098613A1 (en) | Novel microglial cell activation method | |
| Kamran et al. | Gene therapy for the treatment of neurological disorders: central nervous system neoplasms | |
| Li et al. | Recent progress on technologies and applications of transgenic poultry | |
| KR101588474B1 (en) | Method for introducing foreign gene into early embryo of primate animal, and method for production of transgenic primate animal comprising the introduction method | |
| CN110283851A (en) | Target spot MYO9B relevant to malignant pleural effusion and its application | |
| WO2019227882A1 (en) | Construction of mouse model with idiopathic basal ganglia calcification pathogenic gene mutation | |
| US6642369B1 (en) | Nucleic acids involved in the responder phenotype and applications thereof | |
| KR101662734B1 (en) | Method for Enhancing Production of Transgenic Animal by Treatment of Streptolysin O | |
| CN114868707B (en) | Zebra fish model for metabolic encephalopathy and arrhythmia diseases and application thereof | |
| CN106310225B (en) | Application of caspase recruitment domain protein 6 (Card 6) in liver ischemia-reperfusion injury | |
| CN106188266A (en) | Protein B ach1 of a kind of angiogenesis inhibiting and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20890429 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 20890429 Country of ref document: EP Kind code of ref document: A1 |
|
| 32PN | Ep: public notification in the ep bulletin as address of the adressee cannot be established |
Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 10.01.2023) |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 20890429 Country of ref document: EP Kind code of ref document: A1 |