WO1996006927A1 - Transgenic animal expressing a familial form of human amyloid precursor protein - Google Patents
Transgenic animal expressing a familial form of human amyloid precursor protein Download PDFInfo
- Publication number
- WO1996006927A1 WO1996006927A1 PCT/US1995/010920 US9510920W WO9606927A1 WO 1996006927 A1 WO1996006927 A1 WO 1996006927A1 US 9510920 W US9510920 W US 9510920W WO 9606927 A1 WO9606927 A1 WO 9606927A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- app
- human
- animal
- transgenic
- amyloid precursor
- Prior art date
Links
- 101000823051 Homo sapiens Amyloid-beta precursor protein Proteins 0.000 title claims abstract description 36
- 230000009261 transgenic effect Effects 0.000 title claims description 50
- 241001465754 Metazoa Species 0.000 title claims description 48
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 39
- 150000001875 compounds Chemical class 0.000 claims abstract description 17
- 230000014509 gene expression Effects 0.000 claims description 36
- 108700019146 Transgenes Proteins 0.000 claims description 27
- 238000000034 method Methods 0.000 claims description 21
- 101150052863 THY1 gene Proteins 0.000 claims description 13
- 238000004220 aggregation Methods 0.000 claims description 4
- 230000002776 aggregation Effects 0.000 claims description 4
- 238000005259 measurement Methods 0.000 claims description 4
- 241000124008 Mammalia Species 0.000 claims description 3
- 230000004952 protein activity Effects 0.000 claims 2
- 241000283984 Rodentia Species 0.000 claims 1
- 208000024827 Alzheimer disease Diseases 0.000 abstract description 39
- 238000011830 transgenic mouse model Methods 0.000 abstract description 20
- 241000699660 Mus musculus Species 0.000 abstract description 14
- 208000010877 cognitive disease Diseases 0.000 abstract 1
- 101710137189 Amyloid-beta A4 protein Proteins 0.000 description 62
- 101710151993 Amyloid-beta precursor protein Proteins 0.000 description 62
- 102100022704 Amyloid-beta precursor protein Human genes 0.000 description 62
- 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 59
- 210000004556 brain Anatomy 0.000 description 34
- 108020004414 DNA Proteins 0.000 description 30
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 23
- 108020004999 messenger RNA Proteins 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 19
- 239000002299 complementary DNA Substances 0.000 description 15
- 102000046783 human APP Human genes 0.000 description 15
- 102000004169 proteins and genes Human genes 0.000 description 14
- 239000000523 sample Substances 0.000 description 13
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 11
- 108010029485 Protein Isoforms Proteins 0.000 description 11
- 102000001708 Protein Isoforms Human genes 0.000 description 11
- 150000001413 amino acids Chemical class 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 11
- 239000012634 fragment Substances 0.000 description 11
- 238000009396 hybridization Methods 0.000 description 11
- 229940024606 amino acid Drugs 0.000 description 9
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 9
- 239000002953 phosphate buffered saline Substances 0.000 description 9
- 108091008146 restriction endonucleases Proteins 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 241000699670 Mus sp. Species 0.000 description 8
- 230000002068 genetic effect Effects 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 7
- 239000002585 base Substances 0.000 description 7
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 7
- 101150031224 app gene Proteins 0.000 description 6
- 210000004602 germ cell Anatomy 0.000 description 6
- 238000007901 in situ hybridization Methods 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 201000010374 Down Syndrome Diseases 0.000 description 5
- 239000004677 Nylon Substances 0.000 description 5
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 5
- 108091081024 Start codon Proteins 0.000 description 5
- 239000011543 agarose gel Substances 0.000 description 5
- 210000003169 central nervous system Anatomy 0.000 description 5
- 210000000349 chromosome Anatomy 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 210000001320 hippocampus Anatomy 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 238000000520 microinjection Methods 0.000 description 5
- 230000001537 neural effect Effects 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 150000007523 nucleic acids Chemical class 0.000 description 5
- 229920001778 nylon Polymers 0.000 description 5
- 239000002751 oligonucleotide probe Substances 0.000 description 5
- 238000006467 substitution reaction Methods 0.000 description 5
- 230000014621 translational initiation Effects 0.000 description 5
- 208000037259 Amyloid Plaque Diseases 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 108010017826 DNA Polymerase I Proteins 0.000 description 4
- 102000004594 DNA Polymerase I Human genes 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- QWTDNUCVQCZILF-UHFFFAOYSA-N isopentane Chemical compound CCC(C)C QWTDNUCVQCZILF-UHFFFAOYSA-N 0.000 description 4
- 210000002569 neuron Anatomy 0.000 description 4
- 230000007171 neuropathology Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 102000002659 Amyloid Precursor Protein Secretases Human genes 0.000 description 3
- 108010043324 Amyloid Precursor Protein Secretases Proteins 0.000 description 3
- 102000009091 Amyloidogenic Proteins Human genes 0.000 description 3
- 108010048112 Amyloidogenic Proteins Proteins 0.000 description 3
- 101000823042 Mus musculus Amyloid-beta precursor protein Proteins 0.000 description 3
- 238000000636 Northern blotting Methods 0.000 description 3
- 229930040373 Paraformaldehyde Natural products 0.000 description 3
- 206010038997 Retroviral infections Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000002459 blastocyst Anatomy 0.000 description 3
- 210000001109 blastomere Anatomy 0.000 description 3
- 210000005013 brain tissue Anatomy 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 210000002257 embryonic structure Anatomy 0.000 description 3
- 239000013604 expression vector Substances 0.000 description 3
- 208000015181 infectious disease Diseases 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 230000002018 overexpression Effects 0.000 description 3
- 229920002866 paraformaldehyde Polymers 0.000 description 3
- 230000009257 reactivity Effects 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 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 description 2
- 108010090849 Amyloid beta-Peptides Proteins 0.000 description 2
- 102000013455 Amyloid beta-Peptides Human genes 0.000 description 2
- 108010053481 Antifreeze Proteins Proteins 0.000 description 2
- 241000972773 Aulopiformes Species 0.000 description 2
- 108091026890 Coding region Proteins 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 102100034323 Disintegrin and metalloproteinase domain-containing protein 2 Human genes 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101100268553 Homo sapiens APP gene Proteins 0.000 description 2
- 101000780288 Homo sapiens Disintegrin and metalloproteinase domain-containing protein 2 Proteins 0.000 description 2
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 2
- 239000007993 MOPS buffer Substances 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 108091034057 RNA (poly(A)) Proteins 0.000 description 2
- 238000010240 RT-PCR analysis Methods 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- OIRDTQYFTABQOQ-KQYNXXCUSA-N adenosine Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OIRDTQYFTABQOQ-KQYNXXCUSA-N 0.000 description 2
- 210000004727 amygdala Anatomy 0.000 description 2
- 230000003942 amyloidogenic effect Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 230000002528 anti-freeze Effects 0.000 description 2
- 238000000211 autoradiogram Methods 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- AIYUHDOJVYHVIT-UHFFFAOYSA-M caesium chloride Chemical compound [Cl-].[Cs+] AIYUHDOJVYHVIT-UHFFFAOYSA-M 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- SUYVUBYJARFZHO-RRKCRQDMSA-N dATP Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-RRKCRQDMSA-N 0.000 description 2
- SUYVUBYJARFZHO-UHFFFAOYSA-N dATP Natural products C1=NC=2C(N)=NC=NC=2N1C1CC(O)C(COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 SUYVUBYJARFZHO-UHFFFAOYSA-N 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- AFABGHUZZDYHJO-UHFFFAOYSA-N dimethyl butane Natural products CCCC(C)C AFABGHUZZDYHJO-UHFFFAOYSA-N 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 2
- 229960005542 ethidium bromide Drugs 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000010348 incorporation Methods 0.000 description 2
- 238000007373 indentation Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 229960000310 isoleucine Drugs 0.000 description 2
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 2
- 125000000741 isoleucyl group Chemical group [H]N([H])C(C(C([H])([H])[H])C([H])([H])C([H])([H])[H])C(=O)O* 0.000 description 2
- 210000003734 kidney Anatomy 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 210000000478 neocortex Anatomy 0.000 description 2
- 210000002241 neurite Anatomy 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 230000006337 proteolytic cleavage Effects 0.000 description 2
- 235000019515 salmon Nutrition 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 239000004474 valine Substances 0.000 description 2
- OXIKLRTYAYRAOE-CMDGGOBGSA-N (e)-3-(1-benzyl-3-pyridin-3-ylpyrazol-4-yl)prop-2-enoic acid Chemical compound N1=C(C=2C=NC=CC=2)C(/C=C/C(=O)O)=CN1CC1=CC=CC=C1 OXIKLRTYAYRAOE-CMDGGOBGSA-N 0.000 description 1
- 108020005065 3' Flanking Region Proteins 0.000 description 1
- 102100022900 Actin, cytoplasmic 1 Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 102100022524 Alpha-1-antichymotrypsin Human genes 0.000 description 1
- 102000007592 Apolipoproteins Human genes 0.000 description 1
- 108010071619 Apolipoproteins Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
- 102100033215 DNA nucleotidylexotransferase Human genes 0.000 description 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 206010012289 Dementia Diseases 0.000 description 1
- 108010054576 Deoxyribonuclease EcoRI Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108091029865 Exogenous DNA Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 101710132360 Kunitz-type serine protease inhibitor Proteins 0.000 description 1
- 108010021466 Mutant Proteins Proteins 0.000 description 1
- 102000008300 Mutant Proteins Human genes 0.000 description 1
- 108091008604 NGF receptors Proteins 0.000 description 1
- 238000011887 Necropsy Methods 0.000 description 1
- 102000007339 Nerve Growth Factor Receptors Human genes 0.000 description 1
- 208000012902 Nervous system disease Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- 108091000054 Prion Proteins 0.000 description 1
- 102000029797 Prion Human genes 0.000 description 1
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 108020005093 RNA Precursors Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 102100037968 Ribonuclease inhibitor Human genes 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical group [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 208000037280 Trisomy Diseases 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 102000004243 Tubulin Human genes 0.000 description 1
- 108090000704 Tubulin Proteins 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 102000044159 Ubiquitin Human genes 0.000 description 1
- 241001147416 Ursus maritimus Species 0.000 description 1
- PNVLWFYAPWAQMU-CIUDSAMLSA-N Val-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H](N)C(C)C PNVLWFYAPWAQMU-CIUDSAMLSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 108010091628 alpha 1-Antichymotrypsin Proteins 0.000 description 1
- 108010064397 amyloid beta-protein (1-40) Proteins 0.000 description 1
- 210000003295 arcuate nucleus Anatomy 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 210000001130 astrocyte Anatomy 0.000 description 1
- 238000011888 autopsy Methods 0.000 description 1
- 108010058966 bacteriophage T7 induced DNA polymerase Proteins 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 208000036815 beta tubulin Diseases 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Natural products N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 229920002301 cellulose acetate Polymers 0.000 description 1
- 210000003710 cerebral cortex Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 230000003920 cognitive function Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 208000025688 early-onset autosomal dominant Alzheimer disease Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 208000015756 familial Alzheimer disease Diseases 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 238000005558 fluorometry Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000002980 germ line cell Anatomy 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 210000004939 midgestation embryo Anatomy 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 230000000626 neurodegenerative effect Effects 0.000 description 1
- 210000002682 neurofibrillary tangle Anatomy 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000006764 neuronal dysfunction Effects 0.000 description 1
- 230000007121 neuropathological change Effects 0.000 description 1
- 230000000508 neurotrophic effect Effects 0.000 description 1
- 229940046166 oligodeoxynucleotide Drugs 0.000 description 1
- 210000003101 oviduct Anatomy 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 108010024226 placental ribonuclease inhibitor Proteins 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- HJRIWDYVYNNCFY-UHFFFAOYSA-M potassium;dimethylarsinate Chemical compound [K+].C[As](C)([O-])=O HJRIWDYVYNNCFY-UHFFFAOYSA-M 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000002360 prefrontal effect Effects 0.000 description 1
- 210000003814 preoptic area Anatomy 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000002331 protein detection Methods 0.000 description 1
- 239000003531 protein hydrolysate Substances 0.000 description 1
- 210000002637 putamen Anatomy 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000012340 reverse transcriptase PCR Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000011808 rodent model Methods 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 229910021653 sulphate ion Inorganic materials 0.000 description 1
- 210000003863 superior colliculi Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- 210000001103 thalamus Anatomy 0.000 description 1
- 210000001541 thymus gland Anatomy 0.000 description 1
- 231100000820 toxicity test Toxicity 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000011820 transgenic animal model Methods 0.000 description 1
- 108091005703 transmembrane proteins Proteins 0.000 description 1
- 102000035160 transmembrane proteins Human genes 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 210000004340 zona pellucida Anatomy 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0278—Knock-in vertebrates, e.g. humanised 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
- 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
-
- 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
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
- A01K2267/0312—Animal model for Alzheimer's disease
-
- 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
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
Definitions
- the present invention relates to the brain-specific expression of the Familial Alzheimer's disease (V-I) variant of the human amyloid precursor protein in transgenic mice.
- V-I Familial Alzheimer's disease
- Relevant regions of the APP including the signal peptide (Sp), Kunitz protease inhibitor (KPI) and ⁇ A4 domains and the position of the V-I FAD mutation are indicated.
- the position of primers is shown by arrows. The primers were employed across the 66 bp SV40 small t intron (indentation in the physical map) to amplify a 319 bp fragment from the APP 751 FAD iriRNA by RT-PCR.
- FIG. 1 RNA analysis of APP 751 FAD founders.
- Upper panel Northern blot analysis of APP 751 FAD transgenic (Tg) and age-matched, non-transgenic (NTg) Fl mice. Samples (about 4 ⁇ g) of poly A + mRNA from brain (B), kidney (K) and intestine (I) were resolved on a MOPS formaldehyde gel and transferred to a nylon membrane. The RNA was hybridized with 32p labeled transgene specific SV40 sequences. Predominant brain-specific expression was seen in line 14.2 Fl transgenic animals. Line 7.2 and Line 9.3 transgenic animals exhibited a lower level of expression.
- Bottom panel The Northern blot was hybridized with a 32p labeled ⁇ -actin probe and the relative amount of poly A+ mRNA loaded in the different lanes was estimated.
- FIG. 3 RT-PCR analysis of APP751 FAD.
- One microgram of total RNA from various tissues of a perfused transgenic animal (line 14.2) was reverse transcribed and subsequently amplified using AmpliTaq DNA polymerase.
- the amplified products were resolved on a 0.8% agarose gel, blotted to a nylon membrane, and hybridized with S V40 sequences.
- Predominantly APP 751 mRNA expression was found in brain and while expression was negligible in most other tissues.
- Figure 4 Localization of APP 751 FAD RNA by in situ hybridization.
- Computer-generated pseudo-color images of representative in situ hybridization X-ray film autoradiograms show expression of human APP 751 FAD mRNA in various brain areas of the transgenic mice.
- the 14.2 line (rostral to caudal; labeled A, B, C); the 9.3 line (E, F, G); and the 7.2 line (I, J, K).
- the highest level of mRNA expression was found in the brains of mice belonging to the 14.2 line with expression throughout the brain, but was particularly dense in the cortex (C) and hippocampus (h).
- mRNA signals could not be obtained, showing the specificity of the RNA signals obtained with the labeled antisense human APP 751 FAD mRNA probe.
- FIG. 1 APP 751 (V-I) protein analysis by Western blotting.
- the top panel shows reactivity with the 6E10 Ab; the middle panel shows reactivity with CT15 Ab; recognizing human and mouse APP; and the bottom panel shows reactivity with a ⁇ -tubulin control Ab.
- Animal 95 (line 14.2 transgenics) expresses a large quantity of APP 751 protein at levels comparable to the 500 kb YAC derived human APP protein expressed in mice (labeled YAC; Lamb et al., (1993) Nature Genetics 5 * 22-30).
- N+O in the CT15 panel represents the fully modified, glycosylated 751 protein in the line 95 animals.
- Line 9.3 and 7.2 animals (91, 92 and 93, 94, respectively) had lower levels of APP protein.
- Brains from transgenic mice that overexpress the human APP gene were analyzed. Using a monoclonal antibody to the N-terminal end of human APP (Clone 22C1 1 , Boehringer Mannheim; dilution 1 :20), intensely immunoreactive neurons were found in the cortex and hippocampus in the brains of APP751 FAD transgenic 14.2 founder (18 months old).
- Non-transgenic control (15 month old) did not show this staining pattern.
- the intensity of the staining appeared to be age-related.
- This accumulation of APP immunoreactivity in neurons of the transgenic mice appeared to be granular and deposit-like in appearance.
- Figure 7. The 43 amino acid ⁇ -A4 domain of FAD APP
- AD Alzheimer's disease
- Incidence of the disease increases from less than 1 % at age 60-65, to 5% at age 75, to as high as 47% at age 85.
- 60% to 80% of all cases of dementia in persons over age 65 are caused by AD.
- Afflicted individuals exhibit impaired cognitive function and memory.
- Positive identification of AD requires biopsy or autopsy of the brain.
- AD Alzheimer's disease
- senile plaques consist of extracellular deposits containing a ⁇ -amyloid core surrounded by a halo of dystrophic neurites, glia and astrocytes.
- ⁇ - amyloid deposits are present in neocortex blood vessel walls.
- the major component of senile plaques is a 4 kDa peptide referred to as ⁇ A4, that is proteolytically cleaved from a larger 120 kDa amyloid precursor protein (APP).
- APP amyloid precursor protein
- Other components of the plaques include ubiquitin, amyloid P, Apo E, interleukin-1 , and ⁇ - 1 -antichymotrypsin.
- ⁇ A4 amyloid precursor protein
- FAD early onset familial AD
- Genetic analysis of FAD families has established that the disorder is inherited as a dominant autosomal gene defect, which maps to the long arm of chromosome 21 and is closely linked to the APP gene. These findings are consistent with genetic data obtained from the analysis of Down syndrome patients.
- FAD families have also been identified in which an early onset of AD is strictly correlated with the presence of a mutation in exon 17 of the APP gene at amino acid 717 (Val-Ile). This mutation within the transmembrane spanning domain of the APP co- segregates with FAD.
- APP717 FAD families strongly supports the hypothesis that the APP717 gene in these FAD families is directly positioned in the pathway of AD progression.
- the APP gene is approximately 400 kb in length and encodes a glycosylated, transmembrane protein which may be involved in cell-cell interaction.
- the APP gene has at least 19 exons that create at least 5 distinct APP transcripts by alternative splicing.
- the predominant transcripts encode proteins of 695, 751 and 770 amino acids (these major forms of APP are referred as APP 695, APP 751 and APP 770, respectively).
- Transcripts for APP 695 are enriched in the brain.
- APP 751 and APP 770 mRNA species predominate in peripheral tissues. All three isoforms contain the 42 amino acid ⁇ A4 domain. APP isoforms 751 and 770 contain an additional 56 amino acid insert encoding the kunitz type serine protease inhibitor (KPI). APP is proteolytically metabolized by at least two pathways. One pathway involves an ⁇ -secretase cleavage site positioned between Lys 16 and Leu 17 of ⁇ A4 domain; proteolytic cleavage at this site precludes the formation of amyloidogenic ⁇ A4 entity.
- KPI serine protease inhibitor
- the second pathway produces intact, amyloidogenic ⁇ A4 (39-42 amino acids) by proteolytic cleavages at the ⁇ - and ⁇ -secretase cleavage sites of the full-length APP molecule.
- the ⁇ A4 laden senile deposits seen in AD patients are also found in aged humans and other aged mammals including non-human primates, polar bears and dogs. However, other aged mammals, such as laboratory rats and mice, do not normally develop ⁇ A4 deposits.
- the lack of a cost-effective, experimental animal model mimicking human pathogenesis hinders the understanding AD neuropathology and developing therapeutics against AD. Transgenic technology may offer a suitable alternative to this problem.
- transgenic mouse which exhibits neuropathology due to the overexpression of the human FAD APP 751 (V-I) isoform.
- the FAD APP 751 (V-I) isoform is specifically overexpressed in the brain of patients with familial AD and, therefore, represents a useful and novel model, distinct from those established by others.
- the transgenic mice of the present invention are useful in the identification of new targets in AD since the progression of the disease can be followed gradually.
- the mice of the present invention may be used in the identification of compounds that affect the role of FAD APP 751 (V-I) in neuronal dysfunction and compounds that affect formation of ⁇ A4 precipitates and/or ⁇ A4 function.
- APP 751 (V-I) cDNA representing a familial (APP717) form of AD using a strong neuronal-specific promoter has not been attempted by others and is a unique aspect of the animal model described herein.
- the mouse of the present invention differs from others in that it shows a high steady state expression of APP 751 FAD protein by Western blotting, a unique distribution of APP mRNA in the central nervous system by in situ hybridization, and a unique deposition of intraneural protein APP FAD aggregates.
- a transgenic mouse with brain-specific expression of the familial form of the APP 751 isoform with a valine to isoleucine substitution at amino acid 698 (APP 751 isoform numbering; previously introduced as APP717 based on APP 770 isoform numbering) is provided.
- the transgenic mouse of the invention may be used in the study of AD and disorders involving the central nervous system.
- a transgenic mouse with brain-specific expression of the familial form of the APP 751 isoform with a valine to isoleucine substitution at amino acid 698 is provided.
- the transgenic mouse of the invention may be used in the study of AD and disorders involving the central nervous system.
- the term "animal” is used herein to include all vertebrate animals, except humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages.
- a "transgenic animal” is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection or infection with recombinant virus.
- This introduced DNA molecule may be integrated within a chromosome, or it may be extra-chromosomally replicating DNA.
- the term "germ cell-line transgenic animal” refers to a transgenic animal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If such offspring in fact possess some or all of that information, then they, too, are transgenic animals.
- the information may be foreign to the species of animal to which the recipient belongs, foreign only to the particular individual recipient, or genetic information already possessed by the recipient.
- the introduced gene may be differently expressed compared to the native endogenous gene.
- the genes may be obtained by isolating them from genomic sources by preparation of cDNAs from isolated mRNA templates, by directed synthesis, or by some combination thereof.
- the structural gene must be coupled to a promoter in a functional manner.
- Promoter/regulatory sequences may be used to increase, decrease, regulate or designate to certain tissues or to certain stages of development the expression of a gene.
- the promoter need not be a naturally occurring promoter.
- the human Thy-1 (hThy-1) promoter is used.
- the hThy- 1 promoter preferentially allows expression of the gene of interest within the brain (Gordon, J. et al., (1987) Cell, 50, 445-452).
- the "transgenic non-human animals" of the invention are produced by introducing "transgenes" into the germline of the non-human animal.
- the methods enabling the introduction of DNA into cells are generally available and well-known in the art; however, the generation of a particular type of transgenic animal requires experimentation. Different methods of introducing transgenes could be used.
- the zygote is the best target for microinjection. In the mouse, the male pronucleus reaches the size of approximately 20 ⁇ m in diameter, which allows reproducible injection of 1-2 pL of DNA solution.
- the use of zygotes as a target for gene transfer has a major advantage, in most cases, the injected DNA will be incorporated into the host gene before the first cleavage (Brinster, et al., (1985) Proc. Natl. Acad. Sci. USA 82, 4438- 4442).
- transgenic non-human animal will carry the incorporated transgene. Generally, this will also result in the efficient transmission of the transgene to offspring of the founder since 50% of the germ cells will harbor the transgene.
- Microinjection of zygotes is the preferred method for incorporating transgenes in practicing the invention.
- Retroviral infection can also be used to introduce a transgene into a non-human animal.
- the developing non-human embryo can be cultured in vitro to the blastocyst stage.
- blastomeres may be targets for retroviral infection (Jaenich, R. (1976) Proc. Natl. Acad. Sci. USA 73, 1260-1264).
- Efficient infection of the blastomeres is obtained by enzymatic treatment to remove the zona pellucida (Hogan et al., (1986) in Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).
- the viral vector system used to introduce the transgene is typically a replication-defective retrovirus carrying the transgene (Jahner et al., (1985) Proc. Natl. Acad. Sci. USA 82, 6927-6931 ; Van der Putten et al., (1985) Proc. Natl. Acad. Sci. USA 82, 6148-6152). Transfection is easily and efficiently obtained by culturing the blastomeres on a monolayer of virus-producing cells (Van der Putten, supra; Stewart et al., (1987) EMBO J. 6: 383-388). Alternatively, infection can be performed at a later stage.
- Virus or virus- producing cells can be injected into the blastocoele (Jahner et al., ( 1982) Nature 298: 623-628). Most of the founder animals will be mosaic for the transgene since incorporation occurs only in a subset of the cells which formed the transgenic non-human animal. Furthermore, the founder animal may contain retroviral insertions of the transgene at a variety of positions in the genome; these generally segregate in the - 10 -
- ES cells are obtained from pre -implantation embryos cultured in vitro (Evans, M. J., et al., (1981) Nature 292. 154- 156; Bradley, A., et al, (1984) Nature 3Q9, 255-258; Gossler, et al, (1986) Proc. Natl. Acad. Sci. USA 83, 9065-9069; and Robertson, et al, (1986) Nature 322. 445-448).
- Transgenes can be efficiently introduced into ES cells by DNA transfection or by retrovirus-mediated transduction.
- the resulting transformed ES cells can thereafter be combined with blastocysts from a non-human animal.
- the ES cells colonize the embryo and contribute to the germ line of the resulting chimeric animal (For review see Jaenisch, R. (1988) Science 240, 1468-1474).
- DNA as well as its expression are readily available and well-known in the art. Such methods include, but are not limited to DNA (Southern) hybridization to detect the exogenous DNA, polymerase chain reaction (PCR), polyacrylamide gel electrophoresis (PAGE) and Western blots to detect DNA, RNA and protein.
- the methods include immunological and histochemical techniques to detect amyloid precursor protein and pathology associated with Alzheimer's disease.
- a "transgene” is a DNA sequence introduced into the germline of a non-human animal by way of human intervention such as by way of the methods described below.
- a potential therapeutic compound for AD may be detected by measuring its capacity to block the neurodegenerative effect of APP, to block expression of APP, or to evaluate neurotrophic or other neuronally active compounds in these transgenic mice possibly in combination with different genetic backgrounds or transgenes, providing other susceptibility markers (i.e., cytokines, apolipoprotein overexpression and knock out, protease inhibitors, serum amyloid protein, NGF receptor protein, and prion protein transgenic mice).
- susceptibility markers i.e., cytokines, apolipoprotein overexpression and knock out, protease inhibitors, serum amyloid protein, NGF receptor protein, and prion protein transgenic mice.
- Such compounds will be formulated in accordance with known methods to produce pharmaceutically acceptable compositions. Such compositions may be administered to patients in a variety of standard ways.
- a 1.6 kb Bam HI-Bgl II fragment of pBSHTl (encoding the ATG translation initiation codon) was subcloned into the Bam HI site of pTZl 8u to generate pHZ021a.
- PCR amplification was carried out using pHZ021a as template and 20-mer (T7; 5' primer) and 73-mer (oHZ002; 3' primer with disrupted ATG and several convenient cloning sites) ohgonucleotides.
- OHZ002 5 ' ACGTCGACTCTAGAAGATCTTCGACTCGAGATCGATGGTACCCGGGCAGGTT-
- CikAGCTTCTGGGATCTCAGTC SEQ ID NO : 2 .
- SV40 sequences were inserted into this plasmid downstream of the Thy-1 promoter in a two step procedure; first, by inserting a Bgl II linker at the Sma I restriction enzyme site upstream of the SV40 small t intron of pSV2neo (pHZ023; Southern and Berg, ( 1982) Mol, Appl. Genet. 1 :327) and second by isolating the 1.0 kb Bgl II-Bam HI containing SV40 small t intron and polyadenylation site and ligating it to the Bgl II digested pHZ022.
- pHZ024 the neuronal specific human Thy-1 promoter is separated from the SV40 sequences by a multi-purpose cloning site, permitting the insertion of desired gene(s) or their segments.
- a 2.7 kb Sma I-Cla I restriction fragment encoding the full- length human APP 751 cDNA with the late onset FAD mutation (V-I) was obtained by restriction enzyme digestion of plasmid DA- 12 (a gift of Drs. N. Robakis of Mount Sinai School of Medicine and R. Swanson of MRL, West Point). The ends of the Sma I-Cla I restriction enzyme fragment were made blunt with the Klenow fragment of DNA polymerase I. This fragment was ligated into pHZ024 (pHZ024 was digested with Xho I and the Xho I site was made blunt using the Klenow fragment of DNA polymerase I).
- the human APP 751 cDNA (V>I) was thus placed under the control of the hThy-1 promoter, flanked at its 3' end by the SV40 small t intron and poly A addition site.
- the resulting plasmid is referred to as p4.
- the nucleotide sequence of the coding sequence of the APP 751 cDNA insert of plasmid p4 was entirely confirmed to assure that the entire amino acid sequence was as predicted and a full-length polypeptide would be expressed.
- a 79 bp deletion in the 3' non coding extension of the mRNA of APP 751 was noted.
- the p4 expression vector was purified on a CsCl gradient and was digested at a unique Xba I restriction enzyme site and partially restriction enzyme digested with Eco RI to obtain a 7.1 kb fragment, T-APP751 FAD, which does not contain flanking plasmid sequences.
- the 7.1 kb fragment was purified on a preparative 1 % low melting point agarose gel containing 10 ng/ml ethidium bromide.
- the DNA was visualized using minimal exposure to short-wave UV light and the 7.1 kb band was excised, melted at 65-70°C, phenol/chloroform extracted twice, chloroform extracted once and ethanol precipitated in 0.3 M sodium acetate (pH 5.2) and filtered through a pre-rinsed 0.2 ⁇ m cellulose acetate filter.
- the purified 7.1 kb linear DNA containing the human Thy-1 promoter linked to the human APP 751 FAD cDNA and SV40 small t intron and poly A addition sequences was subsequently microinjected (Figure 1 ).
- DNA modifying enzymes were from Boehringer Mannheim, Inc. DNA sequencing was performed using either Sequenase (U.S. Biochemical, Inc.) or a double stranded DNA Cycle Sequencing Kit (BRL, Inc.). Oligodeoxynucleotides were synthesized on ABI DNA Synthesizer model #381 A. PCR was according to Perkin-Elmer, Corp.
- the p4 DNA construct of Example 1 containing the human APP 751 FAD cDNA under the control of the human Thy-1 promoter was microinjected into the pronucleus of one-cell fertilized mouse embryos obtained from superovulated B6SJL females.
- the optimal concentration of the DNA used for microinjection was the LD50 value derived empirically from toxicity test experiments using several dilutions of the gene construct microinjected into mouse embryos. This LD50 value came out to be 7.5 x 10-9 ⁇ g.
- the embryos injected with 7.5 x 10-9 ⁇ g DNA were then surgically reimplanted into the oviducts of pseudopregnant recipient mice and allowed to develop to term.
- postnatal tail samples were taken by clipping approximately 1 cm of the tail for DNA (Southern) blot analysis to determine the presence of the transgene.
- Necropsies and/or biopsies were performed to collect tissue specimens for histological and expression studies.
- Genomic DNA extracted from tail samples using a Proteinase-K lysis method was quantitated by DNA fluorometry.
- genomic DNA was digested with the restriction enzyme Bam HI, size separated on a 0.8% agarose gel, after which the DNA was transferred to a nylon membrane by Southern blotting.
- the filters were hybridized with transgene-specific 32p labeled SV40 sequences. Hybridization was conducted at 65°C in 6 X SSC, 5 X Denhardt's reagent, 50% Dextran sulphate, 1.2 % SDS, 100 mg/ml denatured, sonicated salmon sperm DNA, and 0.1 M Tris (pH 7.4).
- the gel was repeatedly rinsed in DEPC treated water to remove excess formaldehyde. To assure efficient transfer of the RNA, the gel was soaked in 50 mM NaOH and 10 mM NaCl for 20 minutes and neutralized in 100 mM Tris-HCl (pH 7.5) for 30 minutes. Finally, the RNA was transferred to a nylon membrane in 20 X SSC. To identify the APP 751 FAD transgenic transcripts, the filter was hybridized with transgene-specific SV40 sequences. At least a 10-fold increase in the APP 751 RNA expression levels in the brain of the 14.2 transgenic line was observed in comparison to the other transgenic samples, while control brain samples never showed expression of the transgenes (Figure 2).
- APP transgene expression could not be detected in other tissues from the transgenic animals suggesting neuronal-specific expression of the hThy-1 promoter.
- the ability to detect the APP 751 cDNA in Northern blot analysis indicates that the RNA is abundantly expressed in the central nervous system. Previous reports relied on reverse transcriptase PCR technology, presumably due to the low level of expression of the transgene.
- RT-PCR reverse transcription polymerase chain reaction
- PCR amplification was performed in the presence of the 519-5 (CGGGCTCTCCTGATTATTTATCT; SEQ ID NO: 3) and 519-3 primers (AAAGGCATTCCACCA CTGCT; SEQ ID NO: 4) and components of Gene Amp® RNA PCR Kit (Perkin Elmer Cetus Instruments) according to the manufacturers instructions.
- the primers ( Figure 1 , arrows below the physical map) were designed across the 66 bp small t intron ( Figure 1 ; indentation in physical map) to differentiate amplification of the spliced mRNA from intron containing DNA that might contaminate the RNA preparation and to discriminate RNA precursor transcripts from mRNA.
- the amplification profile included a 2 minute incubation at 95°C for 1 cycle; 1 minute at 95°C and 1 minute at 60°C for 35 cycles and a 7 minute extension cycle at 60°C.
- Amplifications were carried out in a Bios thermal cycler.
- the PCR products were resolved on a 1.5% agarose gel in a Tris Acetate-EDTA buffer and transferred to a nylon membrane and hybridized with SV40 sequences (Figure 3).
- An expected RNA derived 319 bp fragment was generated in brain samples from the F2 progeny of all three independently generated transgenic lines (7.2, 9.3 and 14.2; only line 14.2 results are shown in Figure 3). Amplification signals could not be observed with RNA from a non-transgenic control brain sample.
- Sections were thaw mounted on "Probe On" slides (Fisher Scientific), air-dried thoroughly (approximately lh), fixed in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS; pH7.4) for 5 min., rinsed in PBS for 2 min., delipidated and dehydrated in an ethanol series (50, 70 and 95%) (5 min. each and stored in 95% ethanol at +4°C).
- PBS phosphate buffered saline
- the 'antisense' oligonucleotide probe specific for the human APP 751 mRNA was 40 bases long and was complementary to bases 1138-1 177 of the human APP gene (5'-ACT GGC TGC TGT TGT AGG AAT GGC GCT GCC ACA CAC GGC C -3'; SEQ ID NO: 5) (Ponte et al, (1988), Nature, 331. 525-527). It was synthesized on an Applied Biosystems DNA synthesizers (Model 394) and purified on a 8% polyacrylamide/8M urea preparative sequencing gel. When used in in situ hybridization experiments, this probe gave hybridization signal for APP 751 in human and monkey brain sections, but not in mouse brain sections, indicating its specificity for human APP 751 (no cross hybridization with mouse APP 751).
- the APP 751 probe was 3'-end labeled with [35S]deoxyadenosine 5'-( ⁇ -thiotriphosphate) ([35s]dATP) (1415 Ci/mMol) (New England Nuclear) in a 30:1 molar ratio of [35S]dATP:oligonucleotide using terminal deoxynucleotidyl transferase (25 units; Boehringer Manheim) for 15 min. at 37°C in reaction buffer containing 1 M potassium cacodylate, 125 mM Tris-HCl, 1.25 mg/ml bovine serum albumin (pH 6.6) and 25 mM cobalt chloride.
- Radiolabeled oligonucleotide was separated from unincorporated nucleotides using Sephadex G50 spin columns; 2 ⁇ l of 1 M dithiothreitol (DTT ) was added to the eluate to prevent cross linking of sulfur residues.
- DTT dithiothreitol
- Hybridizations of mouse brain sections were carried out essentially as previously described (Sirinathsinghji et al, (1990), Neuroscience, 34, 675-686; Sirinathsinghji and Dunnett (1993), In Molecular imaging in neuroscience (Ed. Sharif, NA), 43-70).
- In situ hybridization was performed on sections from 3 animals from the 14.2 founder (14.2.5.13, 14.2.3.25 and 14.2.3.57) and two control non-transgenic litter mates (14 2.5.12 and 14.2.3.26) and animals from transgenic lines 7.2 and 9.3 and their non-transgenic littermates.
- Autoradiograms (2-day-exposure) of hybridized sections from all three transgenic animals showed dense homogeneous APP751 mRNA expression throughout the brain. Expression was particularly abundant in all cortical areas (prefrontal, cingulate, frontal, occipital, piriform) hippocampus (dentate gyms, CA3, CA2, CA1 fields and amygdala).
- the human APP 751 protein was also identified by Western blotting in lysates from brains from these transgenic mice ( Figure 5). The quantity of protein APP 751 protein identified equaled that of the endogenous mouse APP 751 protein (see Figure legend for details). F. Immunocytochemistry
- mice were deeply anaesthetized and then transcardially perfused with saline, followed by 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS). Brains were removed and post-fixed in 4% paraformaldehyde for 1 h, then placed in 30% sucrose in 0.1 M PBS at 4°C until the brains sank and then frozen in isopentane at -35°C and stored at -70°C until sections were cut. Sections (20 mM) were cut in a cryostat (Reichert) and stored in anti-freeze at -20°C. Sections were removed from anti-freeze and washed through 5 changes of PBS and Triton X100 (0.3%).
- PBS phosphate buffered saline
- transgenic animals of the invention may be used as a source of cells for cell culture.
- Brain tissues of transgenic mice are analyzed for the presence of human amyloid precursor protein by directly analyzing DNA or RNA or by assaying brain tissue for the protein expressed by the gene.
- Cells of brain tissues carrying the gene may be cultured using standard culture techniques that are well-known in the art.
- the transgenic animals and cells derived from the transgenic animals may be used to screen for compounds that modulate expression of human amyloid precursor protein. Modulation may occur at a variety of levels including but not limited to DNA or RNA or protein or combinations thereof.
- One method of determining the ability of a compound to modulate the expression of human amyloid precursor protein in a transgenic non-human animal having cells containing a gene encoding a familial form of the human amyloid precursor protein APP 751 comprises: (a) treating the transgenic animal with the compound; (b) measuring the expression or aggregation of human amyloid precursor protein in the treated animal; and (c) comparing the measurement of step (b) with a control.
- a method of determining the ability of a compound to modulate the expression of human amyloid precursor protein in cells derived from a transgenic animal comprises: (a) treating the cells with the compound; (b) measuring the expression or aggregation of human amyloid precursor protein in the treated cells; and (c) comparing the measurement of step (b) with a control.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Biodiversity & Conservation Biology (AREA)
- General Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/793,558 US6211428B1 (en) | 1994-09-01 | 1995-08-28 | Transgenic mouse expressing a familial form of human amyloid precursor protein |
EP95932340A EP0778886A4 (en) | 1994-09-01 | 1995-08-28 | Transgenic animal expressing a familial form of human amyloid precursor protein |
JP8508912A JPH10504964A (en) | 1994-09-01 | 1995-08-28 | Transgenic animals expressing familial human amyloid precursor protein |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US29987294A | 1994-09-01 | 1994-09-01 | |
US299,872 | 1994-09-01 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1996006927A1 true WO1996006927A1 (en) | 1996-03-07 |
Family
ID=23156672
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1995/010920 WO1996006927A1 (en) | 1994-09-01 | 1995-08-28 | Transgenic animal expressing a familial form of human amyloid precursor protein |
Country Status (5)
Country | Link |
---|---|
US (1) | US6211428B1 (en) |
EP (1) | EP0778886A4 (en) |
JP (1) | JPH10504964A (en) |
CA (1) | CA2198451A1 (en) |
WO (1) | WO1996006927A1 (en) |
Cited By (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5898094A (en) * | 1996-10-21 | 1999-04-27 | University Of South Florida | Transgenic mice expressing APPK670N,M671L and a mutant presenilin transgenes |
WO2000059297A2 (en) * | 1999-04-06 | 2000-10-12 | Harrington Arthritis Research Center | Methods for tracking the progression of alzheimer's disease and identifying treatments using transgenic mice |
WO2001075165A2 (en) * | 2000-03-30 | 2001-10-11 | Elan Pharmaceuticals, Inc. | Screening markers and methods for neurodegenerative disorders |
US6452065B2 (en) * | 1997-05-14 | 2002-09-17 | Merck & Co., Inc. | Transgenic mouse expressing non-native wild-type and familial Alzheimer's Disease mutant presenilin 1 protein on native presenilin 1 null background |
WO2006108201A1 (en) * | 2005-04-14 | 2006-10-19 | Jsw-Research Forschungslabor Gmbh | Promoter for the expression of foreign genes in neuronal cells |
EP1730285A1 (en) * | 2004-04-01 | 2006-12-13 | Neurotech Pharmaceuticals Co., Ltd. | Transgenic mice inducing alzheimer's disease expressing mutant betactf99 |
WO2008068024A2 (en) * | 2006-12-06 | 2008-06-12 | Universität Zürich | Means and methods for isolating and determining novel targets for the treatment of neurodegenerative, neurological or neuropsychiatric disorders and compositions comprising the same |
US7663018B2 (en) | 1996-07-19 | 2010-02-16 | Novartis Ag | Tau hyperphosphorylation in transgenic mice expressing the APP double mutation |
Families Citing this family (54)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6844148B1 (en) * | 1998-09-24 | 2005-01-18 | Pharmacia & Upjohn Company | Alzheimer's disease secretase, APP substrates therefor, and uses therefor |
US20040234976A1 (en) * | 1998-09-24 | 2004-11-25 | Gurney Mark E. | Alzheimer's disease secretase, app substrates therefor, and uses therefor |
US6699671B1 (en) * | 1998-09-24 | 2004-03-02 | Pharmacia & Upjohn Company | Alzheimer's disease secretase, APP substrates therefor, and uses therefor |
NZ527053A (en) * | 1998-09-24 | 2005-04-29 | Upjohn Co | Modified amyloid precursor protein polynucleotides and polypeptides |
US7456007B1 (en) | 1998-12-31 | 2008-11-25 | Elan Pharmaceuticals, Inc. | β-secretase enzyme compositions and methods |
US7115410B1 (en) * | 1999-02-10 | 2006-10-03 | Elan Pharmaceuticals, Inc. | β-secretase enzyme compositions and methods |
EP1165609A2 (en) | 1999-02-10 | 2002-01-02 | Elan Pharmaceuticals, Inc. | Human beta-secretase enzyme, inhibitors and their compositions and uses |
US20090162883A1 (en) * | 1999-09-23 | 2009-06-25 | Pharmacia & Upjohn Company | Alzheimer's Disease Secretase, APP Substrates Thereof, and Uses Thereof |
US7514408B1 (en) | 1999-12-02 | 2009-04-07 | Elan Pharmaceuticals, Inc. | β-secretase enzyme compositions and methods |
WO2003072037A2 (en) * | 2002-02-27 | 2003-09-04 | Pharmacia & Upjohn Company | High-level production of amyloid-beta peptides from imr-32 cells |
US7226771B2 (en) | 2002-04-19 | 2007-06-05 | Diversa Corporation | Phospholipases, nucleic acids encoding them and methods for making and using them |
EA010903B1 (en) | 2002-04-19 | 2008-12-30 | Дайверса Корпорейшн | Phospholifases, nucleic acids encoding them and methodsfor making and using them |
EP3023498B1 (en) | 2003-03-06 | 2018-11-28 | BASF Enzymes LLC | Amylases, nucleic acids encoding them and methods for making and using them |
WO2005032496A2 (en) | 2003-03-07 | 2005-04-14 | Diversa Corporation | Hydrolases, nucleic acids encoding them and mehods for making and using them |
ES2545639T3 (en) | 2003-04-04 | 2015-09-14 | Basf Enzymes Llc | Pectate liases, nucleic acids that encode them and methods for their preparation and use |
CA2525547C (en) * | 2003-05-14 | 2012-07-03 | Torreypines Therapeutics, Inc. | Compounds and uses thereof in modulating amyloid beta |
EP1641910B1 (en) | 2003-07-02 | 2013-02-20 | Verenium Corporation | Glucanases, nucleic acids encoding them and methods for making and using them |
WO2005021714A2 (en) | 2003-08-11 | 2005-03-10 | Diversa Corporation | Laccases, nucleic acids encoding them and methods for making and using them |
CN101432292B (en) | 2004-06-16 | 2013-03-13 | 维莱尼姆公司 | Compositions and methods for enzymatic decolorization of chlorophyll |
US8436006B2 (en) * | 2004-08-06 | 2013-05-07 | Jansssen Pharmaceutica N.V. | 2-amino-quinazoline derivatives useful as inhibitors of β-secretase (BACE) |
US8426429B2 (en) * | 2004-08-06 | 2013-04-23 | Jansssen Pharmaceutica N.V. | 2-amino-quinazoline derivatives useful as inhibitors of β-secretase (BACE) |
US8383637B2 (en) * | 2004-08-06 | 2013-02-26 | Jansssen Pharmaceutica N.V. | 2-amino-quinazoline derivatives useful as inhibitors of β-secretase (BACE) |
EP2886658A1 (en) | 2005-03-10 | 2015-06-24 | BASF Enzymes LLC | Lyase enzymes, nucleic acids encoding them and methods for making and using them |
KR101393679B1 (en) | 2005-03-15 | 2014-05-21 | 비피 코포레이션 노쓰 아메리카 인코포레이티드 | Cellulases, nucleic acids encoding them and methods for making and using them |
US20090324574A1 (en) | 2006-02-02 | 2009-12-31 | Verenium Corporation | Esterases and Related Nucleic Acids and Methods |
US7868022B2 (en) * | 2006-02-06 | 2011-01-11 | Janssen Pharmaceutica Nv | 2-amino-quinoline derivatives useful as inhibitors of β-secretase (BACE) |
WO2007092839A2 (en) * | 2006-02-06 | 2007-08-16 | Janssen Pharmaceutica N.V. | Macrocycle derivatives useful as inhibitors of beta-secretase (bace) |
US7776882B2 (en) * | 2006-02-06 | 2010-08-17 | Baxter Ellen W | 2-amino-3,4-dihydro-quinoline derivatives useful as inhibitors of β-secretase (BACE) |
AU2006338208B2 (en) | 2006-02-10 | 2013-10-24 | Bp Corporation North America Inc. | Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them |
ES2682284T3 (en) | 2006-02-14 | 2018-09-19 | Bp Corporation North America Inc. | Xylanases, nucleic acids that encode them and methods to make and use them |
BRPI0708673A2 (en) | 2006-03-07 | 2011-06-07 | Cargill Inc | A method for making aldolase activity polypeptides and polynucleotides encoding these polypeptides |
BR122017003017B1 (en) | 2006-03-07 | 2018-10-23 | Basf Enzymes Llc | NUCLEIC ACID, EXPRESSION CASSETTE, VECTOR, CLONING VEHICLE, MOTRANSGENIC MICROORGANISM, POLYPEPTIDE, SIGNAL PEPTIDE, COMPOSITION, AND METHODS TO PRODUCE A RECOMBINANT CARIAN CARD A NON-CARBONIZED LIP FOR A NERCARIOUS CARBON TO FORM A CARBON-CARBON CONNECTION, TO MAKE A FOOD, FEED OR DRINK, AND TO CONVERT A BIOMASS ANY LIGNOCELLULOSTIC MATERIAL INTO A FUEL |
MX369001B (en) | 2006-08-04 | 2019-10-24 | Basf Enzymes Llc | Glucanases, nucleic acids encoding them and methods for making and using them. |
CN101558154B (en) | 2006-09-21 | 2016-02-24 | 帝斯曼知识产权资产管理有限公司 | Phospholipid hydrolase, encode their nucleic acid and methods for making and using same thereof |
EP2617816B1 (en) | 2006-09-21 | 2015-11-18 | BASF Enzymes LLC | Phytases, nucleic acids encoding them and methods for making and using them |
EP3540053B1 (en) | 2006-12-21 | 2021-06-02 | BASF Enzymes, LLC | Amylases and glucoamylases, nucleic acids encoding them and methods for making and using them |
US20080188573A1 (en) * | 2007-02-02 | 2008-08-07 | Alcon Research, Ltd. | Use of thy1-fp transgenic mouse for the identification of ophthalmic agents |
NZ584967A (en) | 2007-10-03 | 2012-08-31 | Verenium Corp | Xylanases, nucleic acids encoding them and methods for making and using them |
EP2217721A4 (en) * | 2007-10-29 | 2013-01-09 | Univ California | Osteoarthritis gene therapy |
WO2009088753A1 (en) | 2008-01-03 | 2009-07-16 | Verenium Corporation | Isomerases, nucleic acids encoding them and methods for making and using them |
CN101965397B (en) | 2008-01-03 | 2016-09-28 | 巴斯夫酶有限责任公司 | Transferring enzyme and oxidoreductase, the nucleic acid encoding them and its methods for making and using same |
US8076358B2 (en) * | 2008-01-28 | 2011-12-13 | Janssen Pharmaceutica Nv | 6-substituted-thio-2-amino-quinoline derivatives useful as inhibitors of β-secretase (BACE) |
CA2714008A1 (en) * | 2008-01-29 | 2009-08-06 | Janssen Pharmaceutica N.V. | 2-amino quinoline derivatives useful as inhibitors of .beta.-secretase (bace) |
US8357503B2 (en) | 2008-08-29 | 2013-01-22 | Bunge Oils, Inc. | Hydrolases, nucleic acids encoding them and methods for making and using them |
US8198062B2 (en) | 2008-08-29 | 2012-06-12 | Dsm Ip Assets B.V. | Hydrolases, nucleic acids encoding them and methods for making and using them |
US8153391B2 (en) | 2008-08-29 | 2012-04-10 | Bunge Oils, Inc. | Hydrolases, nucleic acids encoding them and methods for making and using them |
AU2009290563A1 (en) | 2008-09-15 | 2010-03-18 | Genentech, Inc. | Compositions and methods for regulating cell osmolarity |
US20110023152A1 (en) * | 2008-12-04 | 2011-01-27 | Sigma-Aldrich Co. | Genome editing of cognition related genes in animals |
EP2698374B1 (en) | 2009-05-21 | 2016-09-14 | BASF Enzymes LLC | Phytases, nucleic acids encoding them and methods for making and using them |
UA109884C2 (en) | 2009-10-16 | 2015-10-26 | A POLYPEPTIDE THAT HAS THE ACTIVITY OF THE PHOSPHATIDYLINOSYTOL-SPECIFIC PHOSPHOLIPASE C, NUCLEIC ACID, AND METHOD OF METHOD | |
UA111708C2 (en) | 2009-10-16 | 2016-06-10 | Бандж Ойлз, Інк. | METHOD OF OIL REFINING |
GB201204816D0 (en) | 2012-03-19 | 2012-05-02 | Brainco Biopharma S L | Transgenic animal model of mood disorders |
EP3626832A3 (en) | 2014-11-25 | 2020-05-13 | The Brigham and Women's Hospital, Inc. | Method of identifying and treating a person having a predisposition to or afflicted with a cardiometabolic disease |
US10683552B2 (en) | 2014-11-25 | 2020-06-16 | Presidents And Fellows Of Harvard College | Clonal haematopoiesis |
Family Cites Families (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5087571A (en) * | 1984-06-22 | 1992-02-11 | President And Fellows Of Harvard College | Method for providing a cell culture from a transgenic non-human mammal |
EP0451700A1 (en) * | 1990-04-10 | 1991-10-16 | Miles Inc. | Recombinant APP minigenes for expression in transgenic mice as models for Alzheimers's disease |
DE69133372T2 (en) * | 1990-06-15 | 2005-02-24 | Scios Inc., Sunnyvale | Transgenic, non-human mammalian animal showing the amyloid-forming pathology of Alzheimer's disease |
EP0550675A1 (en) * | 1990-09-28 | 1993-07-14 | Cephalon, Inc. | Transgenic animals with alzheimer's amyloid precursor gene |
ES2335720T3 (en) * | 1991-01-21 | 2010-03-31 | Elan Pharmaceuticals, Inc. | TEST AND MODEL FOR ALZHEIMER'S DISEASE. |
US5672805A (en) * | 1991-07-18 | 1997-09-30 | The Regents Of The University Of California | Transgenic mice expressing the neurotoxic C-terminus of β-amyloid precursor protein |
AU2765992A (en) * | 1991-10-03 | 1993-05-03 | Indiana University Foundation | Method for screening for alzheimer's disease |
EP0620849B1 (en) * | 1992-01-07 | 2003-06-25 | Elan Pharmaceuticals, Inc. | Transgenic animal models for alzheimer's disease |
-
1995
- 1995-08-28 US US08/793,558 patent/US6211428B1/en not_active Expired - Fee Related
- 1995-08-28 WO PCT/US1995/010920 patent/WO1996006927A1/en not_active Application Discontinuation
- 1995-08-28 JP JP8508912A patent/JPH10504964A/en active Pending
- 1995-08-28 EP EP95932340A patent/EP0778886A4/en not_active Withdrawn
- 1995-08-28 CA CA002198451A patent/CA2198451A1/en not_active Abandoned
Non-Patent Citations (4)
Title |
---|
CAPORASO G L, ET AL.: "CHLOROQUINE INHIBITS INTRACELLULAR DEGRADATION BUT NOT SECRETION OFALZHEIMER BETA/A4 AMYLOID PRECURSOR PROTEIN", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, NATIONAL ACADEMY OF SCIENCES, US, vol. 89, 1 March 1992 (1992-03-01), US, pages 2252 - 2256, XP002945124, ISSN: 0027-8424, DOI: 10.1073/pnas.89.6.2252 * |
CHARTIER-HARLIN M-C, ET AL.: "EARLY-ONSET ALZHEIMER'S DISEASE CAUSED BY MUTATIONS AT CODON 717 OF THE BETA-AMYLOID PRECURSOR PROTEIN GENE", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 353, 31 October 1991 (1991-10-31), United Kingdom, pages 844 - 846, XP000997063, ISSN: 0028-0836, DOI: 10.1038/353844a0 * |
QUON D., ET AL.: "FORMATION OF BETA-AMYLOID PROTEIN DEPOSITS IN BRAINS OF TRANSGENIC MICE.", NATURE, NATURE PUBLISHING GROUP, UNITED KINGDOM, vol. 352., 18 July 1991 (1991-07-18), United Kingdom, pages 239 - 241., XP002013250, ISSN: 0028-0836, DOI: 10.1038/352239a0 * |
See also references of EP0778886A4 * |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7663018B2 (en) | 1996-07-19 | 2010-02-16 | Novartis Ag | Tau hyperphosphorylation in transgenic mice expressing the APP double mutation |
US5898094A (en) * | 1996-10-21 | 1999-04-27 | University Of South Florida | Transgenic mice expressing APPK670N,M671L and a mutant presenilin transgenes |
US6452065B2 (en) * | 1997-05-14 | 2002-09-17 | Merck & Co., Inc. | Transgenic mouse expressing non-native wild-type and familial Alzheimer's Disease mutant presenilin 1 protein on native presenilin 1 null background |
WO2000059297A2 (en) * | 1999-04-06 | 2000-10-12 | Harrington Arthritis Research Center | Methods for tracking the progression of alzheimer's disease and identifying treatments using transgenic mice |
WO2000059297A3 (en) * | 1999-04-06 | 2001-04-12 | Harrington Arthritis Res Ct | Methods for tracking the progression of alzheimer's disease and identifying treatments using transgenic mice |
US6664442B2 (en) | 2000-03-30 | 2003-12-16 | Elan Pharmaceuticals, Inc. | Selecting compounds to reduce inflammation associated with Alzheimer's disease |
WO2001075165A3 (en) * | 2000-03-30 | 2002-10-31 | Elan Pharm Inc | Screening markers and methods for neurodegenerative disorders |
WO2001075165A2 (en) * | 2000-03-30 | 2001-10-11 | Elan Pharmaceuticals, Inc. | Screening markers and methods for neurodegenerative disorders |
EP1730285A1 (en) * | 2004-04-01 | 2006-12-13 | Neurotech Pharmaceuticals Co., Ltd. | Transgenic mice inducing alzheimer's disease expressing mutant betactf99 |
EP1730285A4 (en) * | 2004-04-01 | 2008-07-16 | Neurotech Pharmaceuticals Co L | Transgenic mice inducing alzheimer's disease expressing mutant betactf99 |
WO2006108201A1 (en) * | 2005-04-14 | 2006-10-19 | Jsw-Research Forschungslabor Gmbh | Promoter for the expression of foreign genes in neuronal cells |
WO2008068024A2 (en) * | 2006-12-06 | 2008-06-12 | Universität Zürich | Means and methods for isolating and determining novel targets for the treatment of neurodegenerative, neurological or neuropsychiatric disorders and compositions comprising the same |
WO2008068024A3 (en) * | 2006-12-06 | 2008-07-24 | Univ Zuerich | Means and methods for isolating and determining novel targets for the treatment of neurodegenerative, neurological or neuropsychiatric disorders and compositions comprising the same |
Also Published As
Publication number | Publication date |
---|---|
EP0778886A1 (en) | 1997-06-18 |
CA2198451A1 (en) | 1996-03-07 |
JPH10504964A (en) | 1998-05-19 |
EP0778886A4 (en) | 2001-05-02 |
US6211428B1 (en) | 2001-04-03 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6211428B1 (en) | Transgenic mouse expressing a familial form of human amyloid precursor protein | |
US6037521A (en) | Transgenic mouse expressing an β-Amyloid transgene | |
AU646877B2 (en) | Transgenic non-human mammal displaying the amyloid-forming pathology of alzheimer's disease | |
AU671093B2 (en) | Transgenic animal models for alzheimer's disease | |
US6187992B1 (en) | Transgenic mouse having a disrupted amyloid precursor protein gene | |
AU652997B2 (en) | Test and model for alzheimer's disease | |
US5912410A (en) | Transgenic non-human mice displaying the amyloid-forming pathology of alzheimer's disease | |
CA2290707C (en) | Apolipoprotein e transgenic animals and assay methods | |
EP0832205A1 (en) | Method for identifying alzheimer's disease therapeutics using transgenic animal models | |
US5604131A (en) | cDNA-genomic DNA hybrid sequence encoding APP770 containing a genomic DNA insert of the KI and OX-2 regions | |
US6734336B1 (en) | Gene-targeted non-human mammal with human fad presenilin mutation and generational offspring | |
EP0724628A1 (en) | Transgenic animal model for cognitive disorders | |
JP5046413B2 (en) | Targeted recombinant non-human mammal with human FAD presenilin mutation and reproductive progeny | |
Kobayashi et al. | Sato et al. | |
WO1994024266A1 (en) | Transgenic animal models for alzheimer's disease | |
AU2001276995A1 (en) | Gene-targeted non-human mammal with human fad presenilin mutation and generational offspring | |
Beech | On the causes of Alzheimer's disease: Investigations using transgenic mouse model systems | |
CA2040077A1 (en) | Recombinant app minigenes for expression in transgenic mice as models for alzheimer's disease | |
Wadsworth et al. | Transgenic mouse expressing APP 770 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): CA JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2198451 Country of ref document: CA |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1995932340 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 08793558 Country of ref document: US |
|
WWP | Wipo information: published in national office |
Ref document number: 1995932340 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 1995932340 Country of ref document: EP |