CN108588125A - A kind of low expression BDNF transgenic mouse models and its construction method and application - Google Patents
A kind of low expression BDNF transgenic mouse models and its construction method and application Download PDFInfo
- Publication number
- CN108588125A CN108588125A CN201810171687.3A CN201810171687A CN108588125A CN 108588125 A CN108588125 A CN 108588125A CN 201810171687 A CN201810171687 A CN 201810171687A CN 108588125 A CN108588125 A CN 108588125A
- Authority
- CN
- China
- Prior art keywords
- bdnf
- mouse
- transgenic
- pcr
- expression
- 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.)
- Withdrawn
Links
- 108090000715 Brain-derived neurotrophic factor Proteins 0.000 title claims abstract description 138
- 102000004219 Brain-derived neurotrophic factor Human genes 0.000 title claims abstract description 132
- 230000014509 gene expression Effects 0.000 title claims abstract description 116
- 238000011830 transgenic mouse model Methods 0.000 title claims abstract description 68
- 238000010276 construction Methods 0.000 title claims abstract description 19
- 241000699660 Mus musculus Species 0.000 claims abstract description 33
- 238000000034 method Methods 0.000 claims abstract description 33
- 230000032683 aging Effects 0.000 claims abstract description 22
- 210000004556 brain Anatomy 0.000 claims abstract description 22
- 238000000520 microinjection Methods 0.000 claims abstract description 21
- 239000013612 plasmid Substances 0.000 claims abstract description 20
- 239000012634 fragment Substances 0.000 claims abstract description 19
- 230000009261 transgenic effect Effects 0.000 claims abstract description 15
- 230000030279 gene silencing Effects 0.000 claims abstract description 4
- 238000001890 transfection Methods 0.000 claims abstract description 4
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 54
- 241000699670 Mus sp. Species 0.000 claims description 48
- 238000001514 detection method Methods 0.000 claims description 33
- 235000013601 eggs Nutrition 0.000 claims description 31
- 210000001320 hippocampus Anatomy 0.000 claims description 31
- 108090000623 proteins and genes Proteins 0.000 claims description 30
- 210000004681 ovum Anatomy 0.000 claims description 24
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 claims description 22
- 102000002322 Egg Proteins Human genes 0.000 claims description 21
- 108010000912 Egg Proteins Proteins 0.000 claims description 21
- 210000004027 cell Anatomy 0.000 claims description 19
- 210000001519 tissue Anatomy 0.000 claims description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 238000003757 reverse transcription PCR Methods 0.000 claims description 13
- 239000000243 solution Substances 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- 229910002092 carbon dioxide Inorganic materials 0.000 claims description 11
- 239000001569 carbon dioxide Substances 0.000 claims description 11
- 238000002474 experimental method Methods 0.000 claims description 10
- 239000002773 nucleotide Substances 0.000 claims description 10
- 125000003729 nucleotide group Chemical group 0.000 claims description 10
- 102000004169 proteins and genes Human genes 0.000 claims description 10
- 108700019146 Transgenes Proteins 0.000 claims description 9
- 239000002299 complementary DNA Substances 0.000 claims description 9
- 239000000499 gel Substances 0.000 claims description 9
- 238000002347 injection Methods 0.000 claims description 9
- 239000007924 injection Substances 0.000 claims description 9
- 230000003287 optical effect Effects 0.000 claims description 9
- 210000001672 ovary Anatomy 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 102100031181 Glyceraldehyde-3-phosphate dehydrogenase Human genes 0.000 claims description 8
- 230000002159 abnormal effect Effects 0.000 claims description 8
- 230000015572 biosynthetic process Effects 0.000 claims description 8
- 108020004445 glyceraldehyde-3-phosphate dehydrogenase Proteins 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 8
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 claims description 8
- 206010002091 Anaesthesia Diseases 0.000 claims description 7
- 230000037005 anaesthesia Effects 0.000 claims description 7
- 210000003128 head Anatomy 0.000 claims description 7
- 230000006698 induction Effects 0.000 claims description 7
- 230000002401 inhibitory effect Effects 0.000 claims description 7
- 238000011740 C57BL/6 mouse Methods 0.000 claims description 6
- 239000003708 ampul Substances 0.000 claims description 6
- 239000003153 chemical reaction reagent Substances 0.000 claims description 6
- 239000011521 glass Substances 0.000 claims description 6
- 230000016087 ovulation Effects 0.000 claims description 6
- 238000003786 synthesis reaction Methods 0.000 claims description 6
- 210000003710 cerebral cortex Anatomy 0.000 claims description 5
- 238000001962 electrophoresis Methods 0.000 claims description 5
- 239000011780 sodium chloride Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 4
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 4
- 244000144987 brood Species 0.000 claims description 4
- 238000000605 extraction Methods 0.000 claims description 4
- 239000006166 lysate Substances 0.000 claims description 4
- 230000013011 mating Effects 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 239000000047 product Substances 0.000 claims description 4
- 210000002966 serum Anatomy 0.000 claims description 4
- 238000008157 ELISA kit Methods 0.000 claims description 3
- 102100034343 Integrase Human genes 0.000 claims description 3
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 claims description 3
- 210000001015 abdomen Anatomy 0.000 claims description 3
- 210000000683 abdominal cavity Anatomy 0.000 claims description 3
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 239000013604 expression vector Substances 0.000 claims description 3
- 210000003195 fascia Anatomy 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 3
- 239000001963 growth medium Substances 0.000 claims description 3
- 230000036541 health Effects 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 210000003205 muscle Anatomy 0.000 claims description 3
- 210000003101 oviduct Anatomy 0.000 claims description 3
- 239000002245 particle Substances 0.000 claims description 3
- 229920003023 plastic Polymers 0.000 claims description 3
- 210000004508 polar body Anatomy 0.000 claims description 3
- 238000010839 reverse transcription Methods 0.000 claims description 3
- 230000002441 reversible effect Effects 0.000 claims description 3
- 210000003491 skin Anatomy 0.000 claims description 3
- 238000011144 upstream manufacturing Methods 0.000 claims description 3
- 210000004291 uterus Anatomy 0.000 claims description 3
- -1 30%TritonX-100 Chemical compound 0.000 claims description 2
- 238000013461 design Methods 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 229940079593 drug Drugs 0.000 claims description 2
- 239000000523 sample Substances 0.000 claims 3
- 239000013614 RNA sample Substances 0.000 claims 1
- 238000005119 centrifugation Methods 0.000 claims 1
- 210000004907 gland Anatomy 0.000 claims 1
- 239000005556 hormone Substances 0.000 claims 1
- 229940088597 hormone Drugs 0.000 claims 1
- 230000001737 promoting effect Effects 0.000 claims 1
- 230000001568 sexual effect Effects 0.000 claims 1
- 238000011160 research Methods 0.000 abstract description 14
- 238000013459 approach Methods 0.000 abstract description 4
- 108020004459 Small interfering RNA Proteins 0.000 abstract 1
- 230000009456 molecular mechanism Effects 0.000 abstract 1
- 101000725076 Homo sapiens Cytochrome c oxidase subunit 5A, mitochondrial Proteins 0.000 description 78
- 102100027563 Cytochrome c oxidase subunit 5A, mitochondrial Human genes 0.000 description 77
- 108020004414 DNA Proteins 0.000 description 18
- 230000000694 effects Effects 0.000 description 15
- 230000015654 memory Effects 0.000 description 14
- 208000024827 Alzheimer disease Diseases 0.000 description 13
- 210000003470 mitochondria Anatomy 0.000 description 13
- 230000000971 hippocampal effect Effects 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 10
- 238000005516 engineering process Methods 0.000 description 9
- 238000004043 dyeing Methods 0.000 description 8
- 210000002569 neuron Anatomy 0.000 description 8
- 230000008569 process Effects 0.000 description 8
- 230000008859 change Effects 0.000 description 7
- 102000019149 MAP kinase activity proteins Human genes 0.000 description 6
- 108040008097 MAP kinase activity proteins Proteins 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 230000026731 phosphorylation Effects 0.000 description 6
- 238000006366 phosphorylation reaction Methods 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 241000700159 Rattus Species 0.000 description 5
- 210000004295 hippocampal neuron Anatomy 0.000 description 5
- 238000010166 immunofluorescence Methods 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 4
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 4
- 108010025020 Nerve Growth Factor Proteins 0.000 description 4
- 102000007072 Nerve Growth Factors Human genes 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 238000009395 breeding Methods 0.000 description 4
- 230000001488 breeding effect Effects 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 230000027928 long-term synaptic potentiation Effects 0.000 description 4
- 239000003900 neurotrophic factor Substances 0.000 description 4
- 230000002018 overexpression Effects 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 3
- 206010001497 Agitation Diseases 0.000 description 3
- 102000005636 Cyclic AMP Response Element-Binding Protein Human genes 0.000 description 3
- 108010045171 Cyclic AMP Response Element-Binding Protein Proteins 0.000 description 3
- 102400000267 Rhomboid-related protein 2, N-terminal fragment Human genes 0.000 description 3
- 101800000645 Rhomboid-related protein 2, N-terminal fragment Proteins 0.000 description 3
- 240000003186 Stachytarpheta cayennensis Species 0.000 description 3
- 235000009233 Stachytarpheta cayennensis Nutrition 0.000 description 3
- 101800000716 Tumor necrosis factor, membrane form Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 230000002490 cerebral effect Effects 0.000 description 3
- 230000019771 cognition Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 230000008021 deposition Effects 0.000 description 3
- 230000004064 dysfunction Effects 0.000 description 3
- 230000005611 electricity Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 210000005036 nerve Anatomy 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 230000008506 pathogenesis Effects 0.000 description 3
- 230000001242 postsynaptic effect Effects 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 208000037259 Amyloid Plaque Diseases 0.000 description 2
- 102000013918 Apolipoproteins E Human genes 0.000 description 2
- 108010025628 Apolipoproteins E Proteins 0.000 description 2
- 101150035467 BDNF gene Proteins 0.000 description 2
- 108010081589 Becaplermin Proteins 0.000 description 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 2
- 102000000634 Cytochrome c oxidase subunit IV Human genes 0.000 description 2
- 108090000365 Cytochrome-c oxidases Proteins 0.000 description 2
- 101001092197 Homo sapiens RNA binding protein fox-1 homolog 3 Proteins 0.000 description 2
- 102000043136 MAP kinase family Human genes 0.000 description 2
- 108091054455 MAP kinase family Proteins 0.000 description 2
- 101150056950 Ntrk2 gene Proteins 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 108010036933 Presenilin-1 Proteins 0.000 description 2
- 102000012412 Presenilin-1 Human genes 0.000 description 2
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 2
- 102100035530 RNA binding protein fox-1 homolog 3 Human genes 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- DZHSAHHDTRWUTF-SIQRNXPUSA-N amyloid-beta polypeptide 42 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)NCC(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)CC)C(C)C)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O)C(C)C)C(C)C)C1=CC=CC=C1 DZHSAHHDTRWUTF-SIQRNXPUSA-N 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
- 229910052791 calcium Inorganic materials 0.000 description 2
- 239000011575 calcium Substances 0.000 description 2
- 229960004407 chorionic gonadotrophin Drugs 0.000 description 2
- 230000007278 cognition impairment Effects 0.000 description 2
- 230000003920 cognitive function Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000006386 memory function Effects 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 210000002682 neurofibrillary tangle Anatomy 0.000 description 2
- 230000006576 neuronal survival Effects 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000009738 saturating Methods 0.000 description 2
- 230000009182 swimming Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- BWSIKGOGLDNQBZ-LURJTMIESA-N (2s)-2-(methoxymethyl)pyrrolidin-1-amine Chemical compound COC[C@@H]1CCCN1N BWSIKGOGLDNQBZ-LURJTMIESA-N 0.000 description 1
- 231100000582 ATP assay Toxicity 0.000 description 1
- 208000000044 Amnesia Diseases 0.000 description 1
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 1
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 1
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- 101150104471 COX5A gene Proteins 0.000 description 1
- 102000000584 Calmodulin Human genes 0.000 description 1
- 108010041952 Calmodulin Proteins 0.000 description 1
- 102000052052 Casein Kinase II Human genes 0.000 description 1
- 108010010919 Casein Kinase II Proteins 0.000 description 1
- 102100039289 Glial fibrillary acidic protein Human genes 0.000 description 1
- 101710193519 Glial fibrillary acidic protein Proteins 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 208000026139 Memory disease Diseases 0.000 description 1
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 1
- 235000003140 Panax quinquefolius Nutrition 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 208000027089 Parkinsonian disease Diseases 0.000 description 1
- 206010034010 Parkinsonism Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 238000001949 anaesthesia Methods 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000035578 autophosphorylation Effects 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940077737 brain-derived neurotrophic factor Drugs 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001713 cholinergic effect Effects 0.000 description 1
- 230000037326 chronic stress Effects 0.000 description 1
- 238000005352 clarification Methods 0.000 description 1
- 230000003931 cognitive performance Effects 0.000 description 1
- 230000001427 coherent effect Effects 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 210000001787 dendrite Anatomy 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 238000006471 dimerization reaction Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 238000002001 electrophysiology Methods 0.000 description 1
- 230000007831 electrophysiology Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 235000008434 ginseng Nutrition 0.000 description 1
- 210000005046 glial fibrillary acidic protein Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 230000000745 ion overload Effects 0.000 description 1
- 230000002427 irreversible effect Effects 0.000 description 1
- 238000009940 knitting Methods 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 238000007422 luminescence assay Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000006984 memory degeneration Effects 0.000 description 1
- 230000007074 memory dysfunction Effects 0.000 description 1
- 208000023060 memory loss Diseases 0.000 description 1
- 230000003340 mental effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 239000012120 mounting media Substances 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 230000007996 neuronal plasticity Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000000505 pernicious effect Effects 0.000 description 1
- 125000001095 phosphatidyl group Chemical group 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000012797 qualification Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 230000009758 senescence Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 102000013498 tau Proteins Human genes 0.000 description 1
- 108010026424 tau Proteins Proteins 0.000 description 1
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000003936 working memory Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0318—Animal model for neurodegenerative disease, e.g. non- Alzheimer's
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Environmental Sciences (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Physics & Mathematics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of low expression BDNF transgenic mouse models and its construction method and application, which includes the following steps：Then it is best to filter out silencing efficiency with the silence plasmid transfection 293T cells built with the method for RT PCR for structure CMV EmGFP siRNA BDNF low expression transgenic fragments；It is with AvrII that the highest silence plasmid of jamming effectiveness is linear, transgenic fragment is obtained, adjustment concentration to 5ng/ μ L is used for microinjection；The transgenic fragment built is linearized, transgenic mice is made with microinjection；PCR identifies positive transgenic mouse.The present invention obtains low expression BDNF transgenic mouse models for the first time, provides new research tool to illustrate brain aging morbidity molecular mechanism, and open new approaches.
Description
Technical field
The invention belongs to animal model technical fields, specifically, be related to a kind of low expression BDNF transgenic mouse models and
Its construction method and application.
Background technology
It is the adaptability that the aging state for adapting to aggravate with the age is made, irreversible change that brain aging, which is a kind of body,
Become, this process normally results in the forfeiture of the decline and part memory of learning ability.Brain aging is developed to towards pernicious direction
Certain phase is possible to that some common and intractable diseases can be evolved into, such as Alzheimer disease (AD), parkinsonism
(PD) and a series of other relevant clinical diseases of aging.Such situation also results in more and more national and social machine
The attention of structure, and a large amount of manpower, financial resources, material resources have been put into, carry out a large amount of research work in Neuscience related field.
Past 1990-1999 was once described as " 10 years of brain research " by U.S. government, was exactly the real picture of each side's input.But this
The high-cost input of sample but produces little effect, although taking countless mental and physical efforts, up to the present, for preventing and treating brain aging
And its measure of relevant disease is still extremely limited, far can not meet the demand of the aging society to grow stronger day by day.
Senescence-accelerated Mouse's (Senescence-Accelerated Mouse, SAM) are that a kind of world of research aging is public
Recognize animal model, be the naturally-aged animal by mostly being obtained for closed crossing, genetic background is AKR/J mouse.SAM is main
Including two major classes, i.e. quick aging system (SAM-prone, SAMP) and resistance to rapid aging system (SAM-resistant, SAMR).
And as the representative in natural aging strain, the P8 systems (SAMP8) in quick aging system, because its early stage just shows cognitive function
Defect and age-related ability of learning and memory loss and be widely used in brain aging relational learning memory mechanism
Research, become the main aging animal research models of experimental study.
Brain aging process causes a series of pathological change into exhibition, wherein most attractive is exactly to cause learning and memory
Dysfunction, and this also be exactly AD to one of the main harm of patient, seriously affect patients ' life quality and dignity.There is scholar logical
Cross to human cases analysis and experimental animal model the study found that the decline of memory is begun to early in the initial period of aging
.It is important that a large amount of clinical and laboratory research data shows that hippocampus has all played in study, memory and cognitive process
Effect, be an important learning and memory function maincenter.Therefore, the long-term potentiation (LTP) of hippocampal cell records quilt
It is widely used as the cell and molecular model of research memory, is especially applied in the correlative study of AD pathogenesis and treatment.
And the pathogenesis in relation to AD, it may be said that be that opinions vary, let a hundred schools contend, be still one in neuroscience field
Undecided mystery, comprehensive study report, so far, everybody is concentrated mainly on following side at more generally acknowledged morbidity theory
Face：1) abnormal deposition of beta-amyloid protein；2) tau protein abnormal phosphorylation is caused neurofibrillary tangles
(NFTs)；3) AD pathogenesis related genes theory, including beta amyloid precursor protein (APP) gene, presenilin 1 (PS1) gene, morning
2 (PS2) genes of old element, apo E (ApoE) gene occur abnormal etc.；4) cholinergic damages theory；5) intracellular Ca2+
Ion overload theory；6) radical damage theory；7) immune reaction theory etc..Wherein, beta-amyloid protein is in learning and memory
The abnormal deposition of related brain areas is acknowledged as the final link that all causes of disease cause AD to fall ill, and the exception of beta-amyloid protein
The senile plaque (SP) that deposition is formed also becomes the important pathological diagnosis foundation that AD makes a definite diagnosis.
And newest research is thought, almost each neurodegenerative disease and relevant diseases develop to certain phase and all can
There is mitochondria dysfunction.Mitochondria dysfunction seems the dependent event as nerve retrograde affection, with generation, AD
It is no exception.
As one of neurotrophic factor (neurotrophic factors, NTFs) family member, brain source nerve battalion
It is a kind of possessing numerous biological activities small point to support the factor (brain-derived neurotrophic factor, BDNF)
Sub- albumen all plays a role maintaining the various aspects such as holding of neuronal survival, aixs cylinder formation guiding, cellular morphology.BDNF
Molecular weight be 12.4kDa, be a kind of basic protein, the assignment of genes gene mapping is in 11p13, and precursor has 247 amino acid residues, through turning over
The ripe basic protein that post-processing forms 119 amino acid residues composition is translated, isoelectric point 9.99 has 3 pairs of disulfide bond in chain,
Exist in the form of dimer in vivo.BDNF is the Major Members of neurotrophic factor (NT) family.It is played by receptor TrkB
Effect.BDNF is combined to be allowed to activate with TfkB and is carried out in two steps, when the Receptor dimerization of ligand induction, second is that intracellular region
The autophosphorylation of tyrosine residue.Activation is interacted by physical efficiency and multiple intracellular proteins and makes its phosphorylation.These are lived
The protein and then activation Ras/ mitogen-activated protein kinases (MAPK) signal transduction path and extracellular signal of change, which are adjusted, to swash
Enzyme is (such as：Mitrogen-activated protein, Erk) phosphorylation, so that the calcium concentration of intracellular is increased, then calcium/calmodulin activated to rely on
Property kinases and casein kinase 2, CREB phosphorylations further activate phosphatidyl to swash -3 kinases of inositol.This process meeting
Because the presence of the endogenous material of TfkB activation can be blocked further to complicate.
The experimental results have confirmed that BDNF is being recognized and the important function in learning and memory formation, maintenance in recent years.
It is interesting that research report brain aging process significantly lowered with NTFs expressions, and NTFs expressions it is notable under
It is typically the arch-criminal for causing brain aging related pathologies and changing to adjust.As key molecule, BDNF is by maintaining neuronal survival, adjusting
Neural plasticity influences the pathogenic process that the links such as learning and memory participate in Alzheimer disease (AD).Forefathers study report hair
There is abnormal expression and publication in AD patient and experimental animal model intracerebral in existing BDNF and its specific receptor TrkB.But at present
Until, mechanism of action of the mitochondrial defects in AD morbidities is unclear, if BDNF/Erk signals take part in is with mitochondria
The brain aging process at center not yet illustrates, and has not been reported both at home and abroad.
Invention content
In view of this, the present invention provides a kind of low expression BDNF transgenic mouse models and its construction method and applications.
In order to solve the above-mentioned technical problem, the invention discloses a kind of structure sides of low expression BDNF transgenic mouse models
Method includes the following steps：
The structure of step 1, CMV-EmGFP-siRNA-BDNF low expression transgenic fragments：Silence expression vector CMV-
EmGFP-siRNA-BDNF purchases are used in combination the software that the said firm website provides to be directed to target gene BDNF from Invitrogen companies
(GeneID:12064) target site, wherein target site CCAAGTGTAATCCCATGGGTT designs the plasmid of silence, then
Plasmid construction is completed by calm and peaceful company of Sino-U.S.；
Step 2, with the silence plasmid transfection 293T cells built, then filter out silencing efficiency with the method for RT-PCR
Best；
It is step 3, with AvrII that the highest silence plasmid of jamming effectiveness is linear, transgenic fragment is obtained, adjustment concentration is extremely
5ng/ μ L are used for microinjection；
Step 4 linearizes the transgenic fragment built, and transgenic mice is made with microinjection；PCR is identified
Positive transgenic mouse.
Optionally, it includes following experimental procedure that the microinjection in step 4, which makes transgenic mouse approach,：
A, super several ovulation induction operations, i.e., first day injection pregnant mare serum gonadotrop(h)in (PMSG) 10IU/ only, are injected after 48 hours
Human chorionic gonadotrophin 10IU/ is only；Meanwhile it choosing 6 weeks C57BL/6 mouse of health male and receiving ovulation induction behaviour with above-mentioned
The female mice 1 of work:1 mates mating, observes cloudy bolt situation, has the mouse of cloudy bolt to propose for use, spare as ovum mouse；
B, ovum is taken：It will upwards be placed for ovum mouse anesthesia postabdomen, preserved skin exposes abdomen, is successively detached with scissors and tweezers
Skin, fascia, muscle, exposure ovary, fallopian tubal and uterus, detach fallopian tubal, fallopian tubal are placed into M2 culture mediums with tweezers
In, the ampulla of fallopian tubal is opened under disecting microscope, and ovum is made to flow in culture solution；The saturating of l mg/ml is added in culture solution
Bright matter acid enzyme is used in combination M2 culture mediums to rinse 3-4 times, removes granular cell；It is observed under the microscope, to fertilized eggs and other
Cell is differentiated that because second polar body is discharged in fertilized eggs, and unfertilized egg and other ovum for becoming abnormal morphology can be easily
It is distinguished；The fertilized eggs chosen move on in the plastic ware for containing M2 culture medium drops, move in carbon dioxide incubator and cultivate directly
Until fertilized eggs are suitble to injection；
C. microinjection：The transgene carrier segment of linearisation is injected into the fertilization of C57BL/6 mouse by microinjection
It in ovum masculonucleus, observes under the microscope, selects that cell is full, and oolemma is clear, the high-visible fertilized eggs of male pronucleus wait for
With；It is made the culture drop and transgene carrier segment solution drop of 20 fertilized eggs on glass slide, is fixed to objective table
On, fertilized eggs are fixed with ovum suction pipe is held, glass pipette injects transgene carrier segment solution drop, it is slowly injected
Masculonucleus collect fertilized eggs, a night are cultivated in 37 DEG C of carbon dioxide incubators after injection；
D. it transplants：Pseudopregnant mouse is anaesthetized, operation takes out ovary and connects fallopian tubal, is fixed with fatty tweezer, under the microscope
It finds fallopian tubal to be open, under microscope, chooses and be split into bicelluar fertilized eggs, it is spare；Above-mentioned fertilized eggs are drawn, by grafts
Mouth is inserted into fallopian tubal mouth, is gently blown into the liquid in grafts, it is seen that ampulla of uterine tube expands and can be clearly seen that three
Bubble is transplanted successfully；Ovary is put back into abdominal cavity, layer-by-layer suture together with fallopian tubal.
Optionally, the temperature of the carbon dioxide incubator is 37 DEG C, contains 5% carbon dioxide, 95% air.
Optionally, the PCR identification positive transgenic mouse in the step 4 are specially：
I), PCR methods identify F1 generation positive transgenic mouse, and positive is as experimental group, and negative patient is as brood wild type pair
According to；
Ii it) draws materials：The F1 generation positive transgenic mouse male and female that each strain head builds mouse are unlimited, each strain 7, after anesthesia
Rapid taking-up cerebral cortex and hippocampus, each position are divided into two parts, and portion is detected for RT-PCR, another is used for albumen table
It is respectively put into clean EP pipes after precooling normal saline flushing is clean up to detection and freezes or carry out immediately experiment detection for -80 DEG C.
Iii) RT-PCR methods detect F1 generation positive BDNF low expression trangenic mices：After taking tissue, by 150mg/1.5ml
The ratio of TRIZOL is put into the homogenizer of the TRIZOL reagents of pre-add precooling, and homogenate is not to viscous without particle under ice bath, gradually
It centrifuges, extraction RNA；After the RNA newly extracted the processed distilled water dissolvings of DEPC, UV spectrophotometer measuring RNA
The purity and concentration of sample；Take 2 μ g RNA for reverse transcription reaction, with Reverse Transcriptase kit using RNA as templated synthesis cDNA
Chain uses RevertAidTMM-MuLV Reverse Transciptase enzyme systems, the cDNA of synthesis are directly used in PCR
Detection reaction；
Add deionized water to supply 25 μ l reaction systems, 94 DEG C 5 minutes, 94 DEG C 1 minute, 55 DEG C 1 minute, 72 DEG C 1 minute altogether
35 cycle, last 72 DEG C 5 minutes, complete PCR detection reaction；PCR products carry out 1% agarose gel electrophoresis detection；With
GAPDH is reference gene, and same gene amplified production carries out on same gel when electrophoresis detection, solidifying with BIO-RAD companies
Gel images are absorbed under glue imager UV mode and carry out electrophoretic band integral optical density analysis, calculate each group BDNF genes
The ratio of band and GAPDH integral optical density values obtains the relative expression quantity of each sample B DNF genes.
Iv) the expression of the detection of Elisa methods the F1 generation positive BDNF low expression trangenic mice cortex and hippocampus BDNF.It takes
After tissue, each tissue is put into containing pH 7.4,0.05M Tris-HCl, 0.5M EDTA, 30%TritonX-100, NaCl,
It is homogenized under ice bath in the lysate of 10%SDS and 1mM PMSF, until being visible by naked eyes tissue block；It centrifuges, take supernatant, BCA
Kit detects protein content；BDNF-ELISA kit kits detect the BDNF contents of each sample；
It takes individual mouse genotypes cortex and hippocampus to carry out the detection of expression of BDNF, finally determines the inhibition of BDNF expression rates
The highest strain of rate, mass propagation are spare.
Optionally, PCR detections reaction system is specially：Each sample takes 1.5 μ l of cDNA templates, it is added 2 ×
PCR Master Mix (Fermentals) 12.5 μ l, each 0.5 μ l of upstream and downstream primer.
Optionally, the sense primer：5'TGTGACAGTATTAGCGAGTGGGT 3', nucleotide sequence such as SEQ
Shown in ID NO.3；
Downstream primer：5'TACGATTGGGTAGTTCGGCATT 3', nucleotide sequence is as shown in SEQ ID NO.4.
The invention also discloses a kind of low expression BDNF transgenic mouse models that above-mentioned construction method obtains.
Prevent in preparation the invention also discloses a kind of above-mentioned low expression BDNF transgenic mouse models or treatment brain is old
Application in chemical drug object.
Compared with prior art, the present invention can be obtained including following technique effect：
1) present invention obtains whole body low expression BDNF transgenic mice new lines for the first time, believes for research BDNF and downstream
The gene function of number access provides research mode tool in each tract disease of whole body；
2) present invention obtains low expression BDNF transgenic mouse models for the first time, has tentatively probed into BDNF/ERK signal paths and has existed
Possibility effect in brain aging cognition dysfunction process centered on mitochondria and mechanism, to illustrate brain aging morbidity molecule
Mechanism provides new research tool, and opens new approaches.
Certainly, it implements any of the products of the present invention it is not absolutely required to while reaching all the above technique effect.
Description of the drawings
Attached drawing described herein is used to provide further understanding of the present invention, and constitutes the part of the present invention, this hair
Bright illustrative embodiments and their description are not constituted improper limitations of the present invention for explaining the present invention.In the accompanying drawings：
Fig. 1 is COX5A over-express vectors schematic diagram and COX5A transgenic mice PCR testing results of the present invention；Wherein,
A and b is COX5A over-express vector schematic diagrames；C is COX5A transgenic mice PCR testing results；
Fig. 2 is detection of expression of the COX5A of the present invention in trangenic mice cerebral tissue；Wherein, a, COX5A are small in transgenosis
Gene expression dose in mouse cerebral tissue；Expression of b, the COX5A albumen in transgenic mice cerebral tissue；
Fig. 3 is BDNF silences structure cell-based screening electrophoretogram of the present invention；293T cellular identifications BDNF is used in vitro
Expression inhibiting efficiency of the low expression transgenic fragment to BDNF；
Fig. 4 is BDNF low expressions trangenic mice (BDNF-DO) structure figures of the present invention；Wherein, a, low expression BDNF carrier structures
Build schematic diagram；B, low expression BDNF transgenic mice genomic DNAs detect PCR electrophoretograms；2000 molecular weight standard of M, DNA；
9,39,34,21 respectively headed by build mouse number Founder 9,39,34,21；WT, wild-type mice；
Fig. 5 is detection of expression of the BDNF of the present invention in BDNF-DO trangenic mice brains；Wherein, a, BDNF gene exist
Expression in BDNF-DO trangenic mice cerebral cortexes；B, bdnf protein is in BDNF-DO trangenic mice cerebral cortexes
Expression；9,39,34,21 respectively headed by build mouse number Founder 9,39,34,21；WT, wild-type mice；* vs WT, P<
0.05；
Fig. 6 is double cross COX5A-UP/BDNF-DO mouse PCR qualification figures of the present invention；2000 molecular weight standard of M, DNA；B+,
Low expression BDNF trangenic mices；C+/B+ is overexpressed COX5A and low expression BDNF double cross mouse；WT, wild-type mice；
Fig. 7 is double cross transgenic mice water maze evaluation result of the present invention；Wherein, a is overexpressed COX5A and low expression
BDNF double cross mouse escape latency measurement results；WT, wild-type mice；COX+ is overexpressed COX5A transgenosis groups；B+ is low
Express BDNF transgenosis groups；COX+/B+ is overexpressed COX5A and low expression BDNF double cross groups；B, each group trangenic mice are being withdrawn from
In the residence time testing result of target quadrant after platform；WT, wild-type mice；COX+ is overexpressed COX5A transgenosis groups；B
+, low expression BDNF transgenosis groups；COX+/B+ is overexpressed COX5A and low expression BDNF double cross groups；O, phase reversed octant；R, it is right
Side quadrant；T, target quadrant；L, left hand quadrant；* vs WT, P<0.05；#vs is overexpressed COX5A transgenosis groups, P<0.05；
Fig. 8 is that the present invention is overexpressed COX5A/ low expression BDNF transgenic mice LTP testing results；Wherein, a, to start
Input-output curve when record；B, WT, COX5A transgenic mices, low expression BDNF transgenic mices and double miscellaneous are overexpressed
Hand over transgenic mice hippocampal slices fEPSP wave representative illustrations；C and d, WT, overexpression COX5A turn base after giving continuous train
Because of mouse, the change and quantitative analysis of low expression BDNF transgenic mices and double cross transgenic mice hippocampal slices fEPSP；
WT, wild-type mice；COX+ is overexpressed COX5A transgenosis groups；B+, low expression BDNF transgenosis groups；COX+/B+ is overexpressed
COX5A and low expression BDNF double cross groups；* vs WT, P<0.05；* vs are overexpressed COX5A transgenosis groups, P<0.05；
Fig. 9 is COX5A of the present invention and common locations of the BDNF in trangenic mice hippocampal cell；Wherein, a-d, in hippocampus
COX5A and neuron common location figure；E-h, COX5A and BDNF common location figures in hippocampus；A and e, COX5A immunofluorescence dyeing are (red
Color)；B, NeuN immunofluorescence dyeing (green)；C and g, DAPI immunofluorescence dyeing (blue)；F, BDNF immunofluorescence dyeing
(green)；D and h, the picture after merging.400 times of amplification factor；Scale：5μm；
Figure 10 is the change that the present invention is overexpressed COX5A/ low expressions BDNF trangenic mices hippocampal neuron and spinous process；Its
In, a-d, a1-d1, wild type, overexpression COX5A transgenosis group, low expression BDNF transgenosis group and overexpression COX5A/ are low
Express BDNF double cross trangenic mice hippocampus Gorky's colored graphs；WT, wild-type mice；COX+ is overexpressed COX5A transgenosis
Group；B+, low expression BDNF transgenosis groups；COX+/B+ is overexpressed COX5A and low expression BDNF double cross groups；E, hippocampal neural
The quantitative analysis of cellular bulk area, protrusion distal end branch amount, proximal end branch amount, dendron complexity；Wherein, dendron complexity：Tree
The ratio of prominent tip quantity and level-one branch quantity, i.e. protrusion distal end branch amount/proximal end branch amount, ratio is higher, illustrates dendron
Branch is more；* vs WT, P<0.05；* vs are overexpressed COX5A transgenosis groups, P<0.05；Amplification factor：A-d, 200 times；a1-
D1,400 times；
Figure 11 is that the present invention is overexpressed COX5A/ low expression BDNF trangenic mice hippocampus mitochondria activities and ATP contents,
In, a is overexpressed COX5A/ low expression BDNF trangenic mice hippocampus mitochondria activity Cco measurement results；It is low to be overexpressed COX5A/ by b
Express BDNF trangenic mice hippocampus ATP assay results；WT, wild-type mice；COX+ is overexpressed COX5A transgenosis groups；B
+, low expression BDNF transgenosis groups；COX+/B+ is overexpressed COX5A and low expression BDNF double cross groups；* vs WT, P<0.05；**
Vs is overexpressed COX5A transgenosis groups, P<0.05；
Figure 12 is that COX5A of the present invention is played a role by BDNF/ERK accesses, wherein a, c, BDNF and its coherent signal
The gene (a) and albumen (c) of pathway molecule ERK1/2 is in Different Month (6 monthly ages and 18 monthly ages) trangenic mice cortex and hippocampus
In expression variation；B, d, BDNF and its gene (b) of associated signal paths molecules ERK 1/2 and albumen (d) are in Different Month (6
Monthly age and 18 monthly ages) quantitative analysis expressed in trangenic mice hippocampus；WT, wild-type mice；COX+ is overexpressed COX5A and turns base
Because of group.
Specific implementation mode
Carry out the embodiment that the present invention will be described in detail below in conjunction with embodiment, thereby to the present invention how application technology hand
Section solves technical problem and reaches the realization process of technical effect to fully understand and implement.
Embodiment 1 is overexpressed the structure of COX5A transgenic mouse models：
The construction method includes the following steps：
Step 1, PCR clonal expansion target gene COX5A (GeneID:12858) opening code-reading frame (ORF), amplification is drawn
Object sequence is as follows：
Sense primer is 5 ' GTCAATGGGTGGAGTATTTACG 3 ', and nucleotide sequence is as shown in SEQ ID NO.1；
Downstream primer is 5 ' GCTTATATAGACCTCCCACCGT 3 ', and nucleotide sequence is as shown in SEQ ID NO.2；
Target fragment 386bp.
Step 2, pcDNA3.1 (+)-m-COX5A plasmid constructions.By digestion, DNA fragmentation recycling, connection reaction inscribe
The ORF of enzyme EcoRI and XhoI digestions pcDNA3.1 (+/-) Vector carriers and the target fragment COX5A of insertion.Such as Fig. 1 a-b institutes
Show.
The plasmid vector built is transferred to the extraction that strain carries out DNA by step 3, by largely preparing Plasmid DNA, electricity
Swimming recycles DNA segment with QiagenDNA gel reclaims kits；
Step 4, the gel column purification DNA with SephedexG50, dissolving recycling is carried out with TE solution to DNA；
Step 5, recycling segment, are used for microinjection.With the TE microinjection diluteds of 0.22 μm of membrane filtration
DNA sample, 12000g are centrifuged 2 hours, and the 2/3 of supernatant is dispensed, and are used for microinjection.
Step 6 linearizes the transgenic fragment built, and transgenic mice is made with microinjection；PCR is identified
Positive transgenic mouse；As Fig. 1-a, b show COX5A over-express vector schematic diagrames；
Wherein, it includes following experimental procedure that microinjection, which makes transgenic mouse approach,：
A. super several ovulation induction operations, i.e., pregnant mare serum gonadotrop(h)in (PMSG) (PMSG) 10IU/ (0.2ml/ of injection in first day
Only), human chorionic gonadotrophin (HCG) 10IU/ only (0.2ml/ is only) is injected after 48 hours；Meanwhile choosing health male 6 weeks
Left and right C57BL/6 mouse and the above-mentioned female mice 1 for receiving ovulation induction operation:1 mates mating, observes cloudy bolt situation, there is cloudy bolt
Mouse propose it is for use, it is spare as ovum mouse.
B. ovum is taken.It will upwards be placed for ovum mouse anesthesia postabdomen, preserved skin exposes abdomen, is successively detached with scissors and tweezers
Skin, fascia, muscle, exposure ovary, fallopian tubal and uterus, detach fallopian tubal, fallopian tubal are placed into M2 culture mediums with tweezers
In, the ampulla of fallopian tubal is opened under disecting microscope, and ovum is made to flow in culture solution.The saturating of l mg/ml is added in culture solution
Bright matter acid enzyme is used in combination M2 culture mediums to rinse 3-4 times, removes granular cell.It is observed under the microscope, to fertilized eggs and other
Cell is differentiated that because second polar body is discharged in fertilized eggs, and unfertilized egg and other ovum for becoming abnormal morphology can be easily
It is distinguished.The fertilized eggs chosen move on in the plastic ware (diameter 35mm) for containing M2 culture medium drops, move to carbon dioxide
Culture is until fertilized eggs are suitble to injection in incubator (37 DEG C, 5% carbon dioxide, 95% air).
C. microinjection.The transgene carrier segment of linearisation is injected into the fertilization of C57BL/6 mouse by microinjection
In ovum masculonucleus.It observes under the microscope, selects that cell is full, and oolemma is clear, the high-visible fertilized eggs of male pronucleus wait for
With.It is made on glass slide there are about the culture drop and transgene carrier segment solution drop of 20 or so fertilized eggs, is fixed to
On objective table, fertilized eggs are fixed with ovum suction pipe is held, glass pipette injects transgene carrier segment solution drop, slowly by it
Ground injects masculonucleus.Fertilized eggs are collected after injection, and a night is cultivated in 37 DEG C of carbon dioxide incubators.
D. it transplants.Pseudopregnant mouse is anaesthetized, operation takes out ovary and connects fallopian tubal, is fixed with fatty tweezer, under the microscope
Find fallopian tubal opening.Under microscope, chooses and be split into bicelluar fertilized eggs, it is spare.Above-mentioned fertilized eggs are drawn, by grafts
Mouth is inserted into fallopian tubal mouth, is gently blown into the liquid in grafts, it is seen that ampulla of uterine tube expands and can be clearly seen that three
Bubble is transplanted successfully.Ovary is put back into abdominal cavity, layer-by-layer suture together with fallopian tubal.
Step 7, PCR methods identify that positive transgenic mouse, transgenic mice are marked for 9-14 days in birth with toeclipping, and collection is cut
Under tissue be detected by PCR methods using special primer, detection primer sequence is such as with alkaline lysis method of extracting genomic DNA
Under：
Sense primer is 5 ' GTCAATGGGTGGAGTATTTACG ' 3 ', and nucleotide sequence is as shown in SEQ ID NO.1；
Downstream primer is 5 ' GCTTATATAGACCTCCCACCGT 3 ', and nucleotide sequence is as shown in SEQ ID NO.2；
PCR identifies positive as experimental group, and negative patient is as brood wild type control；From Fig. 1-c it is found that successfully obtaining
The COX5A for expressing 386bp target fragments is overexpressed transgenic mice, totally 4 strains, is respectively designated as according to toe number
Founder N (Founder35,22,26,28) can be examined compared with wild type (WT) in the detection of rat-tail DNA genomes
Measure the Insert Fragment of 386bp；Illustrate to successfully obtain expected Insert Fragment.
Step 8 goes out the highest strain of COX5A expressions with RT-PCR and Western blot technology screenings, establishes
Stable COX5A is overexpressed mouse model；General head, which is built after mouse mates, can obtain several F0 for mouse, to exclude different insertion positions
The influence that point expresses PDGF-BB, the filial generation given birth to for mouse to F0 with RT-PCR and WB technologies (F1 generation mouse) carry out COX5A
Gene and protein expression detection, filter out highest expression strain, breeding of reserving seed for planting.The present invention obtains the COX5A of 4 strains altogether
Transgenosis F0 is overexpressed for mouse, respectively Founder 35,22,26, No. 28.
As shown in Figure 2 a, compared with WT groups, the mRNA of COX5A builds significantly expression increase in mouse in four head；Such as Fig. 2 b
Shown, compared with WT groups, COX5A albumen builds significantly expression increase, *, P in mouse in four head<0.05；
The structure of 2 low expression BDNF transgenic mouse models of embodiment：
Including following experimental procedure：
The structure of step 1, CMV-EmGFP-siRNA-BDNF low expression transgenic fragments：Silence expression vector CMV-
EmGFP-siRNA-BDNF purchases are used in combination the software that the said firm website provides to be directed to target gene BDNF from Invitrogen companies
[BDNF brain derived neurotrophic factor (Mus musculus), GeneID:12064] target site
For (CCAAGTGTAATCCCATGGG TT), the plasmid of silence is designed, plasmid construction is then completed by calm and peaceful company of Sino-U.S..
Step 2, the silence plasmid transfection 293T cells built, it is good then to filter out silencing efficiency with the method for RT-PCR
Structure transfected No. 47 BDNF low expression transgenosis plasmids as shown in figure 3, compared with control group and other 3 interference plasmids
The expression of cell BDNF significantly reduce, give over to follow-up use；C is control group, and 16,26,46,47 be 4 BDNF low expressions
The number of silence plasmid；Wherein, the efficiency highest of No. 47 plasmid interference BDNF expression, gives over to follow-up use；
Step 3, with AvrII that the highest No. 47 silence plasmids of jamming effectiveness is linear, acquisition transgenic fragment (Fig. 4),
Concentration is adjusted to 5ng/ μ L, is used for microinjection.
Step 4 linearizes the transgenic fragment built, makes transgenic mice with microinjection, method is as before
It states；PCR identifies positive transgenic mouse：Go out the highest strain of BDNF expression inhibiting rates with RT-PCR and Elisa technology screenings,
Establish stable BDNF low expression mouse models；General head, which is built after mouse mates, can obtain several F0 for mouse, to exclude different insert
The influence that BDNF is expressed in angle of striking, the filial generation given birth to for mouse to F0 with RT-PCR and Elisa technologies (F1 generation mouse) carry out
The gene and protein expression of BDNF detects, and filters out the strain of minimum expression, breeding of reserving seed for planting.The present invention obtains 4 strains altogether
BDNF low expression transgenosis F0 are for mouse, and toe number is respectively No. 9, No. 39, No. 34, No. 21, as shown in Fig. 4-b；Including following
Experimental procedure：
A.PCR methods identify F1 generation positive transgenic mouse, and positive is as experimental group, and negative patient is as brood wild type pair
According to；
B. it draws materials.The F1 generation positive transgenic mouse male and female that each strain head builds mouse are unlimited, and each strain 7 is fast after anesthesia
Speed takes out cerebral cortex and hippocampus, and each position is divided into two parts that (portion is detected for RT-PCR, another is used for protein expression
Detection), after precooling normal saline flushing is clean, it is respectively put into clean EP pipes and freezes or carry out immediately experiment detection for -80 DEG C.
C.RT-PCR methods detect F1 generation positive BDNF low expression trangenic mices.After taking tissue, by 150mg/1.5ml
The ratio of TRIZOL is put into the homogenizer of the TRIZOL reagents of pre-add precooling, and homogenate is not to viscous without particle under ice bath, gradually
It centrifuges, extraction RNA；After the RNA newly extracted the processed distilled water dissolvings of DEPC, UV spectrophotometer measuring RNA
The purity and concentration of sample；Take 2 μ g RNA for reverse transcription reaction, with Reverse Transcriptase kit using RNA as templated synthesis cDNA
Chain uses RevertAidTMM-MuLV Reverse Transciptase enzyme systems, the cDNA of synthesis can be directly used for
PCR detection reactions.Each sample takes 1.5 μ l of cDNA templates, be added 2 × PCR Master Mix (Fermentals) 12.5 μ l,
Each 0.5 μ l of upstream and downstream primer, primer sequence are：
Sense primer：5'TGTGACAGTATTAGCGAGTGGGT 3', nucleotide sequence is as shown in SEQ ID NO.1；
Downstream primer：5'TACGATTGGGTAGTTCGGCATT 3', nucleotide sequence as shown in SEQ ID NO.2,
Add deionized water to supply 25 μ l reaction systems, 94 DEG C 5 minutes, 94 DEG C 1 minute, 55 DEG C 1 minute, 72 DEG C 1 minute altogether
35 cycle, last 72 DEG C 5 minutes, complete PCR detection reaction；PCR products carry out 1% agarose gel electrophoresis detection；With
GAPDH is reference gene, and same gene amplified production carries out on same gel when electrophoresis detection, solidifying with BIO-RAD companies
Gel images are absorbed under glue imager UV mode and carry out electrophoretic band integral optical density analysis, calculate each group BDNF genes
The ratio of band and GAPDH integral optical density values obtains the relative expression quantity of each sample B DNF genes, as shown in Figure 5 a.
D.Elisa methods detect the expression of the F1 generation positive BDNF low expression trangenic mice cortex and hippocampus BDNF.Take group
After knitting, each tissue is put into containing 0.05M Tris-HCl (pH 7.4, Amresco), 0.5M EDTA (Amresco), 30%
TritonX-100 (Amresco), NaCl (Amresco), the lysate of 10% SDS (Sigma) and 1mM PMSF (Amresco)
It is homogenized under middle ice bath, until being visible by naked eyes tissue block；It centrifuges, take supernatant, BCA (Sigma, St.Louis, MO, USA) examinations
Agent box detects protein content；BDNF-ELISA kit (B＆D) kit detects the BDNF contents of each sample, as shown in Figure 5 b.
It takes individual mouse genotypes cortex and hippocampus to carry out the detection of expression of BDNF, finally determines the inhibition of BDNF expression rates
The highest strain of rate is Founder 39, and mass propagation is spare.It is selected as kind of a mouse strain breeding double cross mouse.With COX5A
It is overexpressed the highest breedings of strain Founder 35 hybridization, builds double cross mouse.
As shown in figure 5, BDNF 4 strains of BDNF low expressions transgenic mice (Founder 39, Founder 9,
Founder 21, Founder 34) cortex and hippocampus detection of expression as a result, BDNF (genes/proteins) expression inhibiting rate meter
Calculate formula：BDNF expression inhibitings rate=O.D. (WT-Founder)/(the O.D.means optical of O.D.WT X 100%
Density), Founder is specific a certain strain, and WT is wild type, and O.D. is integral optical density value.Thus formula calculates
Go out：Compared to other strains, BDNF determines BDNF in the expression inhibiting rate highest of Founder 39, average out to 55.23%
The highest strain of expression inhibiting rate is Founder 39, gives over to subsequent experimental research.Remaining each strain PDGF-BB overexpression rate point
It Wei not Founder 9 41.05%, Founder 21 53.55%, Founder 34 1.2%.
Embodiment 3 is overexpressed the structure of COX5A/ low expression BDNF transgenic mouse models (COX5A-UP/ BDNF-DO)
COX5A is overexpressed highest strain Founder 35 and BDNF low expressions minimum strain Founder 39F1 generations
It mates, is obtained from offspring rat and be overexpressed COX5A and low expression BDNF double cross mouse；The specific steps are two kinds of lines transgenic mouse
Offspring rat is obtained after mating, and genotype detection is carried out to offspring rat, PCR methods are detected simultaneously by COX5A in rat-tail genomic DNA
Be overexpressed the mouse of trangenic mice genotype and BDNF low expression trangenic mice genotype segments, be as overexpressed COX5A with
Low expression BDNF double cross mouse.
It will be appreciated from fig. 6 that be detected simultaneously by above two segment in transgenosis rat-tail DNA, illustrate structure be overexpressed COX5A with
The success of low expression BDNF double cross mouse.
The learning and memory function that embodiment 4Morris water mazes evaluate double cross transgenic mice changes
As shown in Figure 7, compared with wild type, COX5A/ low expression BDNF double cross trangenic mice, low expression are overexpressed
BDNF transgenic mices will take more time searching platform, and its target quadrant residence time after withdrawing from platform is notable
It reduces, and low expression BDNF transgenic mices are even more serious, escape latency extension, target quadrant shorter residence time；With mistake
Expression COX5A groups are compared, and are overexpressed the extension of COX5A/ low expression BDNF double cross trangenic mice escape latencies, target quadrant stops
The time is stayed to shorten.It these results suggest that, be overexpressed the improvement result of the ability of learning and memory mediated after BDNF low expressions by COX5A
It weakens, side illustration COX5A improves effect and closely related (the experimental method ginsengs of BDNF of transgenic mice ability of learning and memory
According to following documents：①Hebda-Bauer EK,Luo J, Watson SJ,Akil H(2007)Female CREB
alphadelta-deficient mice show earlier age-related cognitive deficits than
males.Neuroscience 150:260-272；②Pouzet B, Zhang WN,Feldon J,Rawlins JN(2002)
Hippocampal lesioned rats are able to learn a spatial position using non-
spatial strategies.Behav Brain Res 133(2):279-291)。
5 Electrophysiology technology of embodiment detects hippocampus LTP：
By Fig. 8 a it is found that in the hippocampus CA1 region of record brain piece with the increase of intensity of electric stimulus, the excitability being recorded
The also proportional increasing of postsynaptic potential (fEPSP) signal, illustrates that the cell viability of recording areas brain piece is good, cell state is steady
It is fixed, subsequent experimental can be completed；By Fig. 8 b-8d it is found that giving continuous high intensity electricity after being recorded by 20 minutes quiescent conditions
It stimulates (60 minutes), is overexpressed COX5A Transgenic Mice Brain pieces and shows higher postsynaptic potential (fEPSP), with wild type
It compares, difference has conspicuousness, illustrates that the neural excitability of hippocampus of mice after COX5A is overexpressed increases, reflects indirectly
The raising of memory capability is practised, it is consistent with experimental result before；And low expression BDNF transgenic mices and double cross transgenosis
Mouse hippocampal slices postsynaptic potential (fEPSP) is remarkably decreased, and especially low expression BDNF transgenic mices group, double cross turns base
Because mouse group is compared with low expression BDNF transgenic mice groups, fEPSP increases；Explanation：The mouse Nerve that COX5A is mediated can
Excitability increase is closely related with BDNF, may realize that (experimental method is with reference to following documents by the adjusting to BDNF accesses：
①Hebda-Bauer EK,Luo J,Watson SJ,Akil H(2007)Female CREB alphadelta-
deficient mice show earlier age-related cognitive deficits than males.
Neuroscience 150:260-272；②Pouzet B,Zhang WN,Feldon J,Rawlins JN(2002)
Hippocampal lesioned rats are able to learn a spatial position using non-
spatial strategies.Behav Brain Res 133(2):279-291)。
The morphological change that 6 immunofluorescence dyeing of embodiment evaluates transgenic mice hippocampus detects COX5A and BDNF thin
The sub- positioning of intracellular：
Take COX5A high expression/BDNF low expression double cross F1 generation trangenic mices, general anaesthesia, perfusion materials, position of drawing materials
For entire central nervous system and spinal cord, taking-up tissue, which is placed in 4% paraformaldehyde, fixes 24 hours, gradient sucrose dehydration, system
Make 20- μm of frozen section, follows these steps to complete immunohistochemistry detection：0.01M PBS rinse 5min, totally 3 times；Addition contains
37 DEG C of incubation 30min of closing of 5% sheep blood serum and 0.3% Triton X-100；Primary antibody COX5A (1 is used respectively:200,Santa
Cruz)、NeuN(1:500,Chemicon)、GFAP(1:500,Santa Cruz)、NF-100(1:4,000,
), and BDNF (1 Immunostar:1,000, Chemicon), 4 DEG C of overnight incubations；0.01M PBS are rinsed 3 times, each 5min；
37 DEG C of incubation 2h of fluorescence secondary antibody；Nucleus is redyed with the Antifade Mounting Medium (Beyotime) containing DAPI.
Microscopically observation is taken pictures.
As shown in Figure 9, COX5A and BDNF is collectively resided in neuron, and predominantly in endochylema, i.e., COX5A passes through right
During the adjusting of BDNF has participated in learning and memory of little mouse improvement.
The change of embodiment 7 Golgi dyeing detection transgenic mice hippocampal neuron and spinous process
Concrete operation step is：Random to take each group trangenic mice and littermate control wild-type mice, every group each 3, routine is numb
Liquor-saturated animal；Through the left heart be pre-chilled saline infusions flush three times, each 30ml, rinse net blood until clarification；At once it takes
Go out brain tissue, brain tissue is cut into the tissue block of 0.3 cm thick with aseptic operation knife；Use FD Rapid Golgi stain
Kits (FD Neuro Technologies, Baltimore, MD, USA) kit carries out dyeing detection.The god of Golji dyeing
Through meta appraisal method reference literature report standard (A.N.Hoffman, A.Krigbaum, J.B.Ortiz, A.Mika,
K.M.Hutchinson,H. A.Bimonte-Nelson and C.D.(2011)Conrad Recovery after
chronic stress within spatial reference and working memory domains:
correspondence with hippocampal morphology.European Journal of Neuroscience,
34；1023-1030.), evaluation object can be just selected as by only meeting the hippocampal neuron of the following conditions：I) entire god
It is impregnated with and is coloured by dyestuff through first cell space and protrusion, without what is blocked；Ii) selected neuron and the neuron of surrounding are separated by
A certain distance, it is relatively independent；Iii) selected neuron is located at Hippocampal CA 1.Every animal must possess at least three
Meet the neuron of conditions above, could be used to evaluate.Measure the length of each neuron top (distal end) and Proximal dendrites simultaneously
The quantity of branch is counted.
As shown in Figure 10, COX5A is overexpressed the neuron length and spinous process that can dramatically increase transgenic mice hippocampus
Quantity, this is the material base of learning and memory, also explains COX5A and is overexpressed transgenic mice better than with monthly age wild-type mice
Learning and memory cognitive performance；And under the background for being overexpressed COX5A after BDNF low expressions, double transgenic mouse hippocampal neuron
Number of projection significantly reduces, consistent with the water maze test result of bi-transgenic mice, illustrates that COX5A may pass through adjusting
BDNF realizes the protrusion quantity for improving mouse hippocampal neuron, and then influences the cognitive function using animal.
Embodiment 8 extracts each lines transgenic hippocampus of mice mitochondria, carries out the measurement of mitochondria activity and ATP contents：
Use Mitochondria preparation kit (Sigma, Saint Louis, Missouri, USA) reagent
Box prepare transgenosis hippocampus of mice mitochondria, Cytochrome c Oxidase kit (Sigma, Saint Louis,
Missouri, USA) activity of the kit for detecting Mitochondrial cytochrome c oxidase；ATP Colorimetric/
Luminescence Assay kit(BioVision Incorporated,Milpitas,South Milpitas
Blvd.Milpitas, CA) for detecting ATP contents in mitochondria, operating procedure is indicated according to the specification that reagent quotient provides
It carries out.
As shown in Figure 11, COX5A, which is overexpressed, significantly increases trangenic mice hippocampus mitochondria activity and ATP contents, and
It is overexpressed under the background of COX5A after low expression BDNF, double transgenic mouse hippocampus mitochondria activity reduces, ATP contents are reduced, and says
Bright COX5A plays a role the regulation and control possibly relied on to BDNF.
Embodiment 9COX5A, which plays a role, is to rely on the verification of BDNF signal paths
Take 6 monthly ages, 18 monthly ages COX5A be overexpressed trangenic mice hippocampus, with Western blot methods detect hippocampus in BDNF,
The change of BDNF associated signal paths molecules ERK 1/2 and its phosphorylation level, the specific steps are:Each group hippocampal tissue is put into
Contain 0.05M Tris-HCl (pH 7.4, Amresco), 0.5 M EDTA (Amresco), 30%TritonX-100
(Amresco), even under ice bath in the lysate of NaCl (Amresco), 10%SDS (Sigma) and 1mM PMSF (Amresco)
Slurry, until being visible by naked eyes tissue block；It centrifuges, supernatant, BCA (Sigma, St.Louis, MO, USA) kit is taken to detect egg
Bai Hanliang；Each sample takes 50 μ g Protein Detections BDNF, ERK1/2 and the protein expression of p-ERK1/2,12%SDS-PAGE electricity
Swimming, transferring film, closing, primary antibody antibody incubation include BDNF (1：800, Abcam), (1 ERK1/2:500, Abcam) and p-ERK1/2
(1:1000, Abcam), secondary antibody is incubated, chemoluminescence method detects BDNF, ERK1/2 and p-ERK1/2 relative expression quantity, GAPDH
As internal reference, calculate the ratio of each group destination protein trace band and GAPDH integral optical density values, obtain each sample B DNF,
The relative expression quantity of ERK1/2 and p-ERK1/2 albumen.
As shown in Figure 12, BDNF, BDNF correlation are believed in (6 monthly ages, 18 monthly ages) trangenic mice hippocampus after COX5A is overexpressed
Number pathway molecule ERK1/2 and its phosphorylation level dramatically increase compared with wild type group, especially more aobvious with 18 months age groups
It writes, it is possible to understand that the activity of the increase of age in years and the BDNF signal paths of reduction can be substantially reduced after being overexpressed as COX5A,
Improve cognition, anti-aging effect to play it.
Above description has shown and described several preferred embodiments of invention, but as previously described, it should be understood that invention is not
It is confined to form disclosed herein, is not to be taken as excluding other embodiments, and can be used for various other combinations, modification
And environment, and can be carried out by the above teachings or related fields of technology or knowledge in the scope of the invention is set forth herein
Change.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of invention, then should all be weighed appended by invention
In the protection domain that profit requires.
SEQUENCE LISTING
<110>Kunming Medical University
<120>A kind of construction method of low expression BDNF transgenic mouse models and application
<130> 2018
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>Artificial sequence（Artificial sequence）
<400> 1
gtcaatgggt ggagtattta cg 22
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence（Artificial sequence）
<400> 2
gcttatatag acctcccacc gt 22
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence（Artificial sequence）
<400> 3
tgtgacagta ttagcgagtg ggt 23
<210> 4
<211> 22
<212> DNA
<213>Artificial sequence（Artificial sequence）
<400> 4
tacgattggg tagttcggca tt 22
Claims (8)
1. a kind of construction method of low expression BDNF transgenic mouse models, which is characterized in that include the following steps：
The structure of step 1, CMV-EmGFP-siRNA-BDNF low expression transgenic fragments：Silence expression vector CMV-EmGFP-
SiRNA-BDNF purchases are used in combination the software that the said firm website provides to be directed to target gene BDNF from Invitrogen companies
(GeneID:12064) target site, wherein target site CCAAGTGTAATCCCATGGGTT designs the plasmid of silence, then
Plasmid construction is completed by calm and peaceful company of Sino-U.S.；
Step 2, with the silence plasmid transfection 293T cells built, it is best then to filter out silencing efficiency with the method for RT-PCR
's；
Step 3, with AvrII that the highest silence plasmid of jamming effectiveness is linear, acquisition transgenic fragment, adjustment concentration to 5ng/
μ L are used for microinjection；
Step 4 linearizes the transgenic fragment built, and transgenic mice is made with microinjection；PCR identifications are positive to be turned
DNA rat.
2. construction method according to claim 1, which is characterized in that it is small that the microinjection in step 4 makes transgenosis
Mouse method includes following experimental procedure：
A, super several ovulation induction operations, i.e., first day injection pregnant mare serum gonadotrop(h)in (PMSG) 10IU/ only, human chorionic are injected after 48 hours
Film promoting sexual gland hormone 10IU/ is only；Meanwhile choosing 6 weeks C57BL/6 mouse of health male and the above-mentioned female for receiving ovulation induction operation
Mouse 1:1 mates mating, observes cloudy bolt situation, has the mouse of cloudy bolt to propose for use, spare as ovum mouse；
B, ovum is taken：It will upwards be placed for ovum mouse anesthesia postabdomen, preserved skin exposes abdomen, and skin is successively detached with scissors and tweezers
Skin, fascia, muscle, exposure ovary, fallopian tubal and uterus, detach fallopian tubal, fallopian tubal are placed into M2 culture mediums with tweezers,
The ampulla that fallopian tubal is opened under disecting microscope, makes ovum flow in culture solution；The hyalomitome of 1mg/ml is added in culture solution
Sour enzyme is used in combination M2 culture mediums to rinse 3-4 times, removes granular cell；It is observed under the microscope, to fertilized eggs and other cells
Differentiated, because second polar body is discharged in fertilized eggs, and unfertilized egg and other ovum for becoming abnormal morphology can be easy to carry out
Difference；The fertilized eggs chosen move on in the plastic ware for containing M2 culture medium drops, move in carbon dioxide incubator culture until by
Until smart ovum is suitble to injection；
C. microinjection：The fertilized eggs that the transgene carrier segment of linearisation is injected into C57BL/6 mouse by microinjection are male
It in property protokaryon, observes under the microscope, selects that cell is full, and oolemma is clear, the high-visible fertilized eggs of male pronucleus are for use；
It is made the culture drop and transgene carrier segment solution drop of 20 fertilized eggs on glass slide, is fixed on objective table, uses
It holds ovum suction pipe to be fixed fertilized eggs, glass pipette injects transgene carrier segment solution drop, it is slowly injected to male
Protokaryon collects fertilized eggs, a night is cultivated in 37 DEG C of carbon dioxide incubators after injection；
D. it transplants：Pseudopregnant mouse is anaesthetized, operation takes out ovary and connects fallopian tubal, is fixed with fatty tweezer, is found under the microscope
Fallopian tubal is open, and under microscope, chooses and is split into bicelluar fertilized eggs, spare；Above-mentioned fertilized eggs are drawn, transplanting nozzle is inserted
Entering fallopian tubal mouth, is gently blown into the liquid in grafts, it is seen that ampulla of uterine tube expands and can be clearly seen that three bubbles,
It transplants successfully；Ovary is put back into abdominal cavity, layer-by-layer suture together with fallopian tubal.
3. construction method according to claim 2, which is characterized in that the temperature of the carbon dioxide incubator is 37 DEG C,
Contain 5% carbon dioxide, 95% air.
4. construction method according to claim 1, which is characterized in that the PCR in the step 4 identifies positive transgenic
Mouse is specially：
I), PCR methods identify F1 generation positive transgenic mouse, and positive is as experimental group, and negative patient is as brood wild type control；
Ii it) draws materials：The F1 generation positive transgenic mouse male and female that each strain head builds mouse are unlimited, and each strain 7 takes rapidly after anesthesia
Going out cerebral cortex and hippocampus, each position is divided into two parts, and portion is detected for RT-PCR, another is detected for protein expression,
After precooling normal saline flushing is clean, it is respectively put into clean EP pipes and freezes or carry out immediately experiment detection for -80 DEG C.
Iii) RT-PCR methods detect F1 generation positive BDNF low expression trangenic mices：After taking tissue, by 150mg/1.5ml TRIZOL
Ratio, be put into the homogenizer of the TRIZOL reagents of pre-add precooling, under ice bath homogenate to not viscous without particle, gradually centrifugation point
From, extraction RNA；After the RNA newly extracted the processed distilled water dissolvings of DEPC, UV spectrophotometer measuring RNA sample
Purity and concentration；2 μ g RNA are taken to be used with Reverse Transcriptase kit using RNA as templated synthesis cDNA chains for reverse transcription reaction
It is RevertAidTMThe cDNA of M-MuLV Reverse Transciptase enzyme systems, synthesis is directly used in PCR detection reactions；
Add deionized water to supply 25 μ l reaction systems, 94 DEG C 5 minutes, 94 DEG C 1 minute, 55 DEG C 1 minute, 72 DEG C 1 minute totally 35
Cycle, last 72 DEG C 5 minutes, complete PCR detection reaction；PCR product carries out 1% agarose gel electrophoresis detection；It is with GAPDH
Reference gene, same gene amplified production carries out on same gel when electrophoresis detection, with the gel imager of BIO-RAD companies
Absorb gel images under UV mode and carry out electrophoretic band integral optical density analysis, calculate each group BDNF genes band with
The ratio of GAPDH integral optical density values obtains the relative expression quantity of each sample B DNF genes.
Iv) the expression of the detection of Elisa methods the F1 generation positive BDNF low expression trangenic mice cortex and hippocampus BDNF.Take tissue
Afterwards, each tissue is put into containing pH 7.4,0.05M Tris-HCl, 0.5M EDTA, 30%TritonX-100, NaCl, 10%
It is homogenized under ice bath in the lysate of SDS and 1mM PMSF, until being visible by naked eyes tissue block；It centrifuges, take supernatant, BCA reagents
Box detects protein content；BDNF-ELISA kit kits detect the BDNF contents of each sample；
It takes individual mouse genotypes cortex and hippocampus to carry out the detection of expression of BDNF, finally determines BDNF expression rate inhibiting rate highests
Strain, mass propagation is spare.
5. construction method according to claim 4, which is characterized in that the PCR detects reaction system and is specially：Each
Sample takes 1.5 μ l of cDNA templates, and 2 × PCR Master Mix (Fermentals) 12.5 μ l, each 0.5 μ of upstream and downstream primer is added
l。
6. construction method according to claim 5, which is characterized in that the sense primer：5'
TGTGACAGTATTAGCGAGTGGGT 3', nucleotide sequence is as shown in SEQ ID NO.3；
Downstream primer：5'TACGATTGGGTAGTTCGGCATT 3', nucleotide sequence is as shown in SEQ ID NO.4.
7. the low expression BDNF transgenic mouse models that the construction method in claim 1-6 described in any claim obtains.
8. the low expression BDNF transgenic mouse models described in claim 7 are preparing answering in preventing or treating brain aging drug
With.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810171687.3A CN108588125A (en) | 2018-03-01 | 2018-03-01 | A kind of low expression BDNF transgenic mouse models and its construction method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810171687.3A CN108588125A (en) | 2018-03-01 | 2018-03-01 | A kind of low expression BDNF transgenic mouse models and its construction method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108588125A true CN108588125A (en) | 2018-09-28 |
Family
ID=63625585
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810171687.3A Withdrawn CN108588125A (en) | 2018-03-01 | 2018-03-01 | A kind of low expression BDNF transgenic mouse models and its construction method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108588125A (en) |
-
2018
- 2018-03-01 CN CN201810171687.3A patent/CN108588125A/en not_active Withdrawn
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Liu et al. | Characterization of Wnt signaling components and activation of the Wnt canonical pathway in the murine retina | |
| Petkau et al. | Progranulin expression in the developing and adult murine brain | |
| Rosenmann et al. | A novel transgenic mouse expressing double mutant tau driven by its natural promoter exhibits tauopathy characteristics | |
| Liao et al. | Histamine H1 receptors in neural stem cells are required for the promotion of neurogenesis conferred by H3 receptor antagonism following traumatic brain injury | |
| Hayworth et al. | Induction of neuregulin signaling in mouse schwann cells in vivo mimics responses to denervation | |
| Kang et al. | Regulation of the intermediate filament protein nestin at rodent neuromuscular junctions by innervation and activity | |
| Marcus et al. | Retinal axon divergence in the optic chiasm: midline cells are unaffected by the albino mutation | |
| Chu et al. | Neonatal whisker clipping alters behavior, neuronal structure and neural activity in adult rats | |
| CN109152799A (en) | Pancreatic stem cells and application thereof | |
| CA2392051A1 (en) | Transgenic mice expressing fluorescent protein under the control of the nestin promoter | |
| CN108396036A (en) | A kind of overexpression COX5A transgenic mouse models and its construction method and application | |
| CN108315350A (en) | It is overexpressed COX5A/ low expression BDNF transgenic mouse models and its construction method and application | |
| Li et al. | Intravital imaging of neocortical heterotopia reveals aberrant axonal pathfinding and myelination around ectopic neurons | |
| CN108588125A (en) | A kind of low expression BDNF transgenic mouse models and its construction method and application | |
| US11653636B2 (en) | Method of making a rat model of retinal degeneration and rat model made thereby | |
| KR20180037449A (en) | Brain tumor animal model and the Use thereof | |
| Thomasen et al. | SorCS2 binds progranulin to regulate motor neuron development | |
| CN110373426A (en) | Nonobese diabetes animal model, construction method and its application | |
| Huang et al. | In Vivo Electroporation and Time‐Lapse Imaging of the Rostral Migratory Stream in Developing Rodent Brain | |
| CN103083645A (en) | Application of neuregulin 1 and acceptor thereof as target for preparing and screening anti-epileptic drugs | |
| Yu et al. | Sacral neural crest-independent origin of the enteric nervous system in mouse | |
| Corona et al. | Methods to Assess the Role of Neurogenesis in Reproductive Behaviors of Birds, Rats, and Sheep | |
| Yu | Functional analysis of the cis-regulatory elements I56i, I56ii and I12b that control Dlx gene expression in the developing forebrain of mouse and zebrafish | |
| Ali | Mef2c transcription factor is required for the development of medium spiny neurons of the mouse striatum | |
| Moore | Investigating the role of MLLT11 in the development of cortical hem derivatives and hippocampal neurogenesis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20180928 |