CN106282122A - A kind of method for building up of non-human mammal phobia or its relevant disease animal model and application thereof - Google Patents
A kind of method for building up of non-human mammal phobia or its relevant disease animal model and application thereof Download PDFInfo
- Publication number
- CN106282122A CN106282122A CN201610873603.1A CN201610873603A CN106282122A CN 106282122 A CN106282122 A CN 106282122A CN 201610873603 A CN201610873603 A CN 201610873603A CN 106282122 A CN106282122 A CN 106282122A
- Authority
- CN
- China
- Prior art keywords
- glce
- mice
- phobia
- human mammal
- animal model
- 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
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 title claims abstract description 63
- 206010034912 Phobia Diseases 0.000 title claims abstract description 62
- 208000019899 phobic disease Diseases 0.000 title claims abstract description 62
- 201000010099 disease Diseases 0.000 title claims abstract description 61
- 238000000034 method Methods 0.000 title claims abstract description 41
- 238000010171 animal model Methods 0.000 title claims abstract description 38
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 106
- 230000002779 inactivation Effects 0.000 claims abstract description 54
- 238000012360 testing method Methods 0.000 claims abstract description 51
- 239000003814 drug Substances 0.000 claims abstract description 25
- 210000004027 cell Anatomy 0.000 claims abstract description 22
- 238000012216 screening Methods 0.000 claims abstract description 9
- 238000002360 preparation method Methods 0.000 claims abstract description 8
- 210000004962 mammalian cell Anatomy 0.000 claims abstract description 7
- 210000000577 adipose tissue Anatomy 0.000 claims description 64
- 230000006399 behavior Effects 0.000 claims description 31
- 208000006011 Stroke Diseases 0.000 claims description 21
- 238000011160 research Methods 0.000 claims description 21
- 206010008190 Cerebrovascular accident Diseases 0.000 claims description 18
- 241001465754 Metazoa Species 0.000 claims description 17
- 238000004458 analytical method Methods 0.000 claims description 16
- 208000019901 Anxiety disease Diseases 0.000 claims description 13
- 230000036506 anxiety Effects 0.000 claims description 12
- 150000001875 compounds Chemical class 0.000 claims description 11
- 208000008589 Obesity Diseases 0.000 claims description 8
- 235000020824 obesity Nutrition 0.000 claims description 8
- 241000124008 Mammalia Species 0.000 claims description 7
- 230000001537 neural effect Effects 0.000 claims description 6
- 229940124597 therapeutic agent Drugs 0.000 claims description 6
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 claims description 5
- 102000004195 Isomerases Human genes 0.000 claims description 5
- 108090000769 Isomerases Proteins 0.000 claims description 5
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 claims description 5
- 230000027326 copulation Effects 0.000 claims description 5
- 229940097043 glucuronic acid Drugs 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 4
- 229940124606 potential therapeutic agent Drugs 0.000 claims description 4
- 230000004913 activation Effects 0.000 claims description 3
- 208000010877 cognitive disease Diseases 0.000 claims description 3
- 241000894007 species Species 0.000 claims description 3
- 208000037114 Symptom Flare Up Diseases 0.000 claims description 2
- 230000007529 anxiety like behavior Effects 0.000 claims description 2
- 230000013011 mating Effects 0.000 claims description 2
- 238000010188 recombinant method Methods 0.000 claims description 2
- 238000002474 experimental method Methods 0.000 abstract description 25
- 229940079593 drug Drugs 0.000 abstract description 9
- 206010041250 Social phobia Diseases 0.000 abstract description 8
- 208000008811 Agoraphobia Diseases 0.000 abstract description 3
- 241000699670 Mus sp. Species 0.000 description 247
- 241000699666 Mus <mouse, genus> Species 0.000 description 108
- 238000011740 C57BL/6 mouse Methods 0.000 description 87
- 230000002146 bilateral effect Effects 0.000 description 46
- 230000000694 effects Effects 0.000 description 38
- 108020004414 DNA Proteins 0.000 description 17
- 210000002149 gonad Anatomy 0.000 description 16
- 208000024891 symptom Diseases 0.000 description 16
- 230000002093 peripheral effect Effects 0.000 description 15
- 230000001143 conditioned effect Effects 0.000 description 14
- 210000001596 intra-abdominal fat Anatomy 0.000 description 14
- 238000006243 chemical reaction Methods 0.000 description 12
- 238000003209 gene knockout Methods 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 11
- 230000008859 change Effects 0.000 description 10
- 210000003141 lower extremity Anatomy 0.000 description 10
- 230000011514 reflex Effects 0.000 description 10
- 230000002567 autonomic effect Effects 0.000 description 9
- 230000002829 reductive effect Effects 0.000 description 9
- 206010064571 Gene mutation Diseases 0.000 description 8
- 210000000078 claw Anatomy 0.000 description 8
- 210000003194 forelimb Anatomy 0.000 description 8
- 230000004044 response Effects 0.000 description 8
- 230000006378 damage Effects 0.000 description 7
- 230000007613 environmental effect Effects 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- PKDBCJSWQUOKDO-UHFFFAOYSA-M 2,3,5-triphenyltetrazolium chloride Chemical compound [Cl-].C1=CC=CC=C1C(N=[N+]1C=2C=CC=CC=2)=NN1C1=CC=CC=C1 PKDBCJSWQUOKDO-UHFFFAOYSA-M 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 210000000744 eyelid Anatomy 0.000 description 6
- 230000008685 targeting Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 108010051219 Cre recombinase Proteins 0.000 description 5
- 230000037396 body weight Effects 0.000 description 5
- 229940000406 drug candidate Drugs 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 229920002971 Heparan sulfate Polymers 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 241000700159 Rattus Species 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 230000005611 electricity Effects 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 230000008451 emotion Effects 0.000 description 4
- 238000012048 forced swim test Methods 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 210000003128 head Anatomy 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000012797 qualification Methods 0.000 description 4
- 230000008925 spontaneous activity Effects 0.000 description 4
- 230000000638 stimulation Effects 0.000 description 4
- AHOUBRCZNHFOSL-YOEHRIQHSA-N (+)-Casbol Chemical compound C1=CC(F)=CC=C1[C@H]1[C@H](COC=2C=C3OCOC3=CC=2)CNCC1 AHOUBRCZNHFOSL-YOEHRIQHSA-N 0.000 description 3
- 206010010904 Convulsion Diseases 0.000 description 3
- 108091092195 Intron Proteins 0.000 description 3
- 241001045988 Neogene Species 0.000 description 3
- AHOUBRCZNHFOSL-UHFFFAOYSA-N Paroxetine hydrochloride Natural products C1=CC(F)=CC=C1C1C(COC=2C=C3OCOC3=CC=2)CNCC1 AHOUBRCZNHFOSL-UHFFFAOYSA-N 0.000 description 3
- 208000005392 Spasm Diseases 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000003542 behavioural effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 230000002490 cerebral effect Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000036461 convulsion Effects 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- 230000007794 irritation Effects 0.000 description 3
- 230000035772 mutation Effects 0.000 description 3
- 101150091879 neo gene Proteins 0.000 description 3
- 210000002569 neuron Anatomy 0.000 description 3
- 229960002296 paroxetine Drugs 0.000 description 3
- 239000013600 plasmid vector Substances 0.000 description 3
- 230000009023 proprioceptive sensation Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 230000002269 spontaneous effect Effects 0.000 description 3
- 230000004936 stimulating effect Effects 0.000 description 3
- 230000000007 visual effect Effects 0.000 description 3
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- 208000014094 Dystonic disease Diseases 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 206010029333 Neurosis Diseases 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 210000000467 autonomic pathway Anatomy 0.000 description 2
- 210000002459 blastocyst Anatomy 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000000994 depressogenic effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 208000010118 dystonia Diseases 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 238000011813 knockout mouse model Methods 0.000 description 2
- AEMOLEFTQBMNLQ-CLQWQSTFSA-N l-iduronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@H](O)[C@@H](O)[C@@H]1O AEMOLEFTQBMNLQ-CLQWQSTFSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 208000015238 neurotic disease Diseases 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 239000002773 nucleotide Substances 0.000 description 2
- 125000003729 nucleotide group Chemical group 0.000 description 2
- 230000008506 pathogenesis Effects 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 1
- 108091016585 CD44 antigen Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 1
- 102000015696 Interleukins Human genes 0.000 description 1
- 108010063738 Interleukins Proteins 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- 241000222065 Lycoperdon Species 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102000012288 Phosphopyruvate Hydratase Human genes 0.000 description 1
- 108010022181 Phosphopyruvate Hydratase Proteins 0.000 description 1
- 241000768494 Polymorphum Species 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical group C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 206010041067 Small cell lung cancer Diseases 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 102000013275 Somatomedins Human genes 0.000 description 1
- 108090000054 Syndecan-2 Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- -1 albumen Chemical class 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000006400 anxiety behaviour Effects 0.000 description 1
- 230000023555 blood coagulation Effects 0.000 description 1
- 101150039352 can gene Proteins 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000001638 cerebellum Anatomy 0.000 description 1
- 239000005482 chemotactic factor Substances 0.000 description 1
- 210000000038 chest Anatomy 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
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001149 cognitive effect Effects 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 210000000028 corpus adiposum pararenale Anatomy 0.000 description 1
- 210000000877 corpus callosum Anatomy 0.000 description 1
- 210000001653 corpus striatum Anatomy 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000007357 dehydrogenase reaction Methods 0.000 description 1
- 230000007357 depressive behavior Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 210000003414 extremity Anatomy 0.000 description 1
- 230000008175 fetal development Effects 0.000 description 1
- 239000011888 foil Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000010363 gene targeting Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 210000004013 groin Anatomy 0.000 description 1
- 230000000971 hippocampal effect Effects 0.000 description 1
- 235000020256 human milk Nutrition 0.000 description 1
- 210000004251 human milk Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 230000000302 ischemic effect Effects 0.000 description 1
- 230000009191 jumping Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 210000005075 mammary gland Anatomy 0.000 description 1
- 230000000873 masking effect Effects 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 108091070501 miRNA Proteins 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 210000000956 olfactory bulb Anatomy 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 239000000902 placebo Substances 0.000 description 1
- 229940068196 placebo Drugs 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000002360 prefrontal effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 208000037974 severe injury Diseases 0.000 description 1
- 230000009528 severe injury Effects 0.000 description 1
- 239000002924 silencing RNA Substances 0.000 description 1
- 208000000587 small cell lung carcinoma Diseases 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 210000004003 subcutaneous fat Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000007306 turnover Effects 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
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
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/90—Isomerases (5.)
-
- 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
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous 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
- A01K67/0276—Knock-out vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
- A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
-
- 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
- C12N5/10—Cells modified by introduction of foreign genetic material
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y501/00—Racemaces and epimerases (5.1)
- C12Y501/03—Racemaces and epimerases (5.1) acting on carbohydrates and derivatives (5.1.3)
-
- 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
- A01K2207/00—Modified animals
- A01K2207/12—Animals modified by administration of exogenous 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
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- 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
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/106—Primate
-
- 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
- A01K2227/107—Rabbit
-
- 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
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Environmental Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Animal Husbandry (AREA)
- Cell Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Pathology (AREA)
- Rheumatology (AREA)
- Endocrinology (AREA)
- Toxicology (AREA)
- Urology & Nephrology (AREA)
- Diabetes (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention provides method for building up of a kind of non-human mammal phobia or its relevant disease animal model and application thereof, specifically, the invention provides phobia or the preparation method of its relevant disease animal model of a kind of non-human mammal, the method comprises the following steps: (a) provides the cell of non-human mammal, by the Glce gene inactivation in described cell, obtain the nonhuman mammalian cells of Glce gene inactivation；B () utilizes the cell of the Glce gene inactivation obtained in step (a), prepare phobia or its relevant disease animal model of Glce gene inactivation.The animal model of the present invention is the animal model of a kind of effective phobia or its relevant disease, can be used for studying the diseases such as phobia, social phobia, agoraphobia, it is possible to for screening and the testing experiment of certain drug.
Description
Technical field
The present invention relates to biological technical field, in particular it relates to a kind of non-human mammal phobia or its relevant disease
Method for building up of animal model and application thereof.
Background technology
Phobia is with the terrified symptom a kind of neurosis as main clinical manifestation.Patient is to some specific object or place
Border produces strong and unnecessary frightened emotion, and with obvious anxiety and autonomic nerve symptom, and actively take to avoid
Mode release this uneasiness.The core symptom of phobia is frightened nervous, and because terror causes severe anxiety even up to
Terrified degree.
The pathogenesis of phobia is not yet studied clear at present, lacks safely and effectively treatment means, therefore further investigated
Its pathogeny is the important foundation for the treatment of phobia.Mouse disease model is studying pathogenesis and the drug sieve of human diseases
Choose and serve very important effect.Probing into the sides such as the cognitive function of the mankind, neurodegenerative diseases, neuropsychiatric disease
Face, mouse model has big advantage.
Gene knockout is the complicated Protocols in Molecular Biology grown up the eighties in last century, and it is based on Development of Mouse Embryos
Tire stem cell DNA homology restructuring principle, also referred to as " gene targeting " technology.Thereafter this technique construction several thousands genes is utilized
Mutant mice, these genetic engineering mice are not only that the research of bioscience and medical science brings breakthrough, also in new drug development
Play vital effect.
The method for building up of current existing mice phobia model mostly will be through exogenous drugs or other physics, change
The method learned processes, even through operation means, it is impossible to the constitutional phobia of simulating human completely, and exists such as operation
Complicated, practical operation is more difficult, the modeling time is longer, and effect is unstable, the shortcomings such as individual variation is bigger.
Therefore, constitutional phobia or its relevant disease clinically can preferably be simulated in the urgent need to exploitation one in this area
, there is not operation technique or difference that drug dose causes in disease, its genetic background is highly consistent, can be as grinding between individuality
Study carefully the non-human mammal phobia of phobia or the pathogeny of its relevant disease and new medicament screen or the dynamic of its relevant disease
Object model.
Summary of the invention
It is an object of the invention to provide one and can preferably simulate constitutional phobia or its relevant disease clinically,
There is not operation technique between individuality or difference that drug dose causes, its genetic background is highly consistent, can be as research
The non-human mammal phobia of phobia or the pathogeny of its relevant disease and new medicament screen or the animal of its relevant disease
Model.
First aspect present invention provides phobia or the system of its relevant disease animal model of a kind of non-human mammal
Preparation Method, it is characterised in that comprise the following steps:
A () provides the cell of non-human mammal, by the glucuronic acid C5 isomerase (Glucuronyl in described cell
C5-epimerase, Glce) gene inactivation, obtain the nonhuman mammalian cells of Glce gene inactivation；With
B () utilizes the cell of the Glce gene inactivation obtained in step (a), prepare the phobia of Glce gene inactivation
Or its relevant disease animal model.
In another preference, in step (a), also comprise the steps:
(a1) DNA homology recombinant technique is utilized, by the exon 3 in described Glce gene to exon 5 one or many
Individual exon is rejected or interrupts, and optionally replaces by selection markers, obtains the nonhuman mammalian cells of Glce gene inactivation.
In another preference, in step (b), also comprise the steps:
(b1) nonhuman mammalian cells of the Glce gene inactivation obtained in step (a) is utilized to prepare chimeric inhuman
Mammal；
(b2) by numerous to the chimeric non-human mammal and the normal wild type mating non-human mammals that obtain in step (b1)
Educating, in offspring, screening obtains the heterozygote non-human mammal of Glce gene inactivation；With
(b3) the mutual copulation of heterozygote non-human mammal by obtaining in step (b2) obtains Glce gene inactivation
Homozygote non-human mammal, thus obtain the animal model of the non-human mammal of Glce gene inactivation.
In another preference, in step (b3), also include step (b4): by inhuman for the homozygote of Glce gene inactivation
The neuronal specificity of mammal and same species knocks out instrument non-human mammal and hybridizes, thus it is special to obtain neuron
The animal model of the non-human mammal of the Glce gene inactivation of the opposite sex.
In another preference, described Glce gene inactivation is included, and gene knockout, gene disruption or gene insert.
In another preference, described gene inactivation includes that Glce gene is not expressed, or expression does not has activated Glce egg
In vain.
In another preference, described Glce gene inactivation is to inactivate by lacking or knock out the exon 3 of Glce.
In another preference, described Glce gene inactivation is the Glce gene inactivation of neuronal specificity.
In another preference, described non-human mammal is rodent or primate, be preferably comprised mice,
Rat, rabbit and/or monkey.
In another preference, described non-human mammal is mice, and Glce Loxp/ in step (b4)
Loxp mice and instrument Mus NSE (neuron-specific enolase)-Cre copulation, i.e. obtain at neuronal cell specificity
Glce knock out mice is called for short cKO mice (i.e. neuronal specificity Glce inactivated mice).
In another preference, described selection markers is selected from lower group: resistant gene, fluorescence protein gene or a combination thereof.
In another preference, described selection markers includes neo gene.
In another preference, the animal mould of the non-human mammal of the Glce gene inactivation obtained in described step (b)
In type, compared with wild-type control animals, there is following one or more feature:
(t1) spacious field level of activation reduces；
(t2) desire exploring strange environment reduces；
(t3) frightened sample behavior increases；
(t4) frightened degree increases；
(t5) behavior depression increases；
(t6) Degree of Depression increases；
(t7) anxiety-like behavior increases；
(t8) anxiety degree increases；
(t9) cognitive disorder increases；
(t10) incidence rate of apoplexy increases；
(t11) obese degree of form increases；
(t12) incidence rate of obesity symptom increases；
(t13) fatty tissue weight in wet base increases.
In another preference, described frightened sample behavior is included in the time minimizing exploring middle section in the experiment of spacious field,
Show the desire reduction exploring strange environment；In testing at conditioned fear, audio conditions is stimulated (stimulating without foot shock)
Stiff reaction increase, and environmental condition stimulates the stiff reaction of (stimulating without foot shock) increase, show frightened instead
Should increase.
In another preference, described spacious field level of activation is selected from lower group: the distance of spacious field activity, activity time, activity
Speed or a combination thereof.
In another preference, described behavior depression is selected from lower group: explore the time of middle section in spacious field is tested
Reduce, show explores the desire of strange environment reduce, the increase of dead time in forced swim test, show behavior exhausted
Hope or a combination thereof.
In another preference, the desire of the strange environment of described exploration reduces and refers to explore middle section in spacious field is tested
Time reduces, and shows the desire reduction exploring strange environment.
In another preference, described cognitive disorder increase refers to that the time exploring new object in new object identification is tested subtracts
Less, the time staying in past heritage body increases, explores the time ratio reduction of new object/past heritage body, explores the time of new object always
The exploration time in accounting reduce, the ability showing cognitive new things reduces.
In another preference, described fatty tissue weight in wet base is selected from lower group: visceral adipose tissue weight in wet base, subcutaneus adipose tissue
Weight in wet base or a combination thereof.
In another preference, described visceral adipose tissue weight in wet base is selected from lower group: bilateral gonad peripheral adipose tissue weight in wet base,
Bilateral perirenal adipose tissue weight in wet base or a combination thereof.
In another preference, described subcutaneus adipose tissue weight in wet base includes bilateral inguinal fatty tissue weight in wet base.
Second aspect present invention provides non-human mammal model prepared by a kind of method described in first aspect present invention
Purposes, this model is used as research phobia or the animal model of its relevant disease.
In another preference, described phobia or its relevant disease include: phobia, social phobia, topophobia
Disease or a combination thereof.
Third aspect present invention provides non-human mammal model prepared by a kind of method described in first aspect present invention
Purposes, wherein, this model is used for screening or identify can alleviate or treat phobia or its relevant disease material (treat
Agent).
In another preference, described phobia or its relevant disease include: phobia, social phobia, topophobia
Disease or a combination thereof.
Fourth aspect present invention provides a kind of screen or determines treatment or alleviates the potential of phobia or its relevant disease
The method of therapeutic agent, comprises the following steps:
A test compound, in test group, in the presence of test compound, is applied to first aspect present invention institute by ()
Non-human mammal model prepared by the method for stating, the behavior to the described animal model of test group carries out behavior analysis；And
In not using described test compound and the identical matched group of other conditions, the behavior to the described animal model of matched group is entered
Row behavior analysis；With
B the behavior of test group and control animals model is compared by (), wherein, compared with matched group, if used
The behavior characterizing phobia or its relevant disease in the animal model of test compound is improved, then show this test chemical combination
Thing can be as phobia or the potential therapeutic agent of its relevant disease.
In another preference, described behavior analysis includes: autonomic activities, Y maze experiment, the experiment of spacious field, new thing
Body identification experiment, water maze laboratory, elevated plus-maze test, conditioned fear experiment, forced swim test or a combination thereof.
In another preference, described method is nondiagnostic and non-therapeutic.
In another preference, described method includes step (c), step (b) is screened or the potential therapeutic agent identified is executed
The non-human mammal model prepared for method described in first aspect present invention, thus measure its row to described animal model
For impact.
In another preference, described improvement is the improvement statistically with significant.
Fifth aspect present invention provides a kind of non-human mammal model, by method system described in first aspect present invention
Standby.
In another preference, for Glce gene inactivation, described non-human mammal model be heterozygosis or
Isozygoty.
In another preference, described Glce gene inactivation is the Glce gene inactivation of neuronal specificity.
Sixth aspect present invention provides the purposes of a kind of cell, the glucuronic acid C5 isomerase in described cell
(Glucuronyl C5-epimerase, Glce) gene inactivation or downward, for preparing the phobia building non-human mammal
Or the biological preparation of its relevant disease animal model.
In another preference, described biological preparation is liquid formulation.
Seventh aspect present invention provides the purposes of the deactivator of a kind of Glce gene or its albumen, is used for preparing structure non-
The phobia of people mammal or the preparation of its relevant disease animal model.
In another preference, described deactivator includes inhibitor.
In another preference, described deactivator is selected from lower group: antibody, micromolecular compound, nucleic acid or a combination thereof.
In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and having in below (eg embodiment)
Can be combined with each other between each technical characteristic that body describes, thus constitute new or preferred technical scheme.As space is limited, exist
This tires out the most one by one states.
Accompanying drawing explanation
Fig. 1 shows Glce gene mutation sequential design strategy.
Fig. 2 shows Glce gene knockout targeting vector plasmid map.
Fig. 3 shows the enzyme action qualification result of Glce gene knockout targeting vector plasmid DNA.
Fig. 4 shows the electrophoresis result of 5 ' end PCR primer of ES cloned genomic dna.
Fig. 5 shows the electrophoresis result of 3 ' end PCR primer of ES cloned genomic dna.
Fig. 6 shows Glce transgenic mice (Loxp/+) series jump site PCR result.
Fig. 7 shows Glce no mutant homozygote (Loxp/Loxp Cre), heterozygous mutation (Loxp/+Cre) and Wild
The series jump site PCR result of type (+/+Cre).
Fig. 8 shows spontaneous activity distance, time and the speed of Glce no mutant homozygote mice.
Fig. 9 shows the Glce no mutant homozygote mice movable total distance, time and speed in spacious field is tested.
Figure 10 shows Glce no mutant homozygote mice activity distance, time and speed in spacious center court region.
Figure 11 shows Glce no mutant homozygote mice activity distance, time and speed in neighboring area, spacious field.
Figure 12 shows the stiff reaction that audio conditions is stimulated in conditioned fear is tested by Glce no mutant homozygote mice
Time.
Figure 13 shows the stiff reaction that environmental condition is stimulated in conditioned fear is tested by Glce no mutant homozygote mice
Time.
Figure 14 shows that Glce no mutant homozygote mice probes into open arms, central area in elevated plus-maze test respectively
Territory and the time of closure arm.
Figure 15 shows the Glce no mutant homozygote mice dead time in forced swim test.
Figure 16 shows that apoplexy Behavioral assessment is tested.
Figure 17 shows TTC dyeing detection apoplexy Mice brain tissues infraction situation.
Figure 18 shows the form contrast of Glce no mutant homozygote mice and C57 mice.Wherein, Zuo Tu: be above that C57 is female
Mice, lower for Glce no mutant homozygote female mice；Right figure: be above C57 male mice is lower male little for Glce no mutant homozygote
Mus.
Figure 19 shows the body weight contrast situation of Glce no mutant homozygote mice and C57 mice.
Figure 20 shows the body fat weight in wet base contrast situation of Glce no mutant homozygote mice and C57 mice.
Figure 21 shows the bilateral gonad peripheral adipose tissue weight in wet base contrast feelings of Glce no mutant homozygote mice and C57 mice
Condition.
Figure 22 shows the bilateral perirenal adipose tissue weight in wet base contrast situation of Glce no mutant homozygote mice and C57 mice.
Figure 23 shows the visceral adipose tissue weight in wet base contrast situation of Glce no mutant homozygote mice and C57 mice.
Figure 24 shows bilateral inguinal fatty tissue (the subcutaneous fat group of Glce no mutant homozygote mice and C57 mice
Knit) weight in wet base contrast situation.
Detailed description of the invention
The present inventor through extensively in-depth study, establish the stable phobia of a kind of inheritance stability, phenotype or its
Relevant disease model, it be Glce gene disallowable or inactivation mice or other non-human mammals.The animal mould of the present invention
Type is a kind of effective phobia or its relevant disease animal model, can be used for studying phobia, social phobia, topophobia
The diseases such as disease, it is possible to for screening and the testing experiment of certain drug.
Additionally, the present invention it has been unexpectedly discovered that, reject or inactivate the animal model obtained by Glce gene can also be simultaneously
For studying the diseases such as apoplexy, obesity, anxiety, depression.Complete the present invention on this basis.
Glce gene
Heparan sulfate is a kind of polysaccharide being widely present in cell surface and cytoplasmic matrix, negative as a band
The linear macromolecule of electricity, many cytokines, somatomedin, chemotactic factor and interleukin can the most specifically combine, and then
The physiological process such as fetal development, cell growth, inflammatory reaction, blood coagulation, neoplasm metastasis and virus infection play a role1。
Glucuronic acid C5 isomerase (Glce) is a pass in heparan sulfate proteoglycan sugar chain building-up process
Key enzyme, the D-Glucose aldehydic acid in sugar chain can be tautomerized to L-iduronic acid by it2, substantially increase answering of Heparan sulfate
Miscellaneous degree, and provide more flexibility for sugar chain, L-iduronic acid is the one of the numerous protein molecular of Heparan sulfate identification
Individual must obligato site3。
At the research discovery to 21 example normal galactophore tissues and 74 example breast tumor tissues, the human milk adenoncus of 82%-84%
In tumor tissue, Glce significantly lowers in the expression of mRNA level in-site and protein level or completely loses4.Additionally, at mammary gland
In cancer and lung cancer cell line, process LAN Glce can suppress the propagation of small cell lung cancer and breast cancer cell, and this points out we Glce can
It can be a potential antioncogene5,6。
Glce gene is positioned on No. 9 chromosomes of mouse genome, total length 618 (EnsemblGene ID:
ENSMUSG00000032252, Genebank accession number: 93683).Glce genome sequence includes outside 4 introns and 5 aobvious
Son, Glce gene expression albumen has 3 transcripts, and transcript 1 has 3 exons, 2 introns, and transcript 2 has 5
Exon, 4 introns, transcript 3 has 2 exons, 1 intron.These sequence informations can be found in document or
The public databases such as EnsemblGene, Genebank.
The Glce gene of other species such as the mankind also can be found in the common data such as document or EnsemblGene, Genebank
Storehouse.
Should be understood that term " Glce " also includes the variant form of various naturally occurring Glce gene.Representational example
Including: the nucleotide sequence of the Glce albumen identical with wild type, encoding wild type Glce is encoded because of the degeneracy of codon
The nucleotide sequence of conservative variation's polypeptide of albumen.Additionally, during for other mammals outside mice, this term refers to
The homologue in this mammal of the Glce gene.Such as people, this term refers to Glce (the known mice Glce base of people
Because the cDNA homology degree with mankind's Glce gene is 91.4%, the homology degree of aminoacid sequence is 97.4%).
Glce gene or the deactivator of its albumen
In the present invention, the deactivator of described Glce includes all inactivating or part inactivation.
The deactivator of the Glce albumen of the present invention includes that (a) inhibitor, the example of described inhibitor include (but not limiting
In): micromolecular compound, antibody, antisensenucleic acids, miRNA, siRNA or a combination thereof；(b) Glce gene knock out agent.
Phobia or its relevant disease
Phobia is with the terrified symptom a kind of neurosis as main clinical manifestation.Patient is to some specific object or place
Border produces strong and unnecessary frightened emotion, and with obvious anxiety and autonomic nerve symptom, and actively take to avoid
Mode release this uneasiness.The core symptom of phobia is frightened nervous, and because terror causes severe anxiety even up to
Terrified degree.
In the present invention, described phobia or its relevant disease include (but being not limited to): phobia, social phobia,
And/or agoraphobia.
Gene inactivation
Research for Unknown Function gene can be adopted in many ways, such as, make the gene inactivation required study, and analyzes institute
The character mutation of the genetic modification obtained, and then obtain the function information of this gene.Another advantage of this research method is permissible
Gene function and disease are associated, thus obtain also can obtain while gene function this gene as potential drug or
Disease information that person's drug target can be treated and disease animal model.The method of gene inactivation can pass through gene knockout, gene
Interrupt or the mode of gene insertion completes.Wherein, gene knochout technique is the non-of research human gene's function in entirety
Normal strong means.
Animal model
In the present invention, it is provided that the non-human mammal model of a kind of very effective neuropsychiatric disease.
In the present invention, the example of non-human mammal includes (but being not limited to): mice, rat, rabbit, monkey etc., more preferably
Ground is rat and mice.
As used herein, term " Glce gene inactivation " includes the situation that one or two Glce gene is deactivated, and i.e. wraps
With including Glce genetic heterozygosis and inactivate with isozygotying.Such as, the mice of Glce gene inactivation can be heterozygosis or the mice isozygotied.
In the present invention, can gene knockout or proceed to exogenous gene (or fragment) and make the methods such as Glce gene inactivation prepare
The non-human mammal (such as mice) of Glce gene inactivation.In the art, by gene knockout or proceed to exogenous gene and make
The technology of target gene inactivation is known, and these routine techniquess can be used in the present invention.
In another preference of the present invention, the inactivation of Glce gene is realized by gene knockout.
In another preference of the present invention, the inactivation of Glce gene be by Glce gene insert exogenous gene (or
Fragment) and realize.
In an instantiation of the present invention, can build a construction containing external source Insert Fragment, this construction contains
With the homology arm of the flanking sequence homology of the both sides of the insertion point of target gene (Glce), such that it is able to by homologous recombination high frequency
External source Insert Fragment (or gene) is inserted into Glce genome sequence (especially exon region) by ground, causes mice Glce base
The frameshit of cause, terminate or knock out in advance, thus cause Glce gene delection or inactivation.
With the inventive method obtain isozygoty or the mice of heterozygosis can educate.The Glce gene of inactivation can be lost with Mendel's rule
Pass to progeny mice.
In a preference, the invention provides a kind of Mice homozygous animal pattern lacking Glce gene.
Drug candidate or therapeutic agent
In the present invention, a kind of animal model utilizing the present invention, screening treatment phobia or its relevant disease are additionally provided
Sick drug candidate or the method for therapeutic agent.
In the present invention, drug candidate or therapeutic agent refer to known have certain pharmacological activity or detected can
Can have the material of certain pharmacological activity, include but not limited to nucleic acid, albumen, saccharide, the little molecule of chemosynthesis or divide greatly
Sub-compound, cell etc..The administering mode of drug candidate or therapeutic agent can be administered orally, intravenous injection, lumbar injection, subcutaneous note
Penetrate, canalis spinalis is administered or direct intracerebral injection.
Main advantages of the present invention include:
(1) present invention can preferably simulate the most idiopathic phobia or its relevant disease.
(2) there is not operation technique between individuality or difference that drug dose causes, its genetic background is highly consistent.
(3) can be as the pathogeny of research phobia and the powerful of new medicament screen.
(4) phobia of the present invention or the inheritance stability of its relevant disease animal model, phenotype are stable.
(5) with the inventive method obtain isozygoty or the animal model of heterozygosis can be educated.Transgenic chimeric mice has reproduction
Ability, the Glce gene of inactivation can be with Mendel's law heredity to progeny mice.
(6) phobia or its relevant disease animal model of the present invention shows frightened nervous symptom, therefore can be wide
General for phobia or the drug screening of its relevant disease with test, the disease such as including phobia, social phobia, agoraphobia
Sick.
(7) the invention firstly discloses the animal model obtained by rejecting or inactivation Glce gene can also be simultaneously used for grinding
Study carefully the diseases such as apoplexy, obesity, anxiety, depression.
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are merely to illustrate the present invention
Rather than restriction the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor
Laboratory Press, 1989) condition described in, or according to the condition proposed by manufacturer.Unless otherwise indicated,
Otherwise percentage ratio and number are percentage by weight and parts by weight.
If no special instructions, the material used by embodiment is commercially available prod.
Embodiment 1 obtains the Glce gene mutation homozygote mice carrying Cre recombinase
First Glce mutant gene sequence (Fig. 1) is built.Design Glce gene knockout targeting vector sequence as shown in Figure 1,
Loxp/Loxp allele is inserted into Glce gene 3 exon both sides, inserts neo gene, 5 ' end arms at 3 ' ends
3125bp, 3 ' end arms 3718bp.Fig. 2 is Glce gene knockout targeting vector plasmid map.1. obtain genes of interest (Glce)
Homologous fragment, is cloned into this DNA fragmentation in plasmid vector；2. from recombiant plasmid, excise most of homology of genes of interest
DNA sequence, only stays the two ends of the online plasmid vector of partial sequence；3. neo gene is cloned into genes of interest homology suitable
In the linear plasmid of sequence, it is allowed to be positioned at the centre of residual genes of interest DNA sequence homologous；4. in the outside of genes of interest DNA sequence homologous
Linearisation recombinant plasmid vector, is cloned into hsv-tk gene in this linear carrier.
Gene loci is as shown in the table:
Note: gene location reference numerals is according to " 10kb Up and Down of Glce gene ".
Fig. 3 is that the enzyme action of Glce gene knockout targeting vector plasmid DNA is identified, uses 1Kb DNA ladder.To target practice
Carrier carries out linearisation: 100 μ g Glce-CKO plasmid DNA (purchased from Biovector NTCC) NotI (enzyme dosage: 150U) lines
Property, enzyme action system is 150 μ l, and overnight, after equal-volume phenol chloroform, chloroform process, dehydrated alcohol precipitates in 37 DEG C of digestion, 100 μ l
Aseptic PBS is resuspended standby.ES cell targeting: ES cell SCR012 derives from 129SV/EVStrain male mice is (purchased from the Chinese Academy of Sciences
Sea Experimental Animal Center) embryonic stem cell, linearisation amount of DNA: 35 μ g, electroporation model: Bio-Rad Gene Pulser
(Cat.No.165-2105), electroporation conditions: voltage 240v, electric capacity 500 μ F, actual conduction time 10.5ms, virtual voltage
256v, colony screening condition: 300 μ g/ml G418 and 2 μMs of GanC screen 8 days.Picking resistance clone and offer DNA sample are altogether
96 parts.
Positive ES cells genome identification method:
1.5 ' arm PCR identify
P1 primer is positioned at outside 5 ' arm, and P2 primer is positioned at neo recombination region, distance arm outer primer 8.2kb.
P1 and P2 primer sequence:
P1:GGCATTTGCACTCACATACACAACCCA(gene site:15824-15850)(SEQ ID NO.:1)
P2:GTGCCACTCCCACTGTCCTTTCC(SEQ ID NO.:2)
PCR reaction system (ul):
PCR reaction condition:
PCR instrument: Eppendorf AG 22331Hamburg
Reagent: TaKaRa La Taq treasured biological engineering (Dalian) company limited (Cat:DRR002B)
Molecular weight Marker:MBI GeneRuler 1kb DNA Ladder (brilliant U.S. biological, Cat:SM0311)
2.3 ' arm PCR identify
P4 primer is positioned at outside 3 ' arm, and P3 primer is positioned at neo recombination region, distance arm outer primer 4.7kb.
P4 and P3 primer sequence:
P4:GAGAGGCTTGGAGGCGGTGCTGATCTT(gene site:29603-29629)(SEQ ID NO.:3)
P3:GATATACTATGCCGATGATTAATTGTC(SEQ ID NO.:4)
PCR reaction system (ul):
PCR reaction condition:
PCR instrument: Eppendorf AG 22331Hamburg
Reagent: TaKaRa La Taq treasured biological engineering (Dalian) company limited (Cat:DRR002B)
Molecular weight Marker:MBI GeneRuler 1kb DNA Ladder (brilliant U.S. biological, Cat:SM0311)
ES cell clone qualification result PCR identifies drug resistance ES cell clone 96, and wherein 19 ES clones occur double
Arm homologous recombination.PCR primer is further characterized by through determined dna sequence.Fig. 4 shows that 5 ' end PCR of ES cloned genomic dna produce
The electrophoresis result of thing.Fig. 5 shows the electrophoresis result of 3 ' end PCR primer of ES cloned genomic dna.
Positive ES cloned blastocysts injection:
Microinjection blastaea is originated: the super several rows of C57BL/6J mice (purchased from Shanghai Slac Experimental Animal Co., Ltd.)
Ovum, is developed to blastocyst stage in natural conception body.Inject 60 pieces of embryos, be implanted into 3 pseudopregnant mouse receptor uterus after injection, be subject to
Body is the first-filial generation of C57BL/6J (♂) and CBA (♀) (purchased from Shanghai Slac Experimental Animal Co., Ltd.).Little from be born
Selecting the chimeric rate Mouse feeder more than 50% in Mus extremely to grow up, carry out copulation with C57BL/6J female mice, offspring's Lycoperdon polymorphum Vitt is little
Mus extracted coda gene group DNA carries out PCR qualification (identifying that strategy is ibid), and result is as shown in Figure 6, it is thus achieved that both arms positive F1 generation is little
Mus (Loxp/+).
By F1 generation Mouse feeder to growing up, with NSE-Cre instrument Mus (purchased from Shanghai Slac Experimental Animal Co., Ltd.)
Hybridization, it is thus achieved that F2 is for Loxp/+Cre mice.By F2 for Mouse feeder to growing up, F2 is for the mutual copulation of interior male and female, according to Mendel
Law heredity, it is thus achieved that F3 is for no mutant homozygote (Loxp/Loxp Cre): heterozygous mutation (Loxp/+Cre): Wild type (+/
+ Cre) ratio be about 1:2:1.Extracted coda gene group DNA carries out PCR qualification (identify strategy ibid), result such as Fig. 7 institute
Show.No mutant homozygote mice (Loxp/Loxp Cre) is tested for follow-up Animal Behavior Science.
The frightened behavior analysis of embodiment 2Glce no mutant homozygote mice
2.1 autonomic activities
The Glce gene mutation homozygote mice carrying Cre recombinase is positioned over (110mm X in dark experimental box
110mm X 330mm), the spontaneous activity 5min of test mice.Active situation by infrared photography shooting mice.Use Shanghai
The video tracking software of Ji Liang software Science and Technology Ltd. and analyze software and mice event trace and activity time are carried out point
Analysis.
Research display, compared with adolescence C57BL/6 normal mouse, adolescence Glce no mutant homozygote mice spontaneous
Movable total distance, activity time and moving speed do not have significant difference (Fig. 8).
Result shows, the born habit of adolescence Glce no mutant homozygote mice compared with C57BL/6 normal mouse also
It is not significantly different from.
2.2 spacious field experiments
Being positioned over by mice in bright spacious experimental box (500mm X 500mm X 590mm), the exploration of test mice is lived
Dynamic 5min.Active situation by infrared photography shooting mice.Use the video tracking of Jiliang Software Sci-Tech Co., Ltd., Shanghai
Mice event trace and activity time are analyzed by software and analysis software.
Result shows, compared with adolescence C57BL/6 normal mouse, adolescence Glce no mutant homozygote mice is in spacious field
Total distance, activity time and moving speed movable in experiment are all substantially reduced (Fig. 9).Result shows, adolescence Glce dashes forward
Become that homozygote mice is more movable compared with C57BL/6 normal mouse substantially reduces.
Compared with adolescence C57BL/6 normal mouse, adolescence Glce no mutant homozygote mice is in spacious center court region
Movable distance, activity time and moving speed are all substantially reduced (Figure 10), and result shows, adolescence Glce no mutant homozygote
Mice is explored the activity of strange environment compared with C57BL/6 normal mouse and substantially reduces.
Compared with adolescence C57BL/6 normal mouse, adolescence Glce no mutant homozygote mice is in neighboring area, spacious field
Movable distance, activity time and moving speed are all substantially reduced (Figure 11), and result shows adolescence Glce no mutant homozygote
Mice is more movable compared with C57BL/6 normal mouse to be substantially reduced.
Generally speaking, compared with adolescence C57BL/6 normal mouse, the activity of adolescence Glce no mutant homozygote mice
Substantially reduce, and the activity exploring strange environment substantially reduces, show that adolescence Glce no mutant homozygote mice has brighter
Aobvious frightened emotion.
2.3 conditioned fear experiments
Conditioned fear experiment point is carried out for three days.Mice is positioned in MED conditioned behavior experimental system (U.S.), by red
Outer shooting record mice behavior expression in MED conditioned behavior experimental system.First day, adapt to 3min, give one group
0.01Hz totally 4 stimulations: intensity of sound 85dB, sound frequency 3500Hz, the sonic stimulation conduct of each stimulus duration 30s
Conditional stimulus, the last 1s of each sonic stimulation give 0.4mA, persistent period 1s electricity irritation electric shock vola as unconditional
Stimulating, meanwhile, the specific environment giving to stimulate is as another conditional stimulus.This electricity irritation electric shock vola can cause mouse jump
Get up and produce frightened emotion, the stiff response situation that this certain environmental conditions is stimulated by record mice.Stimulate after terminating, allow little
Mus is at this specific environment rest 2min, and experiment terminates.Second day, mice is positioned in MED conditioned behavior experimental system, does not gives
Giving sonic stimulation and electricity irritation, being only given the certain environmental conditions identical with first day stimulates 8min, and record mice is specific to this
The stiff response situation that environmental condition stimulates.3rd day, by using the wall of the chest residing for elastoplast blank change mice
Wall and bottom, manufacture one from a few days ago stimulate different new specific environments, mice is positioned over MED conditioned behavior experimental system
In, adapt to 3min, giving the specific sound with first day identical parameters stimulates 5min: intensity of sound 85dB, sound frequency
3500Hz, records the mice stiff response situation to this specific sound conditional stimulus.The stiff response situation of mice is carried out point
Analysis.Research finds, compared with adolescence C57BL/6 normal mouse, adolescence Glce no mutant homozygote mice is at conditioned fear
Certain environmental conditions is stimulated the stiff response time produced to dramatically increase (Figure 12) by experiment, and, adolescence Glce suddenlys change
The stiff response time that specific sound conditional stimulus is produced in conditioned fear is tested by homozygote mice dramatically increases (Figure 13).
Result shows, adolescence Glce no mutant homozygote mice has more significantly frightened feelings compared with C57BL/6 normal mouse
Thread.
The anxiety behavior analysis of embodiment 3 Glce no mutant homozygote mice
The Glce gene mutation homozygote mice (hereinafter referred to as Glce no mutant homozygote mice) carrying Cre recombinase is put
Raise in cleaning feeding environment.At mice adolescence (> 3 monthly age) by a series of Animal Behavior Science experiment include autonomic activities,
The anxiety neurosis sample symptom of the experimental evaluation mouse models such as the experiment of spacious field, Elevated plus-maze.
3.1 autonomic activities
Mice is positioned in dark experimental box (110mm X 110mm X 330mm), the spontaneous activity of test mice
5min.Active situation by infrared photography shooting mice.The video tracking using Jiliang Software Sci-Tech Co., Ltd., Shanghai is soft
Mice event trace and activity time are analyzed by part and analysis software.
Research finds, compared with adolescence C57BL/6 normal mouse, and adolescence Glce no mutant homozygote mice spontaneous
Movable total distance, activity time and moving speed do not have significant difference (Fig. 8).
Result shows, the born habit of adolescence Glce no mutant homozygote mice compared with C57BL/6 normal mouse also
It is not significantly different from.
3.2 spacious field experiments
Being positioned over by mice in bright spacious experimental box (500mm X 500mm X 590mm), the exploration of test mice is lived
Dynamic 5min.Active situation by infrared photography shooting mice.Use the video tracking of Jiliang Software Sci-Tech Co., Ltd., Shanghai
Mice event trace and activity time are analyzed by software and analysis software.
Research finds, compared with adolescence C57BL/6 normal mouse, adolescence Glce no mutant homozygote mice is in spacious field
Distance, activity time and the moving speed of middle section activity are all substantially reduced (Figure 10).
Result shows, adolescence Glce no mutant homozygote mice explores strange environment compared with C57BL/6 normal mouse
Activity substantially reduce, and produce more intensive anxiety.
3.3 elevated plus-maze test
Mice is positioned over the long 30cm of open arms single armed, wide 6cm, the long 30cm of closure arm single armed, wide 6cm, high 14.5cm
In Elevated plus-maze, the most about 50cm, by the activity in Elevated plus-maze in photography mice 5min
Situation.Activity to mice is analyzed finding, compared with adolescence C57BL/6 normal mouse, adolescence Glce sudden change is pure
Zygote mice is probed into the time of open arms in elevated plus-maze test and substantially reduces, and the time resting on closure arm significantly increases
Add, be not significantly different from (Figure 14) in middle section residence time.
Result shows, adolescence Glce no mutant homozygote mice is compared with C57BL/6 normal mouse, and anxiety degree is obvious
Increase.Male adolescence Glce no mutant homozygote mice is anxiety compared with female adolescence Glce no mutant homozygote mice
Become apparent from.In addition, the adolescence Glce no mutant homozygote mice producing to a certain degree anxiety also produces to a certain degree simultaneously
Depression.
The Depressive behavior credit analysis of embodiment 4 Glce no mutant homozygote mice
4.1 autonomic activities
The Glce gene mutation homozygote mice carrying Cre recombinase is positioned over (110mm X in dark experimental box
110mm X 330mm), the spontaneous activity 5min of test mice.Active situation by infrared photography shooting mice.Use Shanghai
The video tracking software of Ji Liang software Science and Technology Ltd. and analyze software and mice event trace and activity time are carried out point
Analysis.
Research display, compared with adolescence C57BL/6 normal mouse, adolescence Glce no mutant homozygote mice spontaneous
Movable total distance, activity time and moving speed do not have significant difference (Fig. 8).
Result shows, the born habit of adolescence Glce no mutant homozygote mice compared with C57BL/6 normal mouse also
It is not significantly different from.
4.2 spacious field experiments
Being positioned over by mice in bright spacious experimental box (500mm X 500mm X 590mm), the exploration of test mice is lived
Dynamic 5min.Active situation by infrared photography shooting mice.Use the video tracking of Jiliang Software Sci-Tech Co., Ltd., Shanghai
Mice event trace and activity time are analyzed by software and analysis software.
Result shows, compared with adolescence C57BL/6 normal mouse, adolescence Glce no mutant homozygote mice is in spacious field
Total distance, activity time and moving speed movable in experiment are all substantially reduced (Fig. 9).Result shows, adolescence Glce dashes forward
Become that homozygote mice is more movable compared with C57BL/6 normal mouse substantially reduces.
Compared with adolescence C57BL/6 normal mouse, adolescence Glce no mutant homozygote mice is in spacious center court region
Movable distance, activity time and moving speed are all substantially reduced (Figure 10), and result shows, adolescence Glce mutant homozygous
Sub-mice is explored the activity of strange environment compared with C57BL/6 normal mouse and substantially reduces.
Compared with adolescence C57BL/6 normal mouse, adolescence Glce no mutant homozygote mice is in neighboring area, spacious field
Movable distance, activity time and moving speed are all substantially reduced (Figure 11), and result shows adolescence Glce no mutant homozygote
Mice is more movable compared with C57BL/6 normal mouse to be substantially reduced.
Generally speaking, compared with adolescence C57BL/6 normal mouse, the activity of adolescence Glce no mutant homozygote mice
Substantially reduce, and the activity exploring strange environment substantially reduces, show that adolescence Glce no mutant homozygote mice has certain journey
The depression of degree.
4.3 forced swimming
Being positioned over by mice (water temperature 21-22 DEG C) in the water vat of diameter 12cm, highly 25cm, mice is forced in water temperature relatively
Low water went swimming, the movable 6min of test mice, record the mice dead time in rear 4min.By photography mice
Active situation.The mice activity time is analyzed.Research finds, compared with adolescence C57BL/6 normal mouse, young
The phase Glce no mutant homozygote mice dead time in forced swim test dramatically increases (Figure 15).
Result shows, adolescence Glce no mutant homozygote mice has a certain degree of compared with C57BL/6 normal mouse
Depressed.Female adolescence Glce no mutant homozygote mice compared with male adolescence Glce no mutant homozygote mice without significance difference
Different.In addition, produce to a certain degree depressed adolescence Glce no mutant homozygote mice and also produce a certain degree of Jiao simultaneously
Consider.
The apoplexy behavior analysis of embodiment 5 Glce no mutant homozygote mice
The Glce gene mutation homozygote mice of band Cre recombinase is placed in cleaning feeding environment raise.This mouse species
Model is entering geratic period (about 1.5 years) generation apoplexy sample symptom.Mouse species is evaluated by a series of apoplexy Behaviors survey
The apoplexy sample symptom of model and the order of severity, the evaluation rat stroke symptom that experimental technique was published on Stroke with reference to calendar year 2001
The Behaviors survey (Chen J, et al.Stroke.2001) of a series of systems of the order of severity, including exercise test, sensation
Test, balanced capacity test, the reflection measurement of body, abnormal mobility test etc., specially keep flat test, propose tail survey
Examination, visual tactile test, proprioceptive sensation test, balance beam test, auricle reflex, eyelid closure reflex, startle reflection, tic, spasm, flesh
The performance testings such as dystonia, the behavior to mice is marked.
Glce gene mutation homozygote mice group 40 only 10 occur the apoplexy, apoplexy incidence rate to be in one's old age
25%；It is 5% that matched group 40 mices of C57BL/6 mice group have 2 apoplexy, apoplexy incidence rate occur in one's old age, result
Showing, Glce gene mutation homozygote mice is more easy to apoplexy compared with C57BL/6 mice.
7.1 exercise test.
(1) keep flat test: be positioned on level land by mice, observe whether mice can normally walk, normal walking scores 0
Point；Whether can keep straight on, it is impossible to craspedodrome score 1 point, otherwise score 0 point；Whether turn-take around damage side, turn-take around damage side
Score 1 point, otherwise score 0 point；Whether roll to damage, roll score 1 point, otherwise score 0 point to damage.
(2) propose tail test: mention the tail of mice, observe the situation that mouse limb is movable, if forelimb curves inwardly, pawl
Son is firmly grasped, if mice forelimb curves inwardly, claw firmly grasps score 1 point, otherwise score 0 point；Whether hind leg curves inwardly, claw is grabbed
Tightly, if mouse hind leg curves inwardly, claw firmly grasps score 1 point, otherwise score 0 point；In 30 seconds, head lifts with vertical
Shaft angle degree is more than 10 degree, if in 30 seconds, head lifts mice and vertical axis angle is more than 10 degree of scores 1 point, otherwise score 0 point.
7.2 sensory test.
(1) visual tactile test: grasp mouse body, allow mice forelimb can freely activity, make mice towards estrade
Edge or cage edge, whether by mice quickly near table edge or cage edge, observing mice forelimb can be quick
Table edge or cage edge is accurately caught to protracting and opening claw in time.If mice forelimb can not quickly to extension,
And open claw in time and accurately catch table edge or cage marginal score 1 point, otherwise score 0 point.
(2) proprioceptive sensation test: grasp mouse body, allow mouse hind leg can freely activity, mice forelimb is positioned over
Whether table edge or cage edge, hind leg are unsettled, use tweezers folder mouse hind leg leg muscle, observe mouse hind leg and can
Snapback.If mouse hind leg can not snapback score 1 point, otherwise score 0 point.
7.3 balanced capacity tests.
Mice is positioned over balance beam one end, observes the situation that mice is the most movable on balance beam, mainly include following 7
The situation of kind: whether (1) can keep one's balance, freely walk on balance beam, if mice can keep one's balance, flat
The upper free walking scores 0 point of weighing apparatus wood.
(2) balance beam edge whether is caught, if mice catches balance beam marginal score 1 point.
(3) embrace balance beam, but have a hind leg to drop, if mice embraces balance beam, but have a hind leg to drop score 2
Point.
(4) embrace balance beam, but have two hind legs to drop, or on balance beam rotate, and on balance beam time
Between more than 60 seconds, if mice embraces balance beam, but have two hind legs to drop, or rotate on balance beam, and at balance beam
On time more than 60 seconds scores 3 points.
(5) but attempting keeping balance on balance beam finally drop, the time on balance beam is more than 40 seconds, if mice
But attempting keeping balance on balance beam finally drops, the time on balance beam is more than 40 seconds scores 4 points.
(6) but attempting keeping balance on balance beam finally drop, the time on balance beam is more than 20 seconds, if mice
But attempting keeping balance on balance beam finally drops, the time on balance beam is more than 20 seconds scores 5 points.
(7) being not attempt on balance beam keep balance or hold the desire of balance beam tightly, the time on balance beam is little
Dropped in 20 seconds, if mice is not attempt on balance beam keep balance or hold the desire of balance beam tightly, on balance beam
Time is less than within 20 seconds, dropping score 6 points.
The reflection measurement of 7.4 bodies and the mobility test of exception.
(1) auricle reflex: use cotton swab to stimulate mice auditory meatus, observes whether mice gets rid of head reaction, if being described mice
There is auricle reflex, if there is no auricle reflex, score 1 point without explanation mice.
(2) eyelid closure reflex: using cotton swab to stimulate mice iris, observing whether mice has the reaction closing eyelid, if having
Bright mice has eyelid closure reflex, if not having eyelid closure reflex, score 1 point without explanation mice.
(3) startle reflection: manufacturing a big noise, such as water bottle drops, and observes whether mice has frightened jumping up
Reaction, if being described mice there is the reflection that startles, if not startling reflection, score 1 point without explanation mice.
(4) observe mice whether have a convulsion, the phenomenon such as spasm, myodystonia.If mice is had a convulsion, spasm, flesh
The phenomenons such as dystonia, score 1 point.
Calculating each the PTS being added, PTS 16 is divided into minor injury, PTS 7 12 to be divided into moderate lesion,
PTS 13 18 is divided into severe injury.
Such as, the mice in Figure 16 can not be kept straight on score 1 point in keeping flat test, score 1 point of turn-taking around damage side, to
Damage rolls score 1 point；In proposing tail test, forelimb curves inwardly, claw firmly grasps score 1 point, and hind leg curves inwardly, claw
Promptly score 1 point, in 30 seconds, head lifts and vertical axis angle is more than 10 degree of scores 1 point；Forelimb energy in visual tactile is tested
Quickly accurately catch table edge or cage marginal score 0 point to protracting and opening claw in time；In proprioceptive sensation is tested
Hind leg energy snapback score 0 point；Attempt keeping balance on balance beam at balance beam test small mouse but finally drop,
Time on balance beam is more than 40 seconds scores 4 points；Mice has auricle reflex score 0 point；There is eyelid closure reflex score 0 point；Tool
Startle reflection score 0 point；Mice is had a convulsion score 1 point.Must being divided into of this mice: 1+1+1+1+1+1+0+0+4+0+0+
0+1=11 divides, and is evaluated as moderate apoplexy.
The cerebral tissue infraction of embodiment 6 Glce no mutant homozygote mice is analyzed
Infraction situation by TTC (2,3,5 triphenyltetrazolium chloride) colouring method detection apoplexy Mice brain tissues.
Operating procedure is: directly take brain after anesthesia, quick-freezing about 5 10 minutes in 20 degree of refrigerators, it is simple to section.Section: cut every 1mm
A piece of.Section being placed in TTC, normal concentration is 2%.After covering with masking foil, put into 37 degree of incubator 15 30min, frequently turn over
Beat one's brains sheet, makes brain sheet uniform contact to dyeing liquor.Then 4% paraformaldehyde is used to fix 30min.Take pictures.
TTC is fat-soluble photaesthesia complex, the ischemia infraction of can be used to dye detection mammalian tissues.It is to breathe
The proton acceptor of pyridine nucleotide structure enzyme system in chain, takes on a red color with the dehydrogenase reaction in normal structure, and ischemic tissue
Interior dehydrogenase activity declines, it is impossible to reaction, therefore will not produce change in pale.
As shown in figure 17, result shows result, and after TTC dyes, the normal cerebral tissue of apoplexy mice takes on a red color, and
The cerebral tissue of infraction is pale.Observe olfactory bulb, prefrontal lobe, corpus callosum, hippocampal tissue, striatum, corpus amygdaloideum, hypothalamus, temporo
Leaf, cerebellum, pons, oblongata etc. all have infraction phenomenon.
The morphological analysis of embodiment 7 Glce no mutant homozygote mice
The birth ratio of Glce no mutant homozygote mice meets Mendel's law, and by detecting the body weight of mice, result is such as
Shown in Figure 19.Result shows, compared with adult C57BL/6 normal mouse, and the shape of Glce neuron-specific knock-out mice of growing up
State fatter (Figure 18).
The body weight analysis of embodiment 8 Glce no mutant homozygote mice
Being placed on electronic balance by Glce no mutant homozygote mice and weigh, research is grown up Glce no mutant homozygote mice and becomes
The weight differences of year C57BL/6 normal mouse.
Research finds, compared with adult C57BL/6 normal mouse, and Glce no mutant homozygote mice obesity symptom of growing up
Incidence rate is higher, and body weight has statistically-significant difference (Figure 19) compared with adult C57BL/6 normal mouse.
Result shows, with exceed adult C57BL/6 normal mouse Weight averages more than 20% standard be considered fertile
Fat, the weight average of female adult C57BL/6 normal mouse is 28.7g, the body weight of female adult Glce no mutant homozygote mice
Average out to 34.6g, the incidence rate of female adult Glce no mutant homozygote mice obesity symptom is about 50%, with female adult
C57BL/6 normal mouse compares and has statistically-significant difference (P < 0.01)；The body of male adult C57BL/6 normal mouse
Galassing is 32.2g, and the weight average of male adult Glce no mutant homozygote mice is 39.5g, and male adult Glce sudden change is pure
The incidence rate of zygote mice obesity symptom is about 66%, has statistics and show compared with male adult C57BL/6 normal mouse
Write difference (P < 0.01)；The weight average of adult C57BL/6 normal mouse (comprising male and female) is 30.7g, and Glce sudden change of growing up is pure
The weight average of zygote mice (comprising male and female) is 37.8g, and the incidence rate of Glce no mutant homozygote mice obesity symptom of growing up is total
Meter is about 60%, has statistically-significant difference (P < 0.01) compared with adult C57BL/6 normal mouse.
The above results fully shows, compared with adult C57BL/6 normal mouse, the Glce no mutant homozygote mice that grows up is fertile
The incidence rate of fat symptom is higher.
The body fat analysis of embodiment 9 Glce no mutant homozygote mice
Take fatty tissue after being dissected by Glce no mutant homozygote mice and include bilateral gonad peripheral adipose tissue, Bilateral Renal week
Fatty tissue, bilateral inguinal fatty tissue are placed on electronic balance and weigh, and research is grown up Glce no mutant homozygote mice and becomes
The fatty tissue weight in wet base difference of year C57BL/6 normal mouse.
Research finds (Figure 20), compared with adult C57BL/6 normal mouse, and the body of the Glce no mutant homozygote mice that grows up
Fat weight in wet base increases, and has statistically-significant difference.
Result shows, the body fat weight in wet base average out to 0.94g, female adult Glce of female adult C57BL/6 normal mouse dashes forward
Become the body fat weight in wet base average out to 8.50g of homozygote mice, there is compared with female adult C57BL/6 normal mouse statistics and show
Write difference (P < 0.01)；Body fat weight in wet base average out to 2.98g, the male adult Glce sudden change of male adult C57BL/6 normal mouse
The body fat weight in wet base average out to 7.52g of homozygote mice, has statistically significant compared with male adult C57BL/6 normal mouse
Difference (P < 0.01)；The body fat weight in wet base average out to 2.21g of adult C57BL/6 normal mouse (comprising male and female), Glce sudden change of growing up
The body fat weight in wet base average out to 8.01g of homozygote mice (comprising male and female), has statistics compared with adult C57BL/6 normal mouse
Learn significant difference (P < 0.01).
The above results fully shows, C57BL/6 normal mouse of growing up compares, the body of the Glce no mutant homozygote mice that grows up
Fat weight in wet base increases.
The fatty tissue analysis of embodiment 10 Glce no mutant homozygote mice
The bilateral gonad peripheral adipose tissue of 10.1 Glce no mutant homozygote mices is analyzed
Take bilateral gonad peripheral adipose tissue after being dissected by Glce no mutant homozygote mice to be placed on electronic balance and weigh, grind
Study carefully the bilateral gonad peripheral adipose tissue weight in wet base difference of adult Glce no mutant homozygote mice and adult C57BL/6 normal mouse.
Compared with adult C57BL/6 normal mouse, the bilateral gonad peripheral adipose of the Glce no mutant homozygote mice that grows up
Tissue wet dramatically increases (Figure 21).
Result shows, the bilateral gonad peripheral adipose tissue weight in wet base average out to of female adult C57BL/6 normal mouse
0.54g, the bilateral gonad peripheral adipose tissue weight in wet base average out to 3.10g of female adult Glce no mutant homozygote mice, with female
C57BL/6 normal mouse of growing up compares and has statistically-significant difference (P < 0.01)；Male adult C57BL/6 normal mouse
Bilateral gonad peripheral adipose tissue weight in wet base average out to 1.66g, around the bilateral gonad of male adult Glce no mutant homozygote mice
Fatty tissue weight in wet base average out to 2.85g, have compared with male adult C57BL/6 normal mouse statistically-significant difference (P <
0.01)；The bilateral gonad peripheral adipose tissue weight in wet base average out to 1.24g of adult C57BL/6 normal mouse (comprising male and female), grows up
The bilateral gonad peripheral adipose tissue weight in wet base average out to 2.98g of Glce no mutant homozygote mice (comprising male and female), with adult
C57BL/6 normal mouse compares and has statistically-significant difference (P < 0.01).
The above results fully shows, compared with adult C57BL/6 normal mouse, grow up Glce no mutant homozygote mice
Bilateral gonad peripheral adipose tissue weight in wet base dramatically increases.
The bilateral perirenal adipose tissue of 10.2 Glce no mutant homozygote mices is analyzed
Take bilateral perirenal adipose tissue after being dissected by Glce no mutant homozygote mice to be placed on electronic balance and weigh, study into
The bilateral perirenal adipose tissue weight in wet base difference of year Glce no mutant homozygote mice and adult C57BL/6 normal mouse.
Compared with adult C57BL/6 normal mouse, the bilateral perirenal adipose tissue of the Glce no mutant homozygote mice that grows up
Weight in wet base dramatically increases (Figure 22).
Result shows, the bilateral perirenal adipose tissue weight in wet base average out to 0.36g of female adult C57BL/6 normal mouse, female
Property is grown up the bilateral perirenal adipose tissue weight in wet base average out to 2.06g of Glce no mutant homozygote mice, with female adult C57BL/6 just
Often mice compares and has statistically-significant difference (P < 0.01)；The bilateral perirenal fat of male adult C57BL/6 normal mouse
Tissue wet average out to 1.18g, the bilateral perirenal adipose tissue weight in wet base average out to of male adult Glce no mutant homozygote mice
1.61g, has statistically-significant difference (P < 0.01) compared with male adult C57BL/6 normal mouse；Adult C57BL/6 is just
The often bilateral perirenal adipose tissue weight in wet base average out to 0.87g of mice (comprising male and female), the Glce no mutant homozygote mice that grows up (comprises
Male and female) bilateral perirenal adipose tissue weight in wet base average out to 1.83g, compared with adult C57BL/6 normal mouse, there is statistics
Significant difference (P < 0.01).
The above results fully shows, compared with adult C57BL/6 normal mouse, grow up Glce no mutant homozygote mice
Bilateral perirenal adipose tissue weight in wet base dramatically increases.
The visceral adipose tissue analysis of 10.3 Glce no mutant homozygote mices
Take bilateral gonad peripheral adipose tissue after being dissected by Glce no mutant homozygote mice and bilateral perirenal adipose tissue is put
Weighing on electronic balance, visceral adipose tissue weight in wet base gross weight is bilateral gonad peripheral adipose tissue and bilateral perirenal adipose tissue
Weight in wet base sum, the adult Glce no mutant homozygote mice of research is poor with the visceral adipose tissue weight in wet base of adult C57BL/6 normal mouse
Different.
Compared with adult C57BL/6 normal mouse, the visceral adipose tissue weight in wet base of the Glce no mutant homozygote mice that grows up
Dramatically increase (Figure 23).
Result shows, the visceral adipose tissue weight in wet base average out to 0.90g of female adult C57BL/6 normal mouse, female one-tenth
The visceral adipose tissue weight in wet base average out to 5.16g of year Glce no mutant homozygote mice, with female adult C57BL/6 normal mouse phase
Relatively there is statistically-significant difference (P < 0.01)；The visceral adipose tissue weight in wet base of male adult C57BL/6 normal mouse is average
For 2.84g, the visceral adipose tissue weight in wet base average out to 4.45g of male adult Glce no mutant homozygote mice, with male adult
C57BL/6 normal mouse compares and has statistically-significant difference (P < 0.01)；Adult C57BL/6 normal mouse (comprising male and female)
Visceral adipose tissue weight in wet base average out to 2.11g, grow up Glce no mutant homozygote mice (comprising male and female) visceral adipose tissue
Weight in wet base average out to 4.81g, has statistically-significant difference (P < 0.01) compared with adult C57BL/6 normal mouse.
The above results fully shows, compared with adult C57BL/6 normal mouse, grow up Glce no mutant homozygote mice
Visceral adipose tissue weight in wet base dramatically increases.
The subcutaneus adipose tissue (such as bilateral inguinal fatty tissue) of 10.4 Glce no mutant homozygote mices is analyzed
Take bilateral inguinal fatty tissue after being dissected by Glce no mutant homozygote mice to be placed on electronic balance and weigh, subcutaneous
Fatty tissue weight in wet base is bilateral inguinal fatty tissue weight in wet base sum, and research is grown up Glce no mutant homozygote mice and grows up
The subcutaneus adipose tissue weight in wet base difference of C57BL/6 normal mouse.
Compared with adult C57BL/6 normal mouse, the bilateral inguinal fat group of the Glce no mutant homozygote mice that grows up
Knit (representing subcutaneus adipose tissue) weight in wet base and dramatically increase (Figure 24).
Result shows, the bilateral inguinal fatty tissue weight in wet base average out to 0.04g of female adult C57BL/6 normal mouse,
The bilateral inguinal fatty tissue weight in wet base average out to 3.34g of female adult Glce no mutant homozygote mice, with female adult
C57BL/6 normal mouse compares and has statistically-significant difference (P < 0.01)；The bilateral of male adult C57BL/6 normal mouse
Groin fatty tissue weight in wet base average out to 0.13g, the bilateral inguinal fatty tissue of male adult Glce no mutant homozygote mice
Weight in wet base average out to 3.07g, has statistically-significant difference (P < 0.01) compared with male adult C57BL/6 normal mouse；Become
The bilateral inguinal fatty tissue weight in wet base average out to 0.10g in year C57BL/6 normal mouse (comprising male and female), Glce sudden change of growing up is pure
The bilateral inguinal fatty tissue weight in wet base average out to 3.20g of zygote mice (comprising male and female), with adult C57BL/6 normal mouse phase
Relatively there is statistically-significant difference (P < 0.01).
The above results fully shows, compared with adult C57BL/6 normal mouse, grow up Glce no mutant homozygote mice
Bilateral inguinal fatty tissue (representing subcutaneus adipose tissue) weight in wet base dramatically increases.
Embodiment 11 treatment phobia or the drug verification medicine sorting platform of its relevant disease (such as social phobia)
In the present embodiment, the animal pattern injected in mice Present clinical treatment phobia built to embodiment 1 or its phase
The medicine paroxetine of related disorders, immediately to animal pattern mice autonomic activities, spacious field experiment, conditioned fear experiment in row
It is estimated for learning index.
Result shows, medicine paroxetine increases the autonomic activities of animal pattern mice, increases and explores in spacious field is tested
The time of middle section, substantially reduce and certain environmental conditions is stimulated the stiff response time produced, substantially reduce specific sound
In the stiff response time that sound conditional stimulus produces, illustrate that paroxetine can alleviate fear.
Embodiment 12 utilizes the medicine sorting platform screening for the treatment of phobia or its relevant disease (such as social phobia) to wait
Select medicine
In the present embodiment, plan is by the animal pattern injected in mice treatment phobia built to embodiment 1 or its phase
The medicine of related disorders, immediately to animal pattern mice autonomic activities, spacious field experiment, conditioned fear experiment in behavioristics
Index is estimated.
By with to the animal pattern mice comparison model animal mice of placebo autonomic activities, spacious field test, condition
The difference in behavioral indexes in frightened experiment, it is possible to improve the drug candidate of behavioral indexes, be this phobia or its
The potential medicine of relevant disease.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document by individually
It is incorporated as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention, those skilled in the art can
To make various changes or modifications the present invention, these equivalent form of values fall within the model that the application appended claims is limited equally
Enclose.
List of references:
1.Kreuger J,Spillmann D,Li JP,Lindahl U.Interactions between heparan
sulfate and proteins:the concept of specificity.The Journal of cell biology
2006；174:323-7.
2.Li JP,Gong F,Hagner-McWhirter A,Forsberg E,Abrink M,Kisilevsky R,
Zhang X,Lindahl U.Targeted disruption of a murine glucuronyl C5-epimerase
gene results in heparan sulfate lacking L-iduronic acid and in neonatal
lethality.The Journal of biological chemistry 2003；278:28363-6.
3.Jia J,Maccarana M,Zhang X,Bespalov M,Lindahl U,Li JP.Lack of L-
iduronic acid in heparan sulfate affects interaction with growth factors and
cell signaling.The Journal of biological chemistry 2009；284:15942-50.
4.Grigorieva E,Eshchenko T,Rykova VI,Chernakov A,Zabarovsky E,Sidorov
SV.Decreased expression of human D-glucuronyl C5-epimerase in breast
cancer.International Journal of cancer 2008；122:1172-6.
5.Prudnikova TY,Mostovich LA,Domanitskaya NV,Pavlova TV,Kashuba VI,
Zabarovsky ER,Grigorieva EV.Antiproliferative effect of D-glucuronyl C5-
epimerase in human breast cancer cells.Cancer Cell international 2010；10:27.
6.Grigorieva EV,Prudnikova TY,Domanitskaya NV,Mostovich LA,Pavlova
TV,Kashuba VI,Zabarovsky ER.D-glucuronyl C5-epimerase suppresses small-cell
lung cancer cell proliferation in vitro and tumour growth in vivo.British
Journal of cancer 2011；105:74-82.
Claims (10)
1. the phobia of a non-human mammal or the preparation method of its relevant disease animal model, it is characterised in that include
Following steps:
A () provides the cell of non-human mammal, by glucuronic acid C5 isomerase (the Glucuronyl C5-in described cell
Epimerase, Glce) gene inactivation, obtain the nonhuman mammalian cells of Glce gene inactivation；With
B () utilizes the cell of the Glce gene inactivation obtained in step (a), prepare the phobia of Glce gene inactivation or its
Relevant disease animal model.
2. the method for claim 1, it is characterised in that in step (a), also comprises the steps:
(a1) DNA homology recombinant technique is utilized, outside one or more in the exon 3 in described Glce gene to exon 5
Aobvious son is rejected or interrupts, and optionally replaces by selection markers, obtains the nonhuman mammalian cells of Glce gene inactivation.
3. the method for claim 1, it is characterised in that in step (b), also comprises the steps:
(b1) nonhuman mammalian cells of the Glce gene inactivation obtained in step (a) is utilized to prepare chimeric inhuman suckling
Animal；
(b2) the chimeric non-human mammal and the normal wild type mating non-human mammals that obtain in step (b1) are bred,
In offspring, screening obtains the heterozygote non-human mammal of Glce gene inactivation；With
(b3) the mutual copulation of heterozygote non-human mammal by obtaining in step (b2) obtains the pure of Glce gene inactivation
Zygote non-human mammal, thus obtain the animal model of the non-human mammal of Glce gene inactivation.
4. method as claimed in claim 3, it is characterised in that in step (b3), also include step (b4): by Glce gene
The homozygote non-human mammal of inactivation knocks out instrument non-human mammal with the neuronal specificity of same species and hybridizes,
Thus obtain the animal model of the non-human mammal of the Glce gene inactivation of neuronal specificity.
5. the method for claim 1, it is characterised in that the Glce gene inactivation obtained in described step (b) inhuman
In the animal model of mammal, compared with wild-type control animals, there are the one or more features selected from lower group:
(t1) spacious field level of activation reduces；
(t2) desire exploring strange environment reduces；
(t3) frightened sample behavior increases；
(t4) frightened degree increases；
(t5) behavior depression increases；
(t6) Degree of Depression increases；
(t7) anxiety-like behavior increases；
(t8) anxiety degree increases；
(t9) cognitive disorder increases；
(t10) incidence rate of apoplexy increases；
(t11) obese degree of form increases；
(t12) incidence rate of obesity symptom increases；
(t13) fatty tissue weight in wet base increases.
6. the purposes of non-human mammal model prepared by method described in a claim 1, it is characterised in that this model is used
Make research phobia or the animal model of its relevant disease.
7. the purposes of non-human mammal model prepared by method described in claim 1, wherein, is used for screening by this model
Or identify the material (therapeutic agent) that can alleviate or treat phobia or its relevant disease.
8. the method screened or determine the potential therapeutic agent treating or alleviating phobia or its relevant disease, its feature exists
In, comprise the following steps:
A test compound, in test group, in the presence of test compound, is applied to method system described in claim 1 by ()
Standby non-human mammal model, the behavior to the described animal model of test group carries out behavior analysis；And do not using
In described test compound and the identical matched group of other conditions, the behavior to the described animal model of matched group carries out behavioristics
Analyze；With
B the behavior of test group and control animals model is compared by (), wherein, compared with matched group, if application of survey
The behavior characterizing phobia or its relevant disease in the animal model of examination compound is improved, then show that this test compound can
As phobia or the potential therapeutic agent of its relevant disease.
9. the purposes of a cell, it is characterised in that glucuronic acid C5 isomerase (the Glucuronyl C5-in described cell
Epimerase, Glce) gene inactivation or downward, move for preparing the phobia building non-human mammal or its relevant disease
The biological preparation of object model.
10. the purposes of the deactivator of a Glce gene or its albumen, it is characterised in that be used for preparing structure non-human mammal
Phobia or the preparation of its relevant disease animal model.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610711576 | 2016-08-23 | ||
| CN2016107115768 | 2016-08-23 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106282122A true CN106282122A (en) | 2017-01-04 |
| CN106282122B CN106282122B (en) | 2020-10-02 |
Family
ID=57716627
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610873603.1A Active CN106282122B (en) | 2016-08-23 | 2016-09-29 | Method for establishing animal model of phobia of non-human mammal or related diseases thereof and application thereof |
| CN201610870869.0A Active CN107760713B (en) | 2016-08-23 | 2016-09-29 | Method for establishing animal model of neuropsychiatric disease of non-human mammal and application thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610870869.0A Active CN107760713B (en) | 2016-08-23 | 2016-09-29 | Method for establishing animal model of neuropsychiatric disease of non-human mammal and application thereof |
Country Status (2)
| Country | Link |
|---|---|
| CN (2) | CN106282122B (en) |
| WO (1) | WO2018036490A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018036490A1 (en) * | 2016-08-23 | 2018-03-01 | 中国科学院上海药物研究所 | Method and application for building animal model of non-human mammal neuropsychiatric disorder |
| CN109528197A (en) * | 2018-11-20 | 2019-03-29 | 中国科学院上海生命科学研究院 | The individuation prediction technique and system of across the Species migration carry out mental disease of monkey-people based on brain function map |
| CN111154809A (en) * | 2020-01-09 | 2020-05-15 | 电子科技大学附属医院·四川省人民医院 | Method for constructing glomerular disease model by using gene manipulation technology and application |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106282123B (en) * | 2016-09-29 | 2020-04-17 | 中国科学院上海药物研究所 | Method for establishing non-human mammal cognitive disorder or animal model with related diseases and application thereof |
| CN109813912B (en) * | 2019-01-04 | 2021-12-28 | 深圳大学 | Application of group of serum differential protein combinations in preparation of reagent for detecting autism |
| CN110754431B (en) * | 2019-11-21 | 2021-09-28 | 中国药科大学 | Building method and application of osteoporosis and Alzheimer's disease combined animal model |
| CN115053861A (en) * | 2022-06-30 | 2022-09-16 | 南方医科大学南方医院 | Construction method and application of animal model of schizophrenia based on immune activation |
| CN115281152B (en) * | 2022-08-12 | 2024-03-12 | 浙江中医药大学 | Method for constructing mouse lupus encephalopathy model |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450602A (en) * | 2013-09-17 | 2015-03-25 | 中国科学院遗传与发育生物学研究所 | Non-human mammal animal model of neurological and psychiatric disease and preparation method and application thereof |
| CN107760713A (en) * | 2016-08-23 | 2018-03-06 | 中国科学院上海药物研究所 | A kind of method for building up of non-human mammal neuropsychiatric disease animal model and application thereof |
Family Cites Families (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ITMI20031023A1 (en) * | 2003-05-21 | 2004-11-22 | Umberto Cornelli | GLYCOSAMINOGLICANS WITH AVERAGE MOLECULAR WEIGHT 2400 D AT THE TREATMENT OF EMOTIONAL DYSFUNCTIONS. |
| US9222088B2 (en) * | 2010-10-22 | 2015-12-29 | Curna, Inc. | Treatment of alpha-L-iduronidase (IDUA) related diseases by inhibition of natural antisense transcript to IDUA |
| US20160050895A1 (en) * | 2013-03-13 | 2016-02-25 | Australian Nuclear Science And Technology Organization | Transgenic non-human organisms with non-functional tspo genes |
| WO2015123439A2 (en) * | 2014-02-12 | 2015-08-20 | Duke University | Methods of promoting neuroblast differentiation and treating neuroblastoma and of providing a prognosis for subjects with neuroblastoma |
| CN105112448B (en) * | 2015-08-21 | 2020-06-26 | 同济大学 | Construction method and application of STCH gene knockout animal model |
| CN106139165B (en) * | 2016-08-23 | 2023-10-27 | 中国科学院上海药物研究所 | Method for establishing animal model of non-human mammal obesity or related diseases thereof and application thereof |
| CN106344933B (en) * | 2016-08-23 | 2020-01-17 | 中国科学院上海药物研究所 | Method for establishing animal model of anxiety disorder or related diseases of non-human mammal and application thereof |
| CN106282123B (en) * | 2016-09-29 | 2020-04-17 | 中国科学院上海药物研究所 | Method for establishing non-human mammal cognitive disorder or animal model with related diseases and application thereof |
-
2016
- 2016-09-29 CN CN201610873603.1A patent/CN106282122B/en active Active
- 2016-09-29 CN CN201610870869.0A patent/CN107760713B/en active Active
-
2017
- 2017-08-22 WO PCT/CN2017/098535 patent/WO2018036490A1/en active Application Filing
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450602A (en) * | 2013-09-17 | 2015-03-25 | 中国科学院遗传与发育生物学研究所 | Non-human mammal animal model of neurological and psychiatric disease and preparation method and application thereof |
| CN107760713A (en) * | 2016-08-23 | 2018-03-06 | 中国科学院上海药物研究所 | A kind of method for building up of non-human mammal neuropsychiatric disease animal model and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| ELVIRA,GRIGORIEVA等: "Decreased expression of human D-glucuronyl C5-epimerase in breast cancer", 《INTERNATIONAL JOURNAL OF CANCER》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018036490A1 (en) * | 2016-08-23 | 2018-03-01 | 中国科学院上海药物研究所 | Method and application for building animal model of non-human mammal neuropsychiatric disorder |
| CN109528197A (en) * | 2018-11-20 | 2019-03-29 | 中国科学院上海生命科学研究院 | The individuation prediction technique and system of across the Species migration carry out mental disease of monkey-people based on brain function map |
| CN109528197B (en) * | 2018-11-20 | 2022-07-08 | 中国科学院脑科学与智能技术卓越创新中心 | Individual prediction method and system for mental diseases based on brain function map |
| CN111154809A (en) * | 2020-01-09 | 2020-05-15 | 电子科技大学附属医院·四川省人民医院 | Method for constructing glomerular disease model by using gene manipulation technology and application |
| CN111154809B (en) * | 2020-01-09 | 2020-11-27 | 电子科技大学附属医院·四川省人民医院 | Method for constructing glomerular disease model by using gene manipulation technology and application |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106282122B (en) | 2020-10-02 |
| CN107760713B (en) | 2020-05-05 |
| WO2018036490A1 (en) | 2018-03-01 |
| CN107760713A (en) | 2018-03-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106282122A (en) | A kind of method for building up of non-human mammal phobia or its relevant disease animal model and application thereof | |
| CN106139165A (en) | A kind of method for building up of non-human mammal obesity or its relevant disease animal model and application thereof | |
| Lijam et al. | Social interaction and sensorimotor gating abnormalities in mice lacking Dvl1 | |
| Yang et al. | Postnatal lesion evidence against a primary role for the corpus callosum in mouse sociability | |
| Royle et al. | Behavioural analysis and susceptibility to CNS injury of four inbred strains of mice | |
| Lee et al. | A null mutation of mouse Kcna10 causes significant vestibular and mild hearing dysfunction | |
| Sundberg | The tabby (ta), tabby-c (Tac), and tabby-J (TaJ) mutations, chromosome X | |
| CN106282123A (en) | A kind of method for building up of non-human mammal cognitive disorder or its relevant disease animal model and application thereof | |
| CN106344933B (en) | Method for establishing animal model of anxiety disorder or related diseases of non-human mammal and application thereof | |
| CN107753957A (en) | A kind of method for building up of non-human mammal apoplexy or its relevant disease animal model and application thereof | |
| Bury et al. | Behavioral testing regimens in genetic-based animal models of Parkinson's disease: cogencies and caveats | |
| JP7012310B2 (en) | Mental illness model animals and their manufacturing methods | |
| CN107955818B (en) | Establishing method and application of non-human primate animal model with neurological diseases | |
| CN112111529A (en) | Neurodegenerative disease animal model and establishment and application thereof | |
| US11653636B2 (en) | Method of making a rat model of retinal degeneration and rat model made thereby | |
| CN107974463B (en) | Slc6a11 gene and application of protein thereof | |
| JP5083820B2 (en) | Hairless transgenic animal | |
| Rysakova et al. | Interaction of neurons in the basal and central nuclei of the amygdala in rabbits with active and passive behavioral strategies in emotionally negative situations | |
| JP7493194B1 (en) | Methods for sexing birds, bird species, production methods, and egg populations | |
| JPWO2007043589A1 (en) | Schizophrenia animal model | |
| CN107974464A (en) | The purposes of Slc6a12 genes and its albumen | |
| Tasnim | Distinct forms of spinal cord inhibition regulate tactile reactivity in mouse models of autism spectrum disorders | |
| JP2015021853A (en) | Screening of medicine using psychiatric symptom model animal | |
| Lee | Determining the Roles of Maternal and Peer Interactions in the Benefits of Communal Nesting | |
| JP5959235B2 (en) | New causative factor of spontaneous dermatitis and skin disease model animal |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |