CN110951781A - 一种癫痫动物模型的构建方法及应用 - Google Patents
一种癫痫动物模型的构建方法及应用 Download PDFInfo
- Publication number
- CN110951781A CN110951781A CN201911310888.8A CN201911310888A CN110951781A CN 110951781 A CN110951781 A CN 110951781A CN 201911310888 A CN201911310888 A CN 201911310888A CN 110951781 A CN110951781 A CN 110951781A
- Authority
- CN
- China
- Prior art keywords
- animal
- epilepsy
- animal model
- exon
- mic19
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 238000010171 animal model Methods 0.000 title claims abstract description 20
- 238000010276 construction Methods 0.000 title claims abstract description 16
- 101001121072 Homo sapiens MICOS complex subunit MIC19 Proteins 0.000 claims abstract description 28
- 206010015037 epilepsy Diseases 0.000 claims abstract description 28
- 102100026626 MICOS complex subunit MIC19 Human genes 0.000 claims abstract description 27
- 241001465754 Metazoa Species 0.000 claims abstract description 22
- 238000011705 epilepsy animal model Methods 0.000 claims abstract description 17
- 230000002269 spontaneous effect Effects 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 3
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 52
- 230000001037 epileptic effect Effects 0.000 claims description 24
- 108090000623 proteins and genes Proteins 0.000 claims description 18
- 238000005516 engineering process Methods 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- 239000003814 drug Substances 0.000 claims description 12
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 11
- 229940079593 drug Drugs 0.000 claims description 9
- 229930193140 Neomycin Natural products 0.000 claims description 8
- 210000002459 blastocyst Anatomy 0.000 claims description 8
- 229960004927 neomycin Drugs 0.000 claims description 8
- 238000012216 screening Methods 0.000 claims description 8
- 230000008685 targeting Effects 0.000 claims description 8
- 101150036876 cre gene Proteins 0.000 claims description 7
- 238000011161 development Methods 0.000 claims description 7
- 108700028369 Alleles Proteins 0.000 claims description 6
- 238000011160 research Methods 0.000 claims description 6
- 230000002068 genetic effect Effects 0.000 claims description 4
- 230000008579 epileptogenesis Effects 0.000 claims description 3
- 241000283690 Bos taurus Species 0.000 claims description 2
- 238000010453 CRISPR/Cas method Methods 0.000 claims description 2
- 241000700199 Cavia porcellus Species 0.000 claims description 2
- 241001494479 Pecora Species 0.000 claims description 2
- 241000700159 Rattus Species 0.000 claims description 2
- 238000010459 TALEN Methods 0.000 claims description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 claims description 2
- 210000001161 mammalian embryo Anatomy 0.000 claims description 2
- 230000013011 mating Effects 0.000 claims description 2
- 230000009456 molecular mechanism Effects 0.000 claims description 2
- 210000000130 stem cell Anatomy 0.000 claims description 2
- 241000255588 Tephritidae Species 0.000 claims 1
- 230000006698 induction Effects 0.000 abstract description 9
- 241000699670 Mus sp. Species 0.000 description 52
- 210000004556 brain Anatomy 0.000 description 16
- 230000002438 mitochondrial effect Effects 0.000 description 10
- 238000011740 C57BL/6 mouse Methods 0.000 description 7
- 210000003470 mitochondria Anatomy 0.000 description 7
- 108010051219 Cre recombinase Proteins 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 102000004169 proteins and genes Human genes 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 210000002569 neuron Anatomy 0.000 description 5
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 4
- 102000008730 Nestin Human genes 0.000 description 4
- 108010088225 Nestin Proteins 0.000 description 4
- 230000005856 abnormality Effects 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 230000007246 mechanism Effects 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 238000010172 mouse model Methods 0.000 description 4
- 210000005055 nestin Anatomy 0.000 description 4
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 3
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 229910001424 calcium ion Inorganic materials 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 238000003752 polymerase chain reaction Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- ZKHQWZAMYRWXGA-UHFFFAOYSA-N Adenosine triphosphate Natural products C1=NC=2C(N)=NC=NC=2N1C1OC(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)C(O)C1O ZKHQWZAMYRWXGA-UHFFFAOYSA-N 0.000 description 2
- 206010051290 Central nervous system lesion Diseases 0.000 description 2
- 101000572820 Homo sapiens MICOS complex subunit MIC60 Proteins 0.000 description 2
- 102100026639 MICOS complex subunit MIC60 Human genes 0.000 description 2
- 108010058682 Mitochondrial Proteins Proteins 0.000 description 2
- 102000006404 Mitochondrial Proteins Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 239000012805 animal sample Substances 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 210000001700 mitochondrial membrane Anatomy 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000035806 respiratory chain Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 210000004291 uterus Anatomy 0.000 description 2
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- 230000002407 ATP formation Effects 0.000 description 1
- ZKHQWZAMYRWXGA-KQYNXXCUSA-J ATP(4-) Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-J 0.000 description 1
- 206010001497 Agitation Diseases 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108010061833 Integrases Proteins 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 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
- 241000124008 Mammalia Species 0.000 description 1
- HOKKHZGPKSLGJE-UHFFFAOYSA-N N-methyl-D-aspartic acid Natural products CNC(C(O)=O)CC(O)=O HOKKHZGPKSLGJE-UHFFFAOYSA-N 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 108091081062 Repeated sequence (DNA) Proteins 0.000 description 1
- 101001010097 Shigella phage SfV Bactoprenol-linked glucose translocase Proteins 0.000 description 1
- 108010052160 Site-specific recombinase Proteins 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 235000001014 amino acid Nutrition 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004958 brain cell Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000001964 calcium overload Effects 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000004891 communication Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000002964 excitative effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000004898 mitochondrial function Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000010627 oxidative phosphorylation Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000001242 postsynaptic effect Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000001020 rhythmical effect Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000005062 synaptic transmission Effects 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 230000009261 transgenic effect 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
- 241001515965 unidentified phage Species 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- 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
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- 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
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Environmental Sciences (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
本发明涉及医学和生物实验模型的制备技术领域,尤其涉及一种癫痫动物模型的构建方法及应用。通过动物Mic19特异性敲除的方式构建自发性癫痫的动物模型。采用本发明的构建方法所得到的癫痫动物模型具有更好的稳定性、更低的个体差异、诱导成功率高。
Description
技术领域
本发明涉及医学和生物实验模型的制备技术领域,尤其涉及一种癫痫动物模型的构建方法及应用。
背景技术
癫痫是神经元异常同步放电,其发生机制主要是由于谷氨酸、天冬氨酸和γ-氨基丁酸介导。谷氨酸(Glutamic acid,以下简称Glu)作为神经兴奋性递质,可以被神经突触后膜的Glu受体,即N-甲基-D-天冬氨酸受体(N-methyl-D-aspartic acid receptor,以下简称NMDAR)结合,进而引起钙离子内流,使后续信号转导通路中关键分子的表达与活性发生改变,最终是使脑内兴奋—抑制平衡被破坏。
研究发现线粒体功能失常会引起神经元内钙离子动态失衡,导致钙离子内流,细胞内长期持续的钙超载,会引起细胞结构和功能发生可塑性的改变,进而影响神经元兴奋性和突触的传递,最终导致癫痫的发作;而线粒体是真核生物细胞内能量合成的重要场所,线粒体内膜向线粒体基质处突起形成线粒体嵴,线粒体嵴在线粒体内整齐有序的排列,是线粒体内产生腺嘌呤核苷三磷酸(Adenosine triphosphate,以下简称ATP)的重要场所。线粒体嵴的异常导致氧化磷酸化水平降低,ATP的产生减少,影响线粒体外膜与内膜的连接与通讯,进而影响线粒体蛋白向嵴内的运输。Mic19,在哺乳动物中又称Chchd3(Coiled-Coil-Helix-Coiled-Coil-Helix Domain Containing 3,小鼠中基因ID:66075,基因定位:Chromosome 6,NC_000072.6)。Mic19基因编码的蛋白是线粒体内膜结构调控关键复合物(mitochondrial contact site and cristae organizing system,以下简称MICOS复合物)中唯一一个非膜整合蛋白,在空间上既能直接与线粒体蛋白MIC60多聚体结合,又能与MICOS亚复合物结合,是MIC60多聚体与MICOS亚复合物结合的连接蛋白。MIC19缺失,将导致线粒体嵴形态异常和嵴连接点减少。因此,MIC19对MICOS复合物的组装、稳定性以及维持线粒体嵴的正常形态方面起到至关重要的作用,并对癫痫的发生和变化机制具有重要影响;然而,现有癫痫动物模型大多应用药物诱导,药物诱导存在诱导成功率低、癫痫级别低以及同批次诱导个体间差异大等缺陷,不利于相关科学研究的进展。
发明内容
本发明的目的之一在于提供一种癫痫动物模型的构建方法,诱导成功率较高、癫痫级别较高及同批次诱导个体间差异较小。
本发明的目的之二在于提供一种癫痫动物模型。
本发明的目的之三在于提供一种癫痫动物模型的应用。
本发明实现目的之一所采用的方案是:一种癫痫动物模型的构建方法,通过动物Mic19特异性敲除的方式构建自发性癫痫的动物模型。
优选地,利用Cre-LoxP技术、TALEN技术、ZFN技术或CRISPR/Cas技术进行动物Mic19特异性敲除。
优选地,当利用Cre-LoxP技术进行动物Mic19特异性敲除时,包括以下步骤:
(1)将编码Loxp、FRT-PGK-neomycin-FRT-Loxp的基因序列分别插入到Mic19等位基因载体的外显子的两端的内含子序列,构建形成打靶载体;
(2)将所述步骤(1)中得到的打靶载体显微注射到动物A的胚胎干细胞,筛选含有新霉素抗性的胚胎干细胞;
(3)将所述步骤(2)中筛选的胚胎干细胞注射到动物A的囊胚中,并移植所述囊胚到假孕动物A的子宫发育形成嵌合A;
(4)将所述步骤(3)得到的嵌合A与动物A交配得到杂合A,将所述杂合A与动物A杂交若干代,得到遗传背景较纯的杂合子A;
(5)将所述步骤(4)得到的杂合子A自交产生纯合F0,纯合F0与带Cre基因的动物A杂交得到F1;
(6)将所述步骤(5)得到的F1与纯合F0杂交得到敲除Mic19的癫痫动物A。
优选地,所述步骤(1)中,外显子包括外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8中的任意一个。
优选地,所述步骤(2)中,利用PCR技术筛选含有新霉素抗性的胚胎干细胞。
优选地,动物A包括小鼠、大鼠、果蝇、豚鼠、猪、牛、羊中的任意一种。
本发明实现目的之二所采用的方案是:一种所述的癫痫动物模型的构建方法得到的癫痫动物模型,所述癫痫动物模型为自发性癫痫。
本发明实现目的之三所采用的方案是:一种所述的癫痫动物模型的构建方法得到的癫痫动物模型的应用于研究癫痫发生发展的分子机制,或制备和/或筛选治疗癫痫的药物。
本发明具有以下优点和有益效果:本发明采用动物脑部Mic19特异性敲除的方式构建自发性癫痫的动物模型,相比于药物诱导的癫痫动物模型,采用本发明的构建方法所得到的癫痫动物模型具有更好的稳定性以及更低的同批次诱导个体间差异;对癫痫动物的诱导成功率也高于药物诱导。
采用本发明的构建方法所得到的癫痫动物模型为自发性癫痫。
采用本发明的构建方法所得到的癫痫动物模型有助于深入研究分子水平上癫痫发生发展的具体机制,并且为制备及筛选治疗癫痫的药物提供了有效的动物样本,能更好地满足癫痫疾病研究的需要。
附图说明
图1为本发明实施例的癫痫小鼠模型构建方法的基本流程图;
图2为本发明实施例的Mic19中敲除外显子2的示意图;
图3为本发明实施例的癫痫小鼠和对照小鼠的基因型鉴定图,其中,Mic19+/+(代表的是对照组小鼠)、Mic19+/-(代表的是杂合子)、Mic19-/-(代表的是Mic19敲除小鼠);
图4为本发明实施例的对照小鼠与癫痫小鼠体型示意图;
图5为本发明实施例的对照小鼠的脑电图;
图6为本发明实施例的癫痫小鼠的脑电图。
具体实施方式
为更好的理解本发明,下面的实施例是对本发明的进一步说明,但本发明的内容不仅仅局限于下面的实施例。
如图1所示为本实施例的癫痫动物模型构建方法的基本流程图,本实施例首先利用Cre-LoxP技术将编码FRT-PGK-neomycin-FRT-Loxp的基因序列插入到野生型Mic19等位基因载体的外显子2和外显子3之间的内含子序列,并在所述外显子1之后的内含子序列插入另外一个同向的Loxp序列,构建形成打靶载体。所述编码FRT-PGK-neomycin-FRT-Loxp的基因序列为带有新霉素抗性的基因序列;然后,将所述打靶载体显微注射到C57BL/6品系小鼠的胚胎干细胞,利用聚合酶链式反应技术(Polymerase Chain Reaction,以下简称PCR技术)筛选含有新霉素抗性的小鼠胚胎干细胞;再将所述筛选的小鼠胚胎干细胞注射到C57BL/6品系小鼠的囊胚中,移植所述囊胚到假孕小鼠的子宫发育形成嵌合鼠;接着,所述嵌合鼠与C57BL/6品系小鼠交配得到杂合小鼠,将所述杂合小鼠与C57BL/6品系小鼠杂交若干代,得到遗传背景较纯的杂合子小鼠;杂合子小鼠自交产生纯合小鼠F0,纯合小鼠F0与带Cre基因的小鼠杂交得到小鼠F1;将所述小鼠F1与纯合小鼠F0杂交得到敲除Mic19外显子2的癫痫小鼠和同窝未敲除Mic19外显子2的对照小鼠。
本实施例通过转基因技术得到的小鼠模型的癫痫发病成功率高于药物诱导,避免了药物诱导的不确定性;并且同批次诱导的小鼠个体由同一批小鼠F1与纯合小鼠F0杂交得到,所有癫痫小鼠和对照小鼠的培育条件保持一致,有效降低小鼠个体间差异。其中,所述野生型Mic19等位基因载体和所述编码FRT-PGK-neomycin-FRT-Loxp的基因序列可通过购买获得,FRT代表短肽酶识别靶标位点(short Flippase Recognition Target sites,简称FRT),FRT由34bp构成、是构建在载体上的序列,通过同源重组的方式可以将FRT插入到小鼠基因组,FRT序列是基因报告序列;LoxP序列是由两个13bp反向重复序列和中间间隔的8bp序列共同组成;neomycin序列表示新霉素位点。所述C57BL/6品系小鼠及胚胎干细胞可通过购买获得。所述Cre基因为Cre重组酶基因,Cre重组酶来源于P1噬菌体的酪氨酸重组酶,Cre重组酶基因编码区序列全长1029bp(EMBL数据库登录号X03453),编码由343个氨基酸组成的38kDa单体蛋白,Cre重组酶属于位点特异性重组酶的整合酶家族的成员,能够介导两个LoxP位点(序列)之间的特异性重组,使LoxP位点间的基因序列被删除或重组。所述Nestin-Cre工具鼠为巢蛋白(Nestin)能够调控Cre重组酶表达的工具鼠,即在工具鼠中,将Cre重组酶基因序列插入在巢蛋白(Nestin)调控启动子序列,所述Nestin-Cre工具鼠公司从jackson购买得到。
如图2所示,为本实施例的Mic19中敲除外显子2的示意图,其中,Exon1为外显子1,Exon2为外显子2,并以此类推。如图2所示,利用所述Cre-LoxP技术在小鼠Mic19野生型等位基因中特定的外显子2两侧插入两个同向排列的Loxp位点,通过打靶载体和同源重组形成靶向等位基因;将正确插入同向Loxp位点的打靶载体注射到小鼠的胚胎干细胞(EmbryonicStem Cell,以下简称:ES细胞)中,通过抗性筛选出已发生同源重组的ES细胞;之后将筛选出的ES细胞注射到囊胚中,再移植到假孕小鼠的子宫发育形成嵌合鼠;所述嵌合鼠与C57BL/6品系小鼠交配产生杂合小鼠,将所述杂合小鼠与野生型小鼠杂交10代左右,得到遗传背景较纯的杂合子小鼠,杂合子小鼠自交产生Loxp/Loxp小鼠(纯合小鼠F0),所述Loxp/Loxp小鼠的表型与C57BL/6品系的野生型小鼠一样。所述Loxp/Loxp小鼠再与Nestin-Cre工具鼠杂交,产生的子代小鼠中同时含有Cre和Loxp/Loxp的靶基因,所述子代小鼠中Cre基因表达产生的重组酶介导靶基因两侧的Loxp位点发生切除,结果将一个Loxp位点和靶序列切除,即子代小鼠的Mic19敲除了外显子2,形成了自发性癫痫的小鼠模型;所述子代小鼠中Cre基因未表达,即子代小鼠的Mic19未敲除外显子2,形成了同窝未敲除的对照小鼠。
在通过所述构建方法得到癫痫小鼠和对照小鼠后,本实施例还需对所述癫痫小鼠和对照小鼠进行相关鉴定。所述鉴定方法包括:在本发明实施例的小鼠繁殖过程中,鉴定并记录所有小鼠的基因型,所有基因型统计后符合孟德尔遗传定律,图3所示为本实施例的癫痫小鼠和对照小鼠基因型鉴定图。如图3所示,对照小鼠的基因型如图3中第一泳道所示,癫痫小鼠的基因型如图3中第三泳道所示。每周记录同窝出生的癫痫小鼠和对照小鼠的体重;图4为本发明实施例的对照小鼠与癫痫小鼠体型示意图,可以看出相同培养条件下癫痫小鼠的体型明显小于对照小鼠的体型;之后,对所述癫痫小鼠和对照小鼠同时进行麻醉,将电极植于所述癫痫小鼠和对照小鼠的硬脑膜表面或其他需要记录的相关脑区。所述癫痫小鼠和对照小鼠恢复一周后,通过电极记录小鼠脑细胞群的自发性、节律性电活动,图5示出了本实施例的对照小鼠的脑电图,图6示出了本实施例的癫痫小鼠的脑电图。如图6所示,脑电图结果显示小鼠发病过程中出现高幅慢波,相对于对照组小鼠脑电图有显著差异,再次证明本实施例得到的是癫痫动物模型。再通过统计软件对收集的电活动数据进行分析,得到癫痫小鼠和对照小鼠的神经元细胞电生理活动参数、各脑区之间链接情况参数、脑区不同波段能量参数等多种小鼠脑区物理参数,便于后续深入进行分子水平的分析;完成小鼠脑区物理参数收集后,充分麻醉所述癫痫小鼠和对照小鼠,快速取下所有小鼠脑部进行称重拍照,将每个所述小鼠脑部进行分开保存,分别用于:提取蛋白进行免疫印迹实验,分析线粒体蛋白及呼吸链复合物亚基蛋白的表达量变化;密度梯度离心法提取线粒体,使用不同复合物体系分别检测呼吸链复合物的活性。首先,取下小鼠脑部后,对脑部用多聚甲醛固定、蔗糖脱水、包埋、冰冻切片后进行组织染色,得到的小鼠脑部样本用于观察并分析小鼠脑部结构是否存在重塑以及神经元和胶质细胞的数目和形态是否存在异常;或者,对脑部组织用戊二醛固定、树脂硬化包埋、半薄切片修面和超薄切片支撑铜网,可通过透射电镜观察得到的小鼠脑部样本中线粒体的结构和功能。所述鉴定过程将小鼠脑组织功能与线粒体结构功能的检测联系起来,可以实时动态了解不同时间点小鼠脑部线粒体结构和功能异常导致的脑病变,从而更加直观深入地研究小鼠脑部线粒体结构和功能的异常引起脑病变。
本发明实施例采用了小鼠脑部Mic19特异性敲除的方式构建自发性癫痫的小鼠模型,相比于药物诱导的癫痫动物模型,所述癫痫动物模型具有更好的稳定性以及更低的同批次诱导个体间差异,所述构建方法对癫痫动物的诱导成功率也高于药物诱导;所述癫痫动物模型有助于深入研究分子水平上癫痫病发生发展的具体机制,并且为制备及筛选治疗癫痫的药物提供了有效的动物样本,能更好地满足癫痫疾病研究的需要。
以上所述是本发明的优选实施方式而已,当然不能以此来限定本发明之权利范围,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和变动,这些改进和变动也视为本发明的保护范围。
Claims (8)
1.一种癫痫动物模型的构建方法,其特征在于:通过动物Mic19特异性敲除的方式构建自发性癫痫的动物模型。
2.根据权利要求1所述的癫痫动物模型的构建方法,其特征在于:利用Cre-LoxP技术、TALEN技术、ZFN技术或CRISPR/Cas技术进行动物Mic19特异性敲除。
3.根据权利要求1所述的癫痫动物模型的构建方法,其特征在于:当利用Cre-LoxP技术进行动物Mic19特异性敲除时,包括以下步骤:
(1)将编码Loxp、FRT-PGK-neomycin-FRT-Loxp的基因序列分别插入到Mic19等位基因载体的外显子的两端的内含子序列,构建形成打靶载体;
(2)将所述步骤(1)中得到的打靶载体显微注射到动物A的胚胎干细胞,筛选含有新霉素抗性的胚胎干细胞;
(3)将所述步骤(2)中筛选的胚胎干细胞注射到动物A的囊胚中,并移植所述囊胚到假孕动物A的子宫发育形成嵌合A;
(4)将所述步骤(3)得到的嵌合A与动物A交配得到杂合A,将所述杂合A与动物A杂交若干代,得到遗传背景较纯的杂合子A;
(5)将所述步骤(4)得到的杂合子A自交产生纯合F0,纯合F0与带Cre基因的动物A杂交得到F1;
(6)将所述步骤(5)得到的F1与纯合F0杂交得到敲除Mic19的癫痫动物A。
4.根据权利要求3所述的癫痫动物模型的构建方法,其特征在于:所述步骤(1)中,外显子包括外显子1、外显子2、外显子3、外显子4、外显子5、外显子6、外显子7、外显子8中的任意一个。
5.根据权利要求3所述的癫痫动物模型的构建方法,其特征在于:所述步骤(2)中,利用PCR技术筛选含有新霉素抗性的胚胎干细胞。
6.根据权利要求3所述的癫痫动物模型的构建方法,其特征在于:动物A包括小鼠、大鼠、果蝇、豚鼠、猪、牛、羊中的任意一种。
7.一种如权利要求1-6中任一所述的癫痫动物模型的构建方法得到的癫痫动物模型,其特征在于:所述癫痫动物模型为自发性癫痫。
8.如权利要求1-6中任一所述的癫痫动物模型的构建方法得到的癫痫动物模型应用于研究癫痫发生发展的分子机制,或制备和/或筛选治疗癫痫的药物。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911310888.8A CN110951781B (zh) | 2019-12-18 | 2019-12-18 | 一种癫痫动物模型的构建方法及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201911310888.8A CN110951781B (zh) | 2019-12-18 | 2019-12-18 | 一种癫痫动物模型的构建方法及应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN110951781A true CN110951781A (zh) | 2020-04-03 |
CN110951781B CN110951781B (zh) | 2021-06-04 |
Family
ID=69982615
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201911310888.8A Active CN110951781B (zh) | 2019-12-18 | 2019-12-18 | 一种癫痫动物模型的构建方法及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN110951781B (zh) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112575033A (zh) * | 2020-12-24 | 2021-03-30 | 南京启真基因工程有限公司 | Crispr系统及其在构建scn1a基因突变的癫痫性脑病克隆猪核供体细胞中的应用 |
CN112680453A (zh) * | 2021-01-26 | 2021-04-20 | 南京启真基因工程有限公司 | Crispr系统及其在构建stxbp1突变的癫痫性脑病克隆猪核供体细胞中的应用 |
WO2024087014A1 (zh) * | 2022-10-25 | 2024-05-02 | 中国科学院深圳先进技术研究院 | 一种癫痫动物模型的制备方法及应用 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20160031955A1 (en) * | 2013-01-31 | 2016-02-04 | INSERM (Institut National de la Santé et de la Recherche Médicale | Methods for Treating Mitochondrial Disorders and Neurodegenerative Disorders |
CN109022486A (zh) * | 2018-09-20 | 2018-12-18 | 河北医科大学第二医院 | 一种癫痫动物模型的构建方法 |
CN110049769A (zh) * | 2016-12-04 | 2019-07-23 | 阿拉维·霍拉萨尼·默哈达姆·马塞尔·维克托 | 治疗与线粒体应激相关的疾病的方法 |
-
2019
- 2019-12-18 CN CN201911310888.8A patent/CN110951781B/zh active Active
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20160031955A1 (en) * | 2013-01-31 | 2016-02-04 | INSERM (Institut National de la Santé et de la Recherche Médicale | Methods for Treating Mitochondrial Disorders and Neurodegenerative Disorders |
CN110049769A (zh) * | 2016-12-04 | 2019-07-23 | 阿拉维·霍拉萨尼·默哈达姆·马塞尔·维克托 | 治疗与线粒体应激相关的疾病的方法 |
CN109022486A (zh) * | 2018-09-20 | 2018-12-18 | 河北医科大学第二医院 | 一种癫痫动物模型的构建方法 |
Non-Patent Citations (3)
Title |
---|
GUARANI V等: "QIL1 is a novel mitochondrial protein required for MICOS complex stability and cristae morphology", 《ELIFE》 * |
KUNDAP UP等: "Animal Models of Metabolic Epilepsy and Epilepsy Associated Metabolic Dysfunction: A Systematic Review", 《PHARMACEUTICALS (BASEL)》 * |
王小姗,黄舒扬: "线粒体疾病与癫痫", 《中华医学会第十八次全国神经病学学术会议论文汇编(下)》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN112575033A (zh) * | 2020-12-24 | 2021-03-30 | 南京启真基因工程有限公司 | Crispr系统及其在构建scn1a基因突变的癫痫性脑病克隆猪核供体细胞中的应用 |
CN112575033B (zh) * | 2020-12-24 | 2023-03-10 | 南京启真基因工程有限公司 | Crispr系统及其在构建scn1a基因突变的癫痫性脑病克隆猪核供体细胞中的应用 |
CN112680453A (zh) * | 2021-01-26 | 2021-04-20 | 南京启真基因工程有限公司 | Crispr系统及其在构建stxbp1突变的癫痫性脑病克隆猪核供体细胞中的应用 |
WO2024087014A1 (zh) * | 2022-10-25 | 2024-05-02 | 中国科学院深圳先进技术研究院 | 一种癫痫动物模型的制备方法及应用 |
Also Published As
Publication number | Publication date |
---|---|
CN110951781B (zh) | 2021-06-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110951781B (zh) | 一种癫痫动物模型的构建方法及应用 | |
Nakane et al. | High incidence of ultraviolet-B-or chemical-carcinogen-induced skin tumours in mice lacking the xeroderma pigmentosum group A gene | |
Sleat et al. | A mouse model of classical late-infantile neuronal ceroid lipofuscinosis based on targeted disruption of the CLN2 gene results in a loss of tripeptidyl-peptidase I activity and progressive neurodegeneration | |
JP3679403B2 (ja) | トランスジェニック動物 | |
CN107760713B (zh) | 一种非人哺乳动物神经精神疾病动物模型的建立方法及其用途 | |
Avraham | Mouse models for deafness: lessons for the human inner ear and hearing loss | |
Festing et al. | Laboratory animal genetics and genetic quality control | |
CN105494263B (zh) | 一种产生ho-1/app/psen1三转基因阿尔茨海默病小鼠模型的方法 | |
US9429567B2 (en) | Method for screening substances having weight-regulating action | |
Canales et al. | The mouse, a model organism for biomedical research | |
CN106282123B (zh) | 一种非人哺乳动物认知障碍或其相关疾病动物模型的建立方法及其用途 | |
Schauwecker | Congenic strains provide evidence that a mapped locus on chromosome 15 influences excitotoxic cell death | |
JP2015062407A (ja) | 精神・神経疾患モデル動物 | |
Soewarto et al. | Features and strategies of ENU mouse mutagenesis | |
CN116144658B (zh) | 用于构建神经退行性动物模型的sgRNA及其应用 | |
CN114606270B (zh) | 一种Mia3条件性基因敲除小鼠模型的构建方法 | |
US7642399B2 (en) | Mouse with deficiency of glutamate transporter GLAST function | |
CN114592011B (zh) | 一种ptdss2条件性基因敲除小鼠模型的构建方法 | |
WO2007043589A1 (ja) | 統合失調症モデル動物 | |
CN112553194B (zh) | Kit基因修饰的非人动物的制备方法和应用 | |
CN114990160A (zh) | 一种Navβ2-ICD低表达转基因鼠模型的构建方法及其应用 | |
CN114304068A (zh) | 癫痫动物模型的制备及其应用 | |
CN114480491A (zh) | 一种grin2a基因突变认知障碍小鼠模型的构建和应用 | |
CN117363658A (zh) | Clcn6基因点突变小鼠动物模型的构建方法及应用 | |
D'Angelo et al. | Anatomy, physiological features, genetics and genetic alterations, breeding and strain differences relevant to the choice of the model—Impact of 3Rs |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |