WO2013088110A1 - Animaux transgéniques pour l'annexine a1 - Google Patents
Animaux transgéniques pour l'annexine a1 Download PDFInfo
- Publication number
- WO2013088110A1 WO2013088110A1 PCT/GB2012/000905 GB2012000905W WO2013088110A1 WO 2013088110 A1 WO2013088110 A1 WO 2013088110A1 GB 2012000905 W GB2012000905 W GB 2012000905W WO 2013088110 A1 WO2013088110 A1 WO 2013088110A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- ocd
- human animal
- seq
- nucleic acid
- animal model
- Prior art date
Links
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 66
- 241001465754 Metazoa Species 0.000 title claims abstract description 39
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 115
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 106
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 106
- 208000021384 Obsessive-Compulsive disease Diseases 0.000 claims abstract description 82
- 210000001744 T-lymphocyte Anatomy 0.000 claims abstract description 66
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 60
- 201000010099 disease Diseases 0.000 claims abstract description 58
- 238000000034 method Methods 0.000 claims abstract description 56
- 210000004027 cell Anatomy 0.000 claims abstract description 36
- 238000010171 animal model Methods 0.000 claims abstract description 32
- 238000011820 transgenic animal model Methods 0.000 claims abstract description 16
- 238000011282 treatment Methods 0.000 claims abstract description 15
- 230000001404 mediated effect Effects 0.000 claims abstract description 12
- 210000001161 mammalian embryo Anatomy 0.000 claims abstract description 11
- 238000012216 screening Methods 0.000 claims abstract description 5
- 125000003729 nucleotide group Chemical group 0.000 claims description 174
- 239000002773 nucleotide Substances 0.000 claims description 173
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 39
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 24
- 208000024891 symptom Diseases 0.000 claims description 13
- 239000003550 marker Substances 0.000 claims description 10
- 238000000520 microinjection Methods 0.000 claims description 10
- 208000023275 Autoimmune disease Diseases 0.000 claims description 9
- 210000002459 blastocyst Anatomy 0.000 claims description 9
- 210000001671 embryonic stem cell Anatomy 0.000 claims description 7
- 201000006417 multiple sclerosis Diseases 0.000 claims description 7
- 208000000323 Tourette Syndrome Diseases 0.000 claims description 6
- 208000016620 Tourette disease Diseases 0.000 claims description 6
- 208000022266 body dysmorphic disease Diseases 0.000 claims description 6
- 210000000287 oocyte Anatomy 0.000 claims description 6
- 208000019906 panic disease Diseases 0.000 claims description 6
- 208000028173 post-traumatic stress disease Diseases 0.000 claims description 6
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 claims description 5
- 238000010374 somatic cell nuclear transfer Methods 0.000 claims description 5
- 241000283984 Rodentia Species 0.000 claims description 4
- 201000000596 systemic lupus erythematosus Diseases 0.000 claims description 4
- 208000036640 Asperger disease Diseases 0.000 claims description 3
- 201000006062 Asperger syndrome Diseases 0.000 claims description 3
- 208000006096 Attention Deficit Disorder with Hyperactivity Diseases 0.000 claims description 3
- 208000020925 Bipolar disease Diseases 0.000 claims description 3
- 206010065701 Dermatillomania Diseases 0.000 claims description 3
- 201000001916 Hypochondriasis Diseases 0.000 claims description 3
- 206010033664 Panic attack Diseases 0.000 claims description 3
- 206010041250 Social phobia Diseases 0.000 claims description 3
- 208000022531 anorexia Diseases 0.000 claims description 3
- 206010061428 decreased appetite Diseases 0.000 claims description 3
- 206010012601 diabetes mellitus Diseases 0.000 claims description 3
- 201000000980 schizophrenia Diseases 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 208000002271 trichotillomania Diseases 0.000 claims description 3
- 208000030507 AIDS Diseases 0.000 claims description 2
- 206010000234 Abortion spontaneous Diseases 0.000 claims description 2
- 208000026872 Addison Disease Diseases 0.000 claims description 2
- 201000001320 Atherosclerosis Diseases 0.000 claims description 2
- 208000009137 Behcet syndrome Diseases 0.000 claims description 2
- 206010009900 Colitis ulcerative Diseases 0.000 claims description 2
- 208000009329 Graft vs Host Disease Diseases 0.000 claims description 2
- 208000003807 Graves Disease Diseases 0.000 claims description 2
- 208000015023 Graves' disease Diseases 0.000 claims description 2
- 208000030836 Hashimoto thyroiditis Diseases 0.000 claims description 2
- 208000022559 Inflammatory bowel disease Diseases 0.000 claims description 2
- 201000011152 Pemphigus Diseases 0.000 claims description 2
- 201000004681 Psoriasis Diseases 0.000 claims description 2
- 206010039710 Scleroderma Diseases 0.000 claims description 2
- 208000021386 Sjogren Syndrome Diseases 0.000 claims description 2
- 206010052779 Transplant rejections Diseases 0.000 claims description 2
- 201000006704 Ulcerative Colitis Diseases 0.000 claims description 2
- 206010046851 Uveitis Diseases 0.000 claims description 2
- 230000001363 autoimmune Effects 0.000 claims description 2
- 238000009395 breeding Methods 0.000 claims description 2
- 230000001488 breeding effect Effects 0.000 claims description 2
- 208000025302 chronic primary adrenal insufficiency Diseases 0.000 claims description 2
- 208000024908 graft versus host disease Diseases 0.000 claims description 2
- 208000015994 miscarriage Diseases 0.000 claims description 2
- 201000001976 pemphigus vulgaris Diseases 0.000 claims description 2
- 208000005987 polymyositis Diseases 0.000 claims description 2
- 208000000995 spontaneous abortion Diseases 0.000 claims description 2
- 208000032841 Bulimia Diseases 0.000 claims 2
- 206010006550 Bulimia nervosa Diseases 0.000 claims 2
- 241000124008 Mammalia Species 0.000 claims 2
- 108090000623 proteins and genes Proteins 0.000 description 53
- 239000013598 vector Substances 0.000 description 45
- 241000699670 Mus sp. Species 0.000 description 36
- 102000004169 proteins and genes Human genes 0.000 description 32
- 241000699666 Mus <mouse, genus> Species 0.000 description 27
- 102000001708 Protein Isoforms Human genes 0.000 description 23
- 108010029485 Protein Isoforms Proteins 0.000 description 23
- 108090000765 processed proteins & peptides Proteins 0.000 description 16
- 230000006399 behavior Effects 0.000 description 15
- 108091026890 Coding region Proteins 0.000 description 13
- 108020004705 Codon Proteins 0.000 description 12
- 238000011161 development Methods 0.000 description 12
- 239000012634 fragment Substances 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 11
- 230000014509 gene expression Effects 0.000 description 11
- 238000011830 transgenic mouse model Methods 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 9
- 238000003752 polymerase chain reaction Methods 0.000 description 9
- 241000894007 species Species 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 102000000412 Annexin Human genes 0.000 description 8
- 108050008874 Annexin Proteins 0.000 description 8
- 101100273713 Homo sapiens CD2 gene Proteins 0.000 description 8
- 108091081024 Start codon Proteins 0.000 description 8
- 150000001413 amino acids Chemical class 0.000 description 8
- 108091033319 polynucleotide Proteins 0.000 description 8
- 102000040430 polynucleotide Human genes 0.000 description 8
- 239000002157 polynucleotide Substances 0.000 description 8
- 241000699660 Mus musculus Species 0.000 description 7
- 241000700159 Rattus Species 0.000 description 7
- 239000002299 complementary DNA Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000013604 expression vector Substances 0.000 description 7
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 101000959738 Homo sapiens Annexin A1 Proteins 0.000 description 6
- 102100021126 N-formyl peptide receptor 2 Human genes 0.000 description 6
- 108091034117 Oligonucleotide Proteins 0.000 description 6
- 201000002491 encephalomyelitis Diseases 0.000 description 6
- 102000004196 processed proteins & peptides Human genes 0.000 description 6
- 239000003814 drug Substances 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 210000002257 embryonic structure Anatomy 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 238000002347 injection Methods 0.000 description 5
- 239000007924 injection Substances 0.000 description 5
- 230000002018 overexpression Effects 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 108090000663 Annexin A1 Proteins 0.000 description 4
- 102100040006 Annexin A1 Human genes 0.000 description 4
- 102100025237 T-cell surface antigen CD2 Human genes 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 239000011543 agarose gel Substances 0.000 description 4
- 230000003542 behavioural effect Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 239000004579 marble Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 238000010172 mouse model Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 208000032116 Autoimmune Experimental Encephalomyelitis Diseases 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 3
- 102000011652 Formyl peptide receptors Human genes 0.000 description 3
- 108010076288 Formyl peptide receptors Proteins 0.000 description 3
- 102000003960 Ligases Human genes 0.000 description 3
- 108090000364 Ligases Proteins 0.000 description 3
- 101710091942 N-formyl peptide receptor 2 Proteins 0.000 description 3
- 101000959729 Rattus norvegicus Annexin A1 Proteins 0.000 description 3
- 238000011579 SCID mouse model Methods 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000004590 computer program Methods 0.000 description 3
- 208000012997 experimental autoimmune encephalomyelitis Diseases 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 108091008146 restriction endonucleases Proteins 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- 125000003821 2-(trimethylsilyl)ethoxymethyl group Chemical group [H]C([H])([H])[Si](C([H])([H])[H])(C([H])([H])[H])C([H])([H])C(OC([H])([H])[*])([H])[H] 0.000 description 2
- -1 Altschul et al. Proteins 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 102100025137 Early activation antigen CD69 Human genes 0.000 description 2
- 241000283073 Equus caballus Species 0.000 description 2
- 208000009386 Experimental Arthritis Diseases 0.000 description 2
- 241000282326 Felis catus Species 0.000 description 2
- 102000005720 Glutathione transferase Human genes 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 108091093094 Glycol nucleic acid Proteins 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 101000934374 Homo sapiens Early activation antigen CD69 Proteins 0.000 description 2
- 101001057504 Homo sapiens Interferon-stimulated gene 20 kDa protein Proteins 0.000 description 2
- 101001055144 Homo sapiens Interleukin-2 receptor subunit alpha Proteins 0.000 description 2
- 102100026878 Interleukin-2 receptor subunit alpha Human genes 0.000 description 2
- 108091092195 Intron Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 241001529936 Murinae Species 0.000 description 2
- 101000959737 Mus musculus Annexin A1 Proteins 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 108091093037 Peptide nucleic acid Proteins 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 2
- 108091046915 Threose nucleic acid Proteins 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000000561 anti-psychotic effect Effects 0.000 description 2
- 230000000692 anti-sense effect Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000000975 bioactive effect Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 150000005829 chemical entities Chemical class 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 208000035475 disorder Diseases 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 238000012239 gene modification Methods 0.000 description 2
- 230000005017 genetic modification Effects 0.000 description 2
- 235000013617 genetically modified food Nutrition 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 230000003394 haemopoietic effect Effects 0.000 description 2
- 210000002861 immature t-cell Anatomy 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000010354 integration Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 210000003101 oviduct Anatomy 0.000 description 2
- 235000021317 phosphate Nutrition 0.000 description 2
- 239000013612 plasmid Substances 0.000 description 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 2
- 208000020016 psychiatric disease Diseases 0.000 description 2
- 108010054624 red fluorescent protein Proteins 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000004626 scanning electron microscopy Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 230000002269 spontaneous effect Effects 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 241001515965 unidentified phage Species 0.000 description 2
- 230000004580 weight loss Effects 0.000 description 2
- ORILYTVJVMAKLC-UHFFFAOYSA-N Adamantane Natural products C1C(C2)CC3CC1CC2C3 ORILYTVJVMAKLC-UHFFFAOYSA-N 0.000 description 1
- 229930024421 Adenine Natural products 0.000 description 1
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 1
- 201000010000 Agranulocytosis Diseases 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000004149 Annexin A2 Human genes 0.000 description 1
- 108090000668 Annexin A2 Proteins 0.000 description 1
- 108090000672 Annexin A5 Proteins 0.000 description 1
- 102000004121 Annexin A5 Human genes 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241001674808 Biastes Species 0.000 description 1
- 241000588832 Bordetella pertussis Species 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 206010010099 Combined immunodeficiency Diseases 0.000 description 1
- 206010010219 Compulsions Diseases 0.000 description 1
- 101710108485 Envelope phospholipase F13 Proteins 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 206010015866 Extravasation Diseases 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 101710109169 Formyl peptide receptor 2 Proteins 0.000 description 1
- 241000700662 Fowlpox virus Species 0.000 description 1
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 1
- HVLSXIKZNLPZJJ-TXZCQADKSA-N HA peptide Chemical compound C([C@@H](C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 HVLSXIKZNLPZJJ-TXZCQADKSA-N 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241000829100 Macaca mulatta polyomavirus 1 Species 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- 241001049988 Mycobacterium tuberculosis H37Ra Species 0.000 description 1
- 206010029897 Obsessive thoughts Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 108010081690 Pertussis Toxin Proteins 0.000 description 1
- 102000004160 Phosphoric Monoester Hydrolases Human genes 0.000 description 1
- 108090000608 Phosphoric Monoester Hydrolases Proteins 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 241000701093 Suid alphaherpesvirus 1 Species 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 241000700618 Vaccinia virus Species 0.000 description 1
- 230000021736 acetylation Effects 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 210000005006 adaptive immune system Anatomy 0.000 description 1
- 229960000643 adenine Drugs 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 238000011091 antibody purification Methods 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 230000007529 anxiety like behavior Effects 0.000 description 1
- 239000002249 anxiolytic agent Substances 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001517 counterregulatory effect Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 230000009429 distress Effects 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000036251 extravasation Effects 0.000 description 1
- 101710108492 fMet-Leu-Phe receptor Proteins 0.000 description 1
- 102100021145 fMet-Leu-Phe receptor Human genes 0.000 description 1
- 230000006390 fear memory Effects 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003862 glucocorticoid Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 208000010726 hind limb paralysis Diseases 0.000 description 1
- 230000003284 homeostatic effect Effects 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- BHEPBYXIRTUNPN-UHFFFAOYSA-N hydridophosphorus(.) (triplet) Chemical compound [PH] BHEPBYXIRTUNPN-UHFFFAOYSA-N 0.000 description 1
- 230000037417 hyperactivation Effects 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000010324 immunological assay Methods 0.000 description 1
- 238000001114 immunoprecipitation Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 210000005007 innate immune system Anatomy 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000013507 mapping Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 230000010004 neural pathway Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 208000010713 partial hind limb paralysis Diseases 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000000644 propagated effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 230000028527 righting reflex Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 229940037128 systemic glucocorticoids Drugs 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 238000011816 wild-type C57Bl6 mouse Methods 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
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4721—Lipocortins
-
- 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
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
- A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
-
- 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
- A01K2217/20—Animal model comprising regulated expression system
- A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
-
- 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
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
-
- 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
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0387—Animal model for diseases of the immune system
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/43—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a FLAG-tag
Definitions
- the present invention relates to a transgenic non-human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD and the use of such an animal model to screen for candidate molecules as treatments for OCD and related diseases.
- OCD obsessive compulsive disorder
- Obsessive compulsive disorder is a chronic, relapsing psychiatric affliction with a lifetime prevalence of 1-3%.
- DSM IV Diagnostic and Statistical Manual of Mental Disorders
- the essential features of this disease are recurrent obsessions and/or compulsions (e.g., doubting, checking, washing) that are time consuming (i.e., they take more than 1 hour a day) or cause marked distress or significant impairment.
- the most effective treatments for mental disorders like OCD are antipsychotic and behavioural treatments. Yet, around 30% of the patients are refractory to pharmaco- and behavioural therapy.
- Anx-Al a protein called Annexin-Al (Anx-Al) plays an immunomodulatory role in T cells.
- Anx-Al a protein called Annexin-Al
- Overexpression of Anx-Al in T cells in transgenic mice exacerbates the development of autoimmune diseases and, at the same time, induces the manifestation of signs of OCD and related diseases.
- the present invention provides a transgenic non-human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD wherein the cells of said animal contain an exogenous nucleic acid encoding Annexin-Al and wherein said exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of said animal.
- OCD obsessive compulsive disorder
- the cells of said animal contain an exogenous nucleic acid encoding Annexin-Al and wherein said exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of said animal.
- the present invention provides a transgenic non-human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD wherein the cells of said animal contain an exogenous nucleic acid encoding Annexin-Al and wherein said exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of said animal.
- OCD obsessive compulsive disorder
- transgenic as used herein with respect to transgenic non-human animals is meant to define a non-human animal that contains an exogenous nucleic acid as defined herein.
- transgenic as used herein thus encompasses animals that contain a gene or nucleic acid that is heterologous to the animal (i.e. from a different species of animal) or, more typically, a homologous gene or nucleic acid (i.e. from the same species of animal) that is overexpressed.
- the non-human animal is a non-human mammal.
- the non-human mammal is typically an experimentally useful animal and is therefore typically a rodent, for example a mouse, rat or guinea pig, and is typically a mouse or a rat, most typically a mouse.
- the non-human mammal may be from another mammalian species, such as rabbit, a canine (dog) or feline (cat), or an ungulate species such as an ovine (sheep), porcine (pig), equine (horse), caprine (goat) or bovine (cow).
- the transgenic non-human animal model of the invention is an animal model for obsessive compulsive disorder (OCD) or a disease related to OCD.
- OCD obsessive compulsive disorder
- Diseases related to OCD include trichotillomania, dermatillomania, Tourette's Syndrome (TS), Asperger's syndrome, anorexia, hnlimia, depression, panic disorder, panic attacks, bipolar disorder, hypochondriasis, post- traumatic stress disorder (PTSD), social anxiety disorder, schizophrenia, attention deficit hyperactivity disorder (ADHD) and body dysmorphic disorder (BDD).
- the cells of the transgenic non-human animal of the invention contain an exogenous nucleic acid encoding Annexin-Al .
- exogenous means from outside the animal of interest.
- an "exogenous" nucleic acid that is present in the cells of an animal means a nucleic acid sequence from outside that particular animal.
- An exogenous nucleic acid can be an additional copy of a nucleic acid that is homologous to a nucleic acid present in the animal or a nucleic acid that is not normally present in the animal, for example a nucleic acid having a different sequence from the nucleic acid present in the animal or a nucleic acid from a different species of animal (i.e. a nucleic acid that is heterologous to the animal).
- the exogenous nucleic acid is an additional copy of a nucleic acid that is homologous to a nucleic acid present in the animal.
- the term "endogenous” is the converse of "exogenous” and therefore means from within the animal of interest.
- the transgenic non-human animal of the invention contains an endogenous gene or nucleic acid that is overexpressed.
- overexpressed means that a nucleic acid is expressed at higher than usual levels, for example due to the addition of one or more copies of an exogenous gene or nucleic acid sequence that corresponds to the endogenous gene or nucleic acid sequence, or by activation of an endogenous gene.
- nucleic acid takes its usual meaning in the art.
- a nucleic acid comprises a plurality of nucleotides, which are sometimes referred to as bases (in single stranded nucleic acid molecules) or as base pairs (bp, in double stranded nucleic acid molecules).
- nucleic acid is used interchangeably herein with the term “polynucleotide”.
- a “nucleic acid” or “polynucleotide” as defined herein includes a plurality of oligonucleotides as defined herein.
- Nucleic acids for use in the present invention are typically the naturally-occurring nucleic acids DNA or RNA, but can also be artificial nucleic acids such as PNA (peptide nucleic acid), LNA (locked nucleic acid), UNA (unlocked nucleic acid), GNA (glycol nucleic acid) and TNA (threose nucleic acid).
- Nucleic acids such as DNA for use in the invention can be synthetic or natural.
- Nucleic acids for use in the present invention typically consist of the nucleotides adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), but modified nucleotides can also be used in the present invention.
- a nucleic acid or polynucleotide sequence can be measured in terms of the number of nucleotides it contains.
- the term "kilobase” (kb) means 1000 nucleotides.
- oligonucleotide means a polymer of nucleotides (i.e. at least 2 nucleotides) that is shorter in length than a "nucleic acid" of "polynucleotide” as defined herein.
- an oligonucleotide consists of up to 40 nucleotides or bases, more typically up to 60 nucleotides or bases.
- an oligonucleotide is sufficiently short that it has no secondary or tertiary structure. An oligonucleotide is therefore a fragment of a nucleic acid or polynucleotide.
- gene takes its usual meaning in the art, and includes a DNA or RNA sequence encoding a polypeptide, together with regions preceding and following the coding region, as well as intervening sequences (introns) between individual coding segments (exons).
- the cells of the transgenic non-human animal of the present invention contain an exogenous nucleic acid encoding Annexin-Al.
- an exogenous nucleic acid is a nucleic acid sequence from outside the transgenic non-human animal of the invention.
- Annexins are a group of calcium- and phospholipid-binding cellular proteins and are also known as lipocortins.
- the annexin family has 13 members in humans, including Annexin Ai, Annexin A2 and Annexin A5, and also 13 members in mice.
- Annexin-Al is also known as Annexin-1 and is referred to herein as " ⁇ - ⁇ '.
- Human annexin-1 (Anx-Al) is a 37-kDa protein and was originally described as a mediator of the actions of glucocorticoids. Over the last few years evidence has shown than Anx-Al plays a homeostatic role in the adaptive immune system, in particular T cells, by modulating the strength of T cell receptor (TCR) signalling.
- TCR T cell receptor
- FIG. 1 is a ribbon diagram showing the three- dimensional structure of human Anx-AI .
- Ac.2- 26 acts as a bioactive surrogate of the whole protein (see e.g. Lim et al, Proc Natl Acad Sci USA 95, 14535-9, 1998).
- Anx-AI and its N-terminal derived bioactive peptides mediate their biological effects through members of the formyl peptide receptor (FPR) family.
- the full-length protein Anx-AI exerts its counterregulatory actions on neutrophil extravasation and innate immunity by direct binding and activation of one member of this family, formyl peptide receptor like-1 (FPRL-1), also known as formyl peptide receptor 2 (FPR-2/ALX).
- FPRL-1 formyl peptide receptor like-1
- FPR-2/ALX formyl peptide receptor 2
- the present inventors have previously found that stimulation of T cells in the presence of hr Anx-AI increases T cell activation via stimulation of FPRL-1 FPR-2 ALX (D'Acquisto et al, Blood 109: 1095-1102, 2007).
- the peptide Ac.2-26 binds to a different member of the FPR family, the N-formylpeptide receptor 1 (FPR-1).
- FPR-1 N-formylpeptide receptor 1
- a recent study (Gao et al. Behav Genet. 41(5): 724-733, 2011) has shown that mice deficient for FPR-1 exhibited increased exploratory activity, reduced anxiety- like behavior, and impaired fear memory.
- FPR-1 is the receptor for a number of ligands and there is no evidence in this study to show that the effects demonstrated in the mice were as a result of blocking annexin binding.
- Annexins have homologues in a wide variety of animal species, including mice, rats, cats, dogs and chimpanzees.
- ENSMUST00000025561 Anxal-001 transcript sequence
- ENSMUSP000000255 1 Anxal-001 protein sequence
- ENSMUST00000099584 Anxal-201 transcript sequence
- ENSMUSP00000097180 Anxal-201 protein sequence
- nucleotide and amino acid sequences of one isoform of mouse Annexin-1 (Anx-Al), Anxal-001, are shown in Figure 2A (SEQ ID NOs: 1 and 2 respectively).
- nucleotide and amino acid and sequences of the other isoform of mouse Anx-Al, Anxal-201, are shown in Figure 2B (SEQ ID NOs: 3 and 4 respectively).
- ENSRNOT00000023664 Anxal transcript sequence
- ENSRNOP00000023664 Anxal protein sequence
- ENSRNOT00000054790 D3ZVZ4_RAT transcript sequence
- ENSRNOP00000051674 D3ZVZ4_RAT protein sequence
- nucleotide and amino acid sequences of one isoform of rat Annexin-1 (Anx-Al), Anxal, are shown in Figure 2C (SEQ ID NOs: 5 and 6 respectively).
- nucleotide and amino acid and sequences of the other isoform of rat Anx- Al , D3ZVZ4_RAT, are shown in Figure 2D (SEQ ID NOs: 7 and 8 respectively).
- Anx-Al There are eight human nucleotide sequences which encode Anx-Al. Of these, only four are translated and thus there are four isoforms of Anx-Al, designated AN Al-002, ANXA 1-003, ANXAl-004 and ANXA1-006.
- ENST00000257497 ANXAl-002 transcript sequence
- ENSP00000257497 ANXAl-002 protein sequence
- ENST00000376911 ANXA1-003 transcript sequence
- ENSP00000366109 ANXA1-003 protein sequence
- ENST00000456643 ANXAl-004 transcript sequence
- ENSP00000412489 ANXAl-004 protein sequence
- ENST00000415424 ANXA 1-006 transcript sequence
- ENSP00000414013 A XA1-006 protein sequence
- nucleotide and amino acid sequences of one isoform of human Annexin-1 (Anx-Al), ANXA1-003, are shown in Figure 2E (SEQ ID NOs: 9 and 10 respectively).
- the nucleotide and amino acid sequences of another isoform, ANXAl-002, are shown in Figure 2F (SEQ ID NOs: 1 1 and 12 respectively).
- the nucleotide and amino acid sequences of another isoform, ANXAl-004 are shown in Figure 2G (SEQ ID NOs: 13 and 14 respectively).
- the nucleotide and amino acid sequences of another isoform, ANXA1-006 are shown in Figure 2H (SEQ ID NOs: 15 and 16 respectively).
- isoforms ANXAl-002, ANXAl-004 and ANXA1-006 are either short splice variants of ANXA1-003 or variants of ANXA1-003 with a small number of amino acid changes.
- the nucleotide sequence of the mouse Anx-Al isoform Anxal-001 shown in Figure 2A is as follows:
- the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2A (SEQ ID NO: 1) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2 A (SEQ ID NO: 1).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2A (SEQ ID NO: 1) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2A (SEQ ID NO: 1).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2A (SEQ ID NO: 2) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2A (SEQ ID NO: 2).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 1065 of the nucleotide sequence shown in Figure 2B (SEQ ID NO: 3) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1065 of the nucleotide sequence shown in Figure 2B (SEQ ID NO: 3).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2B (SEQ ID NO: 3) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2B (SEQ ID NO: 3).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2B (SEQ ID NO: 4) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2B (SEQ ID NO: 4).
- the exogenous nucleic acid encoding Annexin-Al is from the same species as the transgenic non-human animal of the invention. Accordingly, in these embodiments of the invention, the transgenic non-human animal is typically a mouse.
- the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2C (SEQ ID NO: 5) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2C (SEQ ID NO: 5).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2C (SEQ ID NO: 5) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2C (SEQ ID NO: 5).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2C (SEQ ID NO: 6) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2C (SEQ ID NO: 6).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 1155 of the nucleotide sequence shown in Figure 2D (SEQ ID NO: 7) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1155 of the nucleotide sequence shown in Figure 2D (SEQ ID NO: 7).
- the Annexin-Al is encoded by a nucleotide sequence comprising (a) the nucleotide sequence shown in Figure 2D (SEQ ID NO: 7) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2D (SEQ ID NO: 7).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2D (SEQ ID NO: 8) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2D (SEQ ID NO: 8).
- the exogenous nucleic acid encoding Annexin-Al is from the same species as the transgenic non-human animal of the invention. Accordingly, in these embodiments of the invention, the transgenic non-human animal is typically a rat.
- the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2E (SEQ ID NO: 9) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2E (SEQ ID NO: 9).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2E (SEQ ID NO: 9) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2E (SEQ ID NO: 9).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2E (SEQ ID NO: 10) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2E (SEQ ID NO: 10).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2F (SEQ ID NO: 11) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2F (SEQ ID NO: 11).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2F (SEQ ID NO: 11) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2F (SEQ ID NO: 11).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2F (SEQ ID NO: 12) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2F (SEQ ID NO: 12).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 612 of the nucleotide sequence shown in Figure 2G (SEQ ID NO: 13) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 612 of the nucleotide sequence shown in Figure 2G (SEQ ID NO: 13).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2G (SEQ ID NO: 13) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2G (SEQ ID NO: 13).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2G (SEQ ID NO: 14) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2G (SEQ ID NO: 1 ).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) nucleotides 4 to 345 of the nucleotide sequence shown in Figure 2H (SEQ ID NO: 15) or (b) a nucleotide sequence having at least 70% identity to nucleotides 4 to 345 of the nucleotide sequence shown in Figure 2H (SEQ ID NO: 15).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) the nucleotide sequence shown in Figure 2H (SEQ ID NO: 15) or (b) a nucleotide sequence having at least 70% identity to the nucleotide sequence shown in Figure 2H (SEQ ID NO: 15).
- the exogenous nucleic acid encoding Annexin-Al comprises (a) a nucleotide sequence encoding the amino acid sequence shown in Figure 2H (SEQ ID NO: 16) or (b) a nucleotide sequence having at least 70% identity to a nucleotide sequence encoding the amino acid sequence shown in Figure 2H (SEQ ID NO: 16).
- the exogenous nucleic acid encoding Annexin-Al has some sequence variations compared to the sequence of an endogenous nucleic acid encoding Annexin-Al.
- the exogenous nucleic acid encoding Annexin-Al comprises a nucleotide sequence having at least 75%, 80%, 82%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity to any one of the nucleotide sequences described above.
- nucleotide sequence requires a start codon (ATG) and a stop codon (TAG, TAA or TGA) in order to be translated into a peptide.
- ATG start codon
- TAA stop codon
- TGA stop codon
- the sequences shown in Figure 2 generally include start and stop codons. Accordingly, the invention encompasses the use of fragments of the sequences shown in Figure 2, most typically those fragments that do not include the start and stop codons.
- nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2 A SEQ ID NO: 1
- nucleotides 4 to 1065 of the nucleotide sequence shown in Figure 2B SEQ ID NO: 3
- nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2C SEQ ID NO: 5
- nucleotides 4 to 1155 of the nucleotide sequence shown in Figure 2D SEQ ID NO: 7
- nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2E SEQ ID NO: 9
- nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2F SEQ ID NO: 11
- nucleotides 4 to 612 of the nucleotide sequence shown in Figure 2G SEQ ID NO: 13
- nucleotides 4 to 345 of the nucleotide sequence shown in Figure 2H SEQ ID NO: 15
- the exogenous nucleic acid encoding Annexin-Al further includes nucleotide sequences that represent genetic elements useful for transcription and/or nucleotide sequences encoding peptide markers or tags, as described in detail below. In these embodiments, further nucleotide sequences will be included after the start codon and/or before the stop codon.
- the exogenous nucleic acid encoding Annexin-Al further encodes a tag, such as an epitope tag, or a marker, such as a fluorescent marker.
- a tag such as an epitope tag
- a marker such as a fluorescent marker.
- the addition of one or more such markers or tags allows the overexpressed Annexin-Al (encoded by the exogenous nucleic acid encoding Annexin-Al) to be differentiated from endogenous Annexin-Al.
- the different Annexin-Al proteins can then be differentiated using biological or physical assay techniques.
- the marker or tag may be at the amino terminus or carboxy termmus of the overexpressed Annexin-Al protein or may be inserted internally with respect to the amino acid sequence of the overexpressed Annexin-Al protein.
- the tag or marker is typically a peptide, typically a peptide consisting of 5 to 250 amino acids. Typically, the peptide consists of 5 to 50, 10 to 60, 20 to 70, 30 to 80, 40 to 90, 50 to 100, 60 to 150, 70 to 200 or 80 to 250 amino acids.
- the tag is an epitope tag.
- An epitope is a short peptide that is a defined amino acid sequence from a protein with a cognate antibody. The skilled person can select such epitopes based on sequences identified as possessing antigenic properties. These tags are particularly useful for western blotting, immunofluorescence and immunoprecipitation experiments, although they also find use in antibody purification.
- the epitope tag has one of the following amino acid sequences: FLAG tag (KDDDDKYD, SEQ ID NO: 17), c-myc (EQKLISEEDL, SEQ ID NO: 18), simian virus V5 (G PIPNPLLGLDST, SEQ ID NO.
- Haemagglutinin (HA) (YPYDVPDYA, SEQ ID NO: 20), ClonelOO (NVRFSTIVRRRA, SEQ ID NO: 21), rabl la (KQMSDRRENDMSPS, SEQ ID NO: 22), DOB (SGNEVSRAVLLPQSC, SEQ ID NO: 23), SG11 (SSLS YTNPAVAATSANL, SEQ ID NO: 24), erbB4 (RSTLQHPDYLQEYST, SEQ ID NO: 25), ARF (VSTLLRWERFPGHRQA, SEQ ID NO: 26), RY (KFQQLVQCLTEFHAALGAYV, SEQ ID NO: 27), WILPEP1 (QEQCQEVWRKRVISAFLKSP, SEQ ID NO: 28) or HAFIO (RLSDKTGPVAQEKS, SEQ ID NO: 29).
- the epitope tag is recognised by its cognate antibody irrespective of whether it is located at the amino terminus, carboxy terminus or in an internal domain of Annexin-Al .
- the nucleic acid encodes a FLAG tag.
- the nucleic acid includes the sequence GATTACAAGGATGACGACGATAAG (SEQ ID NO: 30).
- the tag possesses enzymatic activity that converts a substrate to a form that is readily detectable by an assay, for example a kinase activity specifying phosphorylation of another protein or peptide substrate that could be added to a secreted or excreted analyte along with a phosphate group donor. Detection could be achieved using an immunological assay based on detection by an antibody specifically recognising the phosphorylated version of the tagged reporter protein or alternatively by the use of phosphate radiolabeled with an isotope of phosphorous such as P or P.
- Other enzymic modifications include for example acetylation, sulphation and glycosylation.
- peptide tag that is a catalytic sequence of an enzyme such as Glutathione-S-transferase (GST) where enzyme activity can be detected by means of an activity assay or by antibody reactivity.
- GST Glutathione-S-transferase
- the marker is a fluorescent marker.
- fluorescent markers are used to give visual readout on a protein. Fluorescent markers include green fluorescent protein (GFP) and red fluorescent protein (RFP).
- the nucleotide encoding the marker or tag is contiguous with the coding sequence for the Annexin-Al, such as the coding sequence shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 or SEQ ID NO: 7, and as described above is typically included after the coding sequence but prior to the stop codon (for example TAG, TGA or TAA).
- a linker nucleic acid sequence may be inserted between the two sequences that encodes a short sequence of amino acids.
- the exogenous nucleic acid encoding Annexin-Al further includes one or more nucleotide sequences encoding other genetic elements.
- a Kozak sequence can be included to enhance transcription.
- a suitable Kozak sequence is GCCACC (SEQ ID NO: 31).
- Other genetic elements that could usefully be included will be known to a person skilled in the art.
- FIG. 3B The sequence of Anx-Al FLAG inserted into the mice generated in the Examples is shown in Figure 3B (SEQ ID NO: 32).
- the sequence includes Smal (and isoschizomer Xmal) recognition sites (CCCGGG, SEQ ID NO: 33), Kozak sequence (GCCACC, SEQ ID NO: 31), Anx-Al FLAG start and stop codons (ATG and TAG respectively) as well the coding sequence for the incorporated FLAG epitope (GATTACAAGGATGACGACGATAAG, SEQ ID NO: 30).
- the forward and reverse primers are also shown.
- the resulting expressed protein has the incorporated FLAG epitope (KDDDDKYD, SEQ ID NO: 17) at the C-terminus of the protein.
- Identity is the relationship between two or more polynucleotide sequences or two or more polypeptide sequences, as determined by comparing the sequences, typically along their whole length. In the art, identity also means the degree of sequence relatedness between polynucleotide or polypeptide sequences, as the case may be, as determined by the match between strings of such sequences. While there exist a number of methods to measure identity between two polynucleotide or two polypeptide sequences, methods commonly employed to determine identity are codified in computer programs.
- a global alignment attempts to align every residue in every sequence and thus forces the alignment to span the entire length of all query sequences.
- Global alignments are most useful when the sequences in the query set are similar and of approximately equal size.
- a general global alignment technique is the Needleman-Wunsch algorithm, which is based on dynamic programming.
- local alignments identify regions of similarity within long sequences that can be widely divergent overall. Local alignments are often preferable, but can be more difficult to calculate because of the additional challenge of identifying the regions of similarity. Local alignments are more useful for dissimilar sequences that are suspected to contain regions of similarity or similar sequence motifs within a larger sequence.
- the Smith- Waterman algorithm is a general local alignment method and is also based on dynamic programming. With sufficiently similar sequences, there is no difference between local and global alignments. Hybrid methods, known as semiglobal or "glocal" (short for global-local) methods, attempt to find the best possible alignment that includes the start and end of one or the other sequence. This can be especially useful when the downstream part of one sequence overlaps with the upstream part of the other sequence.
- Preferred computer programs to determine identity between two sequences include, but are not limited to, BLAST (Altschul et al., J. Mol. Biol.
- CiustalW/CIustalX (Thompson et al., Nucleic Acids Research 22 (22): 4673-4680 (1994), latest version is 2.1) and the GCG program package (Devereux et al., Nucleic Acids Research, 12, 387 (1984)).
- the Clustal program can be used to compare both nucleotide and amino acid sequences. This program compares sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of identity analysis are contemplated in the present invention.
- the percent identity of two amino acid sequences or of two nucleic acid sequences can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced in the one sequence for best alignment with the other sequence) and comparing the amino acid residues or nucleotides at corresponding positions.
- the "best alignment" is an alignment of two sequences which results in the highest percent identity.
- Gapped BLAST can be utilised as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402.
- PSI-Blast can be used to perform an iterated search which detects distant relationships between molecules (Id.).
- the nucleotide sequences used in the invention have at least 70% identity, using the default parameters of the BLAST computer program (Atschul et al., J. Mol. Biol. 215, 403- 410 (1990)) provided by HGMP (Human Genome Mapping Project), at the nucleotide level, to the nucleotide sequences described herein. More typically, the nucleotide sequence has at least 75%, 80%, 82%, 83%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% identity, at the nucleotide level, to the nucleotide sequences described herein. In some embodiments, the nucleotide sequence has 83.6%, 93.8% or 97.3% identity to the nucleotide sequence shown in Figure 2A (SEQ ID NO: 1).
- the exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of the transgenic non-human animal of the present invention.
- This expression pattern is typically achieved by the nucleic acid encoding Annexin-Al being under the control of a suitable promoter, i.e. a T cell-specific promoter.
- a suitable T cell-specific promoter for use in the invention is the CD2 promoter.
- This promoter drives expression in both immature and mature T cells.
- the human or mouse CD2 promoter can be used.
- one of the promoters of the human T- cell- or lymphocyte-specific gene, lck can be used. This gene encodes a tyrosine kinase and is a member of the src family .
- the promoters preferentially drive the expression of transgenic genes at the level of immature T cells (thymocytes).
- a promoter for use in the invention can be a constitutive promoter or an inducible promoter.
- the promoter is typically a constitutive promoter, but an inducible promoter can alternatively be used such that the T cell specific overexpression of Annexin-Al occurs in response to a particular stimulus.
- a nucleic acid according to the invention is suitably inserted into a vector for expression in the transgenic non-human animal of the invention.
- the vector is typically an expression vector that contains nucleic acid sequences as defined above.
- vector or "expression vector” generally refers to any nucleic acid vector which may be RNA, DNA or cDNA.
- expression vector may include, among others, chromosomal, episomal, and virus-derived vectors, for example, vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
- any vector suitable to maintain, propagate or express nucleic acid to express a polypeptide in a host can be used for expression in this regard.
- Expression vectors will include various genetic elements, including but not limited to an origin of replication, a suitable promoter as defined above (typically a T cell-specific promoter) and/or enhancer and/or a locus control region (LCR), and also any necessary ribosome binding sites, polyadenylation regions, splice donor and acceptor sites, transcriptional termination sequences, and 5'-flanking non-transcribed sequences that are necessary for expression.
- an expression vector for use in the invention can include a selectable marker to permit isolation of vector containing cells after exposure to the vector.
- the expression vectors can also include selectable markers, such as antibiotic resistance, which enable the vectors to be propagated.
- the expression vector for use in the invention includes an origin of replication, a promoter and an LCR.
- the promoter is typically a T cell-specific promoter, for example a CD2 promoter or an LCK promoter.
- the vector used in the Examples herein is the VACD2 vector having the structure shown in Figure 4.
- the vector includes components of the prokaryotic pBS SK- vector, the human CD2 promoter and LCR, as well as the exogenous nucleic acid encoding Anx-Al .
- any suitable vector can be used.
- the nucleic acid encoding Annexin-Al is typically stably integrated into the genome of the transgenic non-human animal of the invention.
- the cells of the offspring of the transgenic non-human animal of the present invention also contain the same exogenous nucleic acid encoding Annexin-Al as cells of the transgenic non-human animal of the present invention.
- the transgenic non-human animal of the present invention can be prepared by any suitable method known in the art. Suitable methods include but are not limited to pronuclear injection, methods involving transformation of embryonic stem cells and somatic cell nuclear transfer.
- the transgenic non-human animal of the present invention is prepared by the technique of pronuclear microinjection. This technique is well known in the art and was first described by Gordon et al (Proc Natl Acad Sci U S A. 1980 December; 77(12): 7380- 7384). The technique involves directly injecting DNA into the pronucleus of a fertilized egg and then implanting the egg into an animal such as a mouse.
- the DNA encodes Annexin-Al and is under the control of a T cell-specific promoter.
- the DNA is typically in the form of a vector.
- the transgenic non-human animal of the present invention is prepared by transforming embryonic stem cells with a DNA sequence of interest, injecting the embryonic stem cells into the inner cell mass of a blastocyst and then implanting the blastocyst into an animal such as a mouse.
- the DNA encodes Annexin-Al and is under the control of a T cell-specific promoter.
- the DNA is typically in the form of a vector.
- the transgenic non-human animal of the present invention is prepared using a nuclear transfer cloning procedure, typically somatic cell nuclear transfer (SCNT),
- SCNT somatic cell nuclear transfer
- the non-human animal may be subjected to further transgenesis, in which the transgenesis is the introduction of an additional gene or genes or a protein-encoding nucleic acid sequence or sequences that encode proteins other than Annexin-Al.
- the transgenesis may be carried out by any of the methods described herein for producing the transgenic non-human animal of the invention, but the additional exogenous nucleic acid encodes a different protein from Annexin-Al.
- the present invention provides a transgenic non-human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD wherein an exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of said animal.
- OCD obsessive compulsive disorder
- Annexin-Al is overexpressed in human T cells using adenoviral, retroviral or electroporation methods.
- the human T cells are then transferred into an immunodeficient non-human animal such as an immunodeficient mouse, also known as a severe combined immunodeficiency (SCID) mouse.
- SCID mice which lack both T and B lymphocytes and accept xenogenic cells, have been used for human cell transfer for evaluating the pathogenesis of a variety of human diseases.
- the exogenous nucleic acid encoding Annexin-Al typically encodes a human or mouse Annexin- Al as described in detail herein and may have any one of the sequences set out in SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13 or SEQ ID NO: 15 or SEQ ID NO: 1 or SEQ ID NO: 3, or a fragment thereof such as a coding sequence, as described herein.
- the present invention provides a method of producing a transgenic non- human animal embryo, comprising inserting an exogenous nucleic acid encoding Annexin-Al under control of a T cell-specific promoter into a fertilized egg, blastocyst or enucleated oocyte of a non-human animal.
- the method can be carried out using pronuclear microinjection, by transforming embryonic stem cells with a DNA sequence of interest and injecting the embryonic stem cells into the inner cell mass of a blastocyst or by using somatic cell nuclear transfer.
- an embryo is to be construed broadly as being from the blastocyst stage up to birth.
- the blastocyst may be stored, for example by freezing, subsequent to being produced by this method.
- the present invention provides a method of producing a transgenic non- human animal model for obsessive compulsive disorder (OCD) or a disease related to OCD, comprising:
- the non-human animal embryo can be implanted into a female non-human animal by any suitable method, which will be known to a person of skill in the art.
- the method of the third aspect of the invention further comprises breeding from the non-human animal. The result of this method is offspring of the non-human animal of the first aspect of the invention.
- the present invention therefore provides a cell line derived from the transgenic non-human animal model of the first aspect of the invention.
- the cell line will typically be a blood cell line, for example a cell line of any cell derived from the haematopoietic lineage.
- the cell line is a haematopoietic stem cell line.
- the cell line is typically a lymphocyte cell line and is more typically a T cell line.
- an exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of the transgenic non-human animal of the first aspect of the invention.
- T cells derived from the transgenic non-human animal of the first aspect of the invention are useful for scientific study, for example to investigate relationships between T cells and OCD.
- a T cell line can be prepared, for example, by extracting T cells from a transgenic non-human animal model of the first aspect of the invention and then culturing these cells in vitro or by transferring them into "recipient" mice (adoptive cell transfer) to see if signs of OCD can be transferred by these cells.
- T cells can be collected from spleen and lymph nodes and optionally further purified by commercially available kits.
- the transgenic non-human animal model of the present invention is therefore a useful tool for studying the symptoms and progression of OCD and related diseases.
- the invention therefore also extends to the use of tire transgenic non-human animal of the first aspect of the invention for the study of OCD or a disease related to OCD.
- the invention also extends to the use of the transgenic non-human animal of the first aspect of the invention as an animal model for OCD or a disease related to OCD.
- the transgenic non-human animal model of the present invention is a useful tool to investigate the link between T cells and the development of OCD and related diseases.
- the invention therefore also extends to the use of the transgenic non-human animal of the first aspect of the invention to investigate the link between T cells and the development of OCD or a disease related to OCD.
- investigating the gene expression profile of AnxAl 18 T cells should provide information on the specific phenotype of these cells and its impact on known neuronal pathways involved in the development of OCD. The availability of this information might also help to understand the behavioural changes observed in immunocompromised or immunosuppressed patients.
- the transgenic non-buman animal model of the present invention is also a valuable tool to test for drugs for use in the treatment of OCD and related diseases.
- the present invention provides a method of screening a candidate molecule to determine whether said candidate molecule is effective for the treatment of OCD or a disease related to OCD comprising:
- the candidate molecule can be any molecule that is suitable for administering to a human or animal for treatment of a disease.
- the candidate molecule can be a chemical entity or a biological molecule such as an antibody.
- the candidate molecule can, for example, be a T cell modulating drug or an anti-psychotic or anxiolytic drug.
- Step (a) of the fifth aspect of the invention comprises administering the candidate molecule to the non-human transgenic animal model of the first aspect of the invention.
- the candidate molecule can be administered to the animal in any suitable manner, but will typically by administered orally or by injection, for example by intravenous, intraperitoneal or subcutaneous injection.
- the candidate molecule can be formulated with one or more pharmacologically acceptable excipients or diluents for testing in a method according to this aspect of the invention.
- Step (b) of the fifth aspect of the invention comprises determining whether said non-human transgenic animal model exhibits reduced symptoms of OCD or a disease related to OCD after administration of the candidate molecule. This step will be carried out after the candidate molecule is administered to the non-human transgenic animal model. Step (b) of this aspect of the invention is typically carried out by monitoring the behaviour of the non- human transgenic animal model after administration of the candidate molecule. For example, in a transgenic mouse model, the reduction of marble burying behaviour or digging behaviour (both proposed to reflect compulsive behaviour) after administration of the candidate molecule compared to the level of such behaviour prior to administration of the candidate molecule indicates that the candidate molecule is effective at treating OCD or a disease related to OCD.
- the method of the fifth aspect of the invention is a straightforward and cost-effective method for drug screening, since the spontaneous mouse model of OCD and related disorders requires no behavioural training and no pharmacological manipulation.
- the transgenic non- human animal model of the present invention can also be used to test for drugs for use in the treatment of T cell mediated diseases.
- the present invention provides a method of screening a candidate molecule to determine whether said candidate molecule is effective for the treatment of a T cell mediated disease comprising:
- the candidate molecule is effective for the treatment of the T cell mediated disease.
- the T cell mediated disease can be, for example, graft-versus-host disease, graft rejection, atherosclerosis, HIV, AIDS, psoriasis, miscarriage or an autoimmune disease.
- Autoimmune diseases include rheumatoid arthritis (RA), multiple sclerosis (MS) ; systemic lupus erythematosus (SLE), Addison's disease, Grave's disease, scleroderma, polymyositis, diabetes, autoimmune uveoretinitis, ulcerative colitis, pemphigus vulgaris, inflammatory bowel disease, autoimmune thyroiditis, uveitis, Behcet's disease and Sjogren's syndrome.
- RA rheumatoid arthritis
- MS multiple sclerosis
- SLE systemic lupus erythematosus
- Addison's disease Grave's disease, scleroderma, polymyositis, diabetes, autoimmune
- the candidate molecule can be any molecule that is suitable for administering to a human or animal for treatment of a disease.
- the candidate molecule can be a chemical entity or a biological molecule such as an antibody.
- Step (a) of the sixth aspect of the invention is the same as step (a) of the fifth aspect of the invention and is thus as described herein in relation to step (a) of the fifth aspect of the invention.
- Step (b) of the sixth aspect of the invention comprises determining whether said non-human transgenic animal model exhibits reduced symptoms of the T cell mediated disease after administration of the candidate molecule. This step will be carried out after the candidate molecule is administered to the non-human transgenic animal model.
- the symptoms could for example be symptoms of MOG 3-5s-i **d experimental autoimmune encephalomyelitis (EAE) or collagen-induced arthritis (CIA), which are mouse models of multiple sclerosis (MS) and rheumatoid arthritis (RA) respectively.
- the Anx-Al tg mice produced by the present inventors show signs of spontaneous anxiety/OCD behaviour as seen in other genetically modified mice.
- the genetic modification introduced in these mice i.e. overexpression of AnxAl
- T cells T cells that are specifically restricted to T cells. Therefore, these mice provide a unique opportunity to investigate how alteration in T cell functions can in turn influence the development of neuropsychiatric disorders.
- Annexin-1 over-expression is strong, T cell specific, copy-number dependent and genome integration position independent.
- the present invention provides a transgenic mouse model for obsessive compulsive disorder (OCD) or a disease related to OCD wherein the cells of said mouse contain an exogenous nucleic acid encoding Annexin-Al and comprising nucleotides 4 to 1038 of the nucleotide sequence shown in Figure 2A (SEQ ID NO: 1) and wherein said exogenous nucleic acid encoding Annexin-Al is expressed in the T cells of said animal.
- the transgenic mouse overexpresses Annexin-Al in its T cells.
- the exogenous nucleic acid encoding Annexin-Al is under control of the human CD2 promoter.
- the exogenous nucleic acid further comprises a nucleotide sequence encoding a FLAG tag, for example the nucleotide sequence GATTACAAGGATGACGACGATAAG (SEQ ID NO: 30).
- the cells of said mouse contain an exogenous nucleic acid having the sequence shown in Figure 3B (SEQ ID NO: 32).
- the transgenic mouse is generated by inserting C-terminus FLAG-tagged Anx-Al into the mouse genome using the technique of pronuclear microinjection and using a VACD2 cassette vector.
- the C-terminus FLAG-tagged Anx-Al is inserted into the mouse genome as part of a vector, typically the VACD2 vector which is 15425 bp in length and contains the appropriate elements for in vivo expression of the inserted gene in mouse.
- the vector typically includes the human CD2 Locus Control Region (hCD2LCR), the human CD2 promoter and the prokaryotic pBS SK- vector.
- restriction enzymes such as Notl and Sail are used to remove the prokaryotic sequences from the pBS SK- vector and a restriction enzyme such as Smal used to insert the Anx-Al FLAG gene.
- the construct is typically inserted into the mouse genome by pronuclear microinjection. Briefly, the (optionally purified) construct is microinjected into the pronucleus of embryos, typically embryonic day 0.5 (E0.5) embryos. Microinjected embryos that survive injection are transferred to the oviduct of a foster mother, typically an E0.5 pseudopregnant foster mother. Offspring are then born approximately 20 days later.
- FIGURE 1 is a ribbon diagram of annexin-1 structure showing the four annexin repeats and the N-terminal domain.
- FIGURE 2 A shows (i) the nucleotide sequence (SEQ ID NO: 1) and (ii) the amino acid sequence (SEQ ID NO: 2) of mouse Annexin-1 (Anx-Al), isoform Anxal-001.
- Figure 2B shows (i) the nucleotide sequence (SEQ ID NO: 3) and (ii) the amino acid sequence (SEQ ID NO: 4) of mouse Armexin-1 (Anx-Al), isoform Anxal-201.
- Figure 2C shows (i) the nucleotide sequence (SEQ ID NO: 5) and (ii) the amino acid sequence (SEQ ID NO: 6) of rat Annexin-1 (Anx-Al), isoform Anxal .
- Figure 2D shows (i) the nucleotide sequence (SEQ ID NO: 7) and (ii) the amino acid sequence (SEQ ID NO: 8) of rat Annexin-1 (Anx-Al), isoform D3ZVZ4_RAT.
- Figure 2E shows (i) the nucleotide sequence (SEQ ID NO: 9) and (ii) the amino acid sequence (SEQ ID NO: 10) of human Annexin-1 (Anx-Al), isoform ANXA1-003.
- Figure 2F shows (i) the nucleotide sequence (SEQ ID NO: 11) and (ii) the amino acid sequence (SEQ ID NO: 12) of human Annexin-1 (Anx-Al), isoform ANXA1-002.
- Figure 2G shows (i) the nucleotide sequence (SEQ ID NO: 13) and (ii) the amino acid sequence (SEQ ID NO: 14) of human Annexin-1 (Anx-Al), isoform ANXA1-004.
- Figure 2H shows (i) the nucleotide sequence (SEQ ID NO: 15) and (ii) the amino acid sequence (SEQ ID NO: 16) of human Annexin-1 (Anx-Al), isoform ANXA1-006.
- FIGURE 3 shows the generation of the Anx-Al FLAG.
- A Agarose gel. analysis of Anx-Al FLAG PCR product (1041 bp) generated from RAW264.7 cell cDNA.
- B Sequence of Anx-Al FLAG (SEQ ID NO: 32) showing the Anx-Al coding sequence (between start codon and FLAG epitope), the Smal (and isoschizomer Xmal) recognition sites (CCCGGG, SEQ ID NO: 33), Kozak sequence (GCCACC, SEQ ID NO: 31), Anx-Al FLAG start and stop codons (ATG and TAG respectively) as well the coding sequence for the incorporated FLAG epitope (GATTACAAGGATGACGACGATAAG, SEQ ID NO: 30). The forward and reverse primers are also shown.
- the resulting expressed protein has the incorporated FLAG epitope (KDDDDKYD, SEQ ID NO: 17) at the C-terminus of the protein.
- FIGURE 4 (A) shows the VACD2/FLAG Anx-Al construct used for the generation of Anx-Al transgenic mice.
- the VACD2 vector is 15425 bp in length and contains the appropriate elements for in vivo expression of the inserted gene in mouse. Shown here, the human CD2 Locus Control Region (hCD2LCR), the human CD2 promoter and the prokaryotic pBS SK- vector. Enzymes that were used to manipulate the vector prior to microinjection to the mouse genome are shown. Notl and Sail were used to remove the prokaryotic sequences and Smal used to insert the Anx-Al FLAG gene.
- FIG. B is a schematic representation of the VACD2 Anx-Al Flag construct showing the CD2 promoter region, the inserted Anx-Al Flag cDNA and the locus control region (LCR).
- the figure also shows a schematic representation of the Anx-Al Flag with the 4 annexin repeats (I-II-III-IV) and the amino acid sequence of the Flag epitope used to tag the protein.
- FIGURE 5 shows that Anx-Al tg mice exhibit an increase in marble burying behavior.
- B Representative pictures of the experimental setting used for the marble burying test: cage with marble before the experiment (Start), cage after the test with C57 BL6 (C57/BL6) or AnxAl' s (AnxAl tg ) mice.
- FIGURE 6 shows that Anx-Al tg mice exhibit an increased digging behavior.
- B Representative pictures showing the typical behavior C57/BL6 (C57 BL6) or AnxAl' 8 (AnxAl tg ) mice in their home cage.
- FIGURE 7 shows T cell repertoire and response of Anx-Al t T cells.
- A Spleen (top panels) and lymph node lymphocytes from wt C57 BL6 and Anx-Al t8 mice were analysed for CD4 and CD8 expression. Numbers in dot plots indicate the percentage of cells falling in the quadrant for the respective marker (CD4 and CD8). Results are representative of 4 experiments.
- B Naive CD4+ T cells were stimulated with indicated concentrations of plate-bound anti-CD3/CD28 for 12-16 hrs and then analyzed for their profile of CD25 and CD69 activation markers. Numbers in dot plots indicate the percentage of CD25/CD69 double positive. Results are representative of 3 experiments.
- FIGURE 8 shows increased development of MOG35-55-induced EAE in Anx-Al 3 ⁇ 4 mice.
- Example 1 Generation of Anx-Al 3 ⁇ 4 mice
- Transgenic mice overexpressing Anx-Al (Anx-Al tg ) in T cells were generated by inserting C- terminus FLAG-tagged Anx-Al into the mouse genome using a VACD2 cassette vector and the technique of pronuclear microinjection. Briefly, the murine Anx-Al gene was amplified and tagged with the FLAG epitope and then cloned into the pcDNA3.1 vector. The Anx-Al FLAG was recovered from the pcDNA3.1 vector and then ligated into linearised VACD2 vector. The VACD2 Anx-Al FLAG construct was then modified and purified, then inserted into the mouse genome by pronuclear injection. The methods will now be described in detail. Generation of FLAG tagged version of Anx-Al - Polymerase chain reaction (PCR)
- Figure 3B shows the sequence of Anx-Al FLAG (SEQ ID NO: 32) showing the Anx-Al coding sequence, the Smal (and isoschizomer Xmal) recognition sites, Kozak sequence, Anx-Al FLAG start and stop codons as well the coding sequence for the incorporated FLAG epitope. The forward and reverse primers are also shown. Table 1. Primers used for the amplification of Anx-Al FLAG and reaction conditions.
- the CCCGGG sequence (SEQ ID NO: 33) is the recognition site for Smal (and its isoschizomer Xmal), the KOZAK sequence GCCACC (SEQ ID NO: 31) is contiguous with the ATG start codon.
- the sequence CTACTTATCGTCGTCATCCTTGTAATC (SEQ ID NO: 34) includes the coding sequence for the FLAG epitope DYKDDDD (SEQ ID NO: 35) and a stop codon and was incorporated in frame with the Anx-Al coding region. (Bottom) PCR conditions used for the reaction.
- Annexin-1 FLAG cDNA was cloned into the Smal site of pcDNA3.1 (+) vector (Invitrogen).
- the PCR product and the host vector pcDNA3.1 were digested with Smal for 2 hours. Both the insert and the linearized vector digest products were analysed and purified by agarose gel electrophoresis (1 % agarose gels, TAE).
- the two linearized fragments to be ligated were treated with 10 units of bovine alkaline phosphatase (Calf Intestine Phosphatase (CIP) Roche) in 1 Ox enzyme buffer for 1 hour at 37 °C. This dephosphorylates the ends of the fragments (removal of 5' phosphates), prevents re- ligation and improves ligation efficiency.
- CIP-treated DNA were quantified and ligated using high concentrated T4 Ligase enzyme (NEB, UK) and molar ratio of vector to insert 1:3, 1 :1 and 3:1.
- the reaction contained 10-50 ng of vector, 5 x ligase buffer, 5 units of T4 DNA ligase and water up to 20 ⁇ . The reactions were incubated for 16 hours at RT. Mock ligation controls were always included using only the vector or only the insert. Subcloning into the VACD2 cassette
- the VACD2 vector (kindly provided by D.Kioussis, National Institute for Medical Research, London) contains the human CD2 promoter and the locus control region of the human CD2 gene conferring tissue specific expression of the adjacent gene after integration into the mouse genome.
- the Anx-Al FLAG was recovered from pcDNA3.1 vector following Smal digestion that produces 'blunt-ended' fragments as well as by Xmal (isoschizomer) digestion that produces 'sticky-ends'.
- the VACD2 vector was linearised with Smal and Xmal digestion. Both digestions were performed as follows; 1 ⁇ g of DNA (in 5 ⁇ ) was mixed with 1 ⁇ of Smal (or Xmal) enzyme (10 units, NEB, UK), 2 ⁇ ⁇ enzyme buffer and 12 ⁇ of water. The samples were incubated for 1 h at 25 °C and separated on 0.8 % agarose gels. The 15.4 kb linearised fragment of the VACD2 vector and the 1 kb Anx-Al FLAG insert were gel purified.
- the two fragments were quantified and treated with 10 units of CIP. Ligation of blunt ended DNA fragments was performed at molar ratio of vector to insert 1:3, 1:1 and 3:1. The reaction contained 50 ng of vector, 5 x ligase buffer, 5 units of T4 DNA ligase and water. Ligation reactions were incubated at RT for 16 hours. Mock ligation controls were included using only vector and only insert.
- Microinjection in CBAxC57BL/6 mice was performed in the Transgenic core facility of Queen Mary University of London (http://www.icms.qmul.ac.uk/corefacilities/transgenic/index.html).
- the VACD2 Anx-Al FLAG construct was modified by removal of prokaryotic sequences and purified for pronuclear microinjection into the mouse genome.
- the purified construct was microinjected into the pronucleus of embryonic day 0.5 (E0.5) embryos.
- Microinjected embryos that survived injection were transferred to the oviduct of an E0.5 pseudopregnant foster mother. Offspring were then born approximately 20 days later.
- mice Male or female; 6-7 weeks of age were observed for their burying/digging performance in cages filled with 5 cm layer of sawdust bedding, lightly pressed down to make a flat even surface.. Measurements included the latency to start digging and the number of digging bouts. Test duration was 15 min.
- mice Male C57/BL6 mice were immunized with MOG3 3- 5 5 /CFA as previously described (Paschalidis et al., J Neuroinilarnmation. 2009; 6:33). Briefly, mice were immunized subcutaneously on day 0 with 300 ⁇ 1 of emulsion consisting of 300 g of MOG35-55 in PBS combined with an equal volume of CFA containing 300pg heat-killed M. tuberculosis H37Ra. The emulsion was injected in both flanks and followed by an intraperitoneal injection of B. pertussis toxin (500ng 100 ⁇ ) in 100 ⁇ of saline on days 0 and 2. Mice were observed daily for signs of EAE and weight loss.
- B. pertussis toxin 500ng 100 ⁇
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Environmental Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biochemistry (AREA)
- Veterinary Medicine (AREA)
- Microbiology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Physics & Mathematics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
La présente invention concerne un modèle animal non humain transgénique pour un trouble obsessionnel compulsif (TOC) ou une maladie associée à un TOC, les cellules dudit animal contenant un acide nucléique exogène codant pour l'annexine A1 et ledit acide nucléique exogène codant pour l'annexine A1 étant exprimé dans les lymphocytes T dudit animal, et l'utilisation d'un tel modèle animal non humain transgénique pour l'étude d'un TOC ou d'une maladie associée à un TOC. L'invention concerne en outre un procédé de production d'un embryon d'animal non humain transgénique et un procédé de production d'un modèle animal non humain transgénique pour un trouble obsessionnel compulsif (TOC) ou une maladie associée à un TOC, une lignée cellulaire dérivée du modèle animal non humain transgénique de l'invention, et des procédés de criblage de molécules candidates pour déterminer si elles sont efficaces pour le traitement d'un TOC ou d'une maladie associée à un TOC ou pour le traitement d'une maladie médiée par les lymphocytes T.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB201121561A GB201121561D0 (en) | 2011-12-14 | 2011-12-14 | Transgenic animal |
GB1121561.3 | 2011-12-14 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2013088110A1 true WO2013088110A1 (fr) | 2013-06-20 |
Family
ID=45560501
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/GB2012/000905 WO2013088110A1 (fr) | 2011-12-14 | 2012-12-14 | Animaux transgéniques pour l'annexine a1 |
Country Status (2)
Country | Link |
---|---|
GB (1) | GB201121561D0 (fr) |
WO (1) | WO2013088110A1 (fr) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150004164A1 (en) * | 2011-12-14 | 2015-01-01 | Queen Mary & Westfield College, Univ. of London | Use Of Antibody |
USRE47982E1 (en) | 2010-06-09 | 2020-05-12 | Queen Mary & Westfield College, University Of London | Annexin 1 antibody |
US11041019B2 (en) | 2017-02-08 | 2021-06-22 | Medannex Ltd. | Anti human annexin A1 antibody |
CN115362984A (zh) * | 2022-07-07 | 2022-11-22 | 成都中医药大学 | 一种小鼠自身免疫性甲状腺炎模型的构建方法 |
-
2011
- 2011-12-14 GB GB201121561A patent/GB201121561D0/en not_active Ceased
-
2012
- 2012-12-14 WO PCT/GB2012/000905 patent/WO2013088110A1/fr active Application Filing
Non-Patent Citations (25)
Title |
---|
ALTSCHUL ET AL., J. MOI. BIOL., vol. 215, no. 403, 1990 |
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 |
ALTSCHUL ET AL., NUCLEIC ACIDS RES., vol. 25, 1997, pages 3389 - 3402 |
ALTSCHUL ET AL., NUCLEIC ACIDS RESEARCH, vol. 25, no. 17, 1997, pages 3389 - 402 |
ATSCHUL ET AL., J. MOL. BIOL., vol. 215, 1990, pages 403 - 410 |
CAMPBELL ET AL., NATURE, vol. 380, no. 6569, 1996, pages 64 - 6 |
D'ACQUISTO ET AL., BLOOD, vol. 109, 2007, pages 1095 - 1102 |
D'ACQUISTO F ET AL: "Annexin-A1: a pivotal regulator of the innate and adaptive immune systems", BRITISH JOURNAL OF PHARMACOLOGY, NATURE PUBLISHING GROUP, BASINGSTOKE, HANTS; GB, vol. 155, no. 2, 1 September 2008 (2008-09-01), pages 152 - 169, XP002653853, ISSN: 0007-1188, [retrieved on 20080721], DOI: 10.1038/BJP.2008.252 * |
D'ACQUISTO FULVIO ET AL: "Annexin-1 modulates T-cell activation and differentiation", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 109, no. 3, 1 February 2007 (2007-02-01), pages 1095 - 1102, XP002582224, ISSN: 0006-4971 * |
DEVEREUX ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, 1984, pages 387 |
GAO ET AL., BEHAV GENET, vol. 41, no. 5, 2011, pages 724 - 733 |
GORDON ET AL., PROC NATL ACAD SCI USA., vol. 77, no. 12, December 1980 (1980-12-01), pages 7380 - 7384 |
JOHN C D ET AL: "Annexin A1 and the formyl peptide receptor family: neuroendocrine and metabolic aspects", CURRENT OPINION IN PHARMACOLOGY, ELSEVIER SCIENCE PUBLISHERS, NL, vol. 8, no. 6, 1 December 2008 (2008-12-01), pages 765 - 776, XP025669944, ISSN: 1471-4892, [retrieved on 20081024], DOI: 10.1016/J.COPH.2008.09.005 * |
KARLIN; ALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 2264 - 2268 |
KARLIN; ALTSCHUL, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 5873 - 5877 |
LIM ET AL., PROC NATL ACAD SCI USA, vol. 95, 1998, pages 14535 - 9 |
LUNBIAO CUI ET AL: "Overexpression of annexin a1 induced by terephthalic acid calculi in rat bladder cancer", PROTEOMICS, vol. 7, no. 22, 1 November 2007 (2007-11-01), pages 4192 - 4202, XP055054832, ISSN: 1615-9853, DOI: 10.1002/pmic.200700582 * |
NIKOLAOS PASCHALIDIS ET AL: "Role of endogenous Annexin-A1 in the regulation of thymocyte positive and negative selection", CELL CYCLE, vol. 9, no. 4, 15 February 2010 (2010-02-15), pages 785 - 794, XP055054841, ISSN: 1538-4101, DOI: 10.4161/cc.9.4.10673 * |
PASCHALIDIS ET AL., J NEUROINFLAMMATION, vol. 6, 2009, pages 33 |
PEARSON; LIPMAN, PROC. NATL. ACAD. SCI., vol. 85, 1988, pages 2444 - 8 |
THOMPSON ET AL., NUCLEIC ACIDS RESEARCH, vol. 22, no. 22, 1994, pages 4673 - 4680 |
TORELLIS; ROBOTTI, COMPUT. APPL. BIOSCI., vol. 10, 1994, pages 3 - 5 |
WANG L; SIMPSON HB; DULAWA SC, BEHAV PHARMACOL, vol. 20, no. 2, March 2009 (2009-03-01), pages 119 - 33 |
WANG LI ET AL: "Assessing the validity of current mouse genetic models of obsessive-compulsive disorder", BEHAVIOURAL PHARMACOLOGY, RAPID SCIENCE, PUBLISHERS, GB, vol. 20, no. 2, 1 March 2009 (2009-03-01), pages 119 - 133, XP009167440, ISSN: 0955-8810, DOI: 10.1097/FBP.0B013E32832A80AD * |
YING-FU LIU ET AL: "Identification of annexin A1 as a proinvasive and prognostic factor for lung adenocarcinoma", CLINICAL & EXPERIMENTAL METASTASIS, vol. 28, no. 5, 1 June 2011 (2011-06-01), pages 413 - 425, XP055054839, ISSN: 0262-0898, DOI: 10.1007/s10585-011-9380-1 * |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
USRE47982E1 (en) | 2010-06-09 | 2020-05-12 | Queen Mary & Westfield College, University Of London | Annexin 1 antibody |
US20150004164A1 (en) * | 2011-12-14 | 2015-01-01 | Queen Mary & Westfield College, Univ. of London | Use Of Antibody |
US10752677B2 (en) * | 2011-12-14 | 2020-08-25 | Queen Mary & Westfield College, University Of London | Methods of treating obsessive compulsive disorder (OCD) or anxiety using an antibody that binds to annexin-1 |
US11041019B2 (en) | 2017-02-08 | 2021-06-22 | Medannex Ltd. | Anti human annexin A1 antibody |
CN115362984A (zh) * | 2022-07-07 | 2022-11-22 | 成都中医药大学 | 一种小鼠自身免疫性甲状腺炎模型的构建方法 |
CN115362984B (zh) * | 2022-07-07 | 2024-01-16 | 成都中医药大学 | 一种小鼠自身免疫性甲状腺炎模型的构建方法 |
Also Published As
Publication number | Publication date |
---|---|
GB201121561D0 (en) | 2012-01-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP7026678B2 (ja) | C9orf72座位中にヘキサヌクレオチドリピート伸長を有する非ヒト動物 | |
JP5509417B2 (ja) | 魚類のVasa遺伝子を用いた生殖細胞マーカー | |
US6891031B2 (en) | Coordinate cytokine regulatory sequences | |
CN107815465B (zh) | 人源化基因改造动物模型的制备方法及应用 | |
JP2022113700A (ja) | CRISPR-Casゲノム編集に基づく、Fel d1ノックアウト並びに関連組成物及び方法 | |
Hasegawa et al. | Generation of CRISPR/Cas9-mediated bicistronic knock-in ins1-cre driver mice | |
CN104797132B (zh) | 基因敲入非人动物 | |
WO2013088110A1 (fr) | Animaux transgéniques pour l'annexine a1 | |
CN109207524A (zh) | 基于fto基因的人类肥胖症斑马鱼模型的建立与应用 | |
CN108777951B (zh) | 具有包括改造的多样性簇的免疫球蛋白重链可变区的非人动物及其用途 | |
CN106521638A (zh) | 一种基因突变大鼠资源库及其制备方法 | |
Trappe et al. | The Caenorhabditis elegans ortholog of human PHF5a shows a muscle-specific expression domain and is essential for C. elegans morphogenetic development | |
US20200149063A1 (en) | Methods for gender determination and selection of avian embryos in unhatched eggs | |
CN108070614B (zh) | 人源化基因改造动物模型的制备方法及应用 | |
JPH05507850A (ja) | 性決定遺伝子 | |
Michalski et al. | Generation of a new frizzled 2 flox mouse model to clarify its role in development | |
EP2246429A1 (fr) | Modèle mammifère non humain de l'épilepsie | |
EP1790724A1 (fr) | Animal non-humain assommé | |
CN114747541B (zh) | 一种psgl-1人源化非人类动物模型的构建方法及应用 | |
JP2005500835A (ja) | Pervスクリーニング法およびその使用 | |
JP7502805B2 (ja) | 補体媒介性溶解に対する細胞感受性を増加させる方法 | |
KR20190038613A (ko) | 유전자 변형된 면역결핍 비-인간 동물에서의 개선된 인간 적혈구 생존에 관한 방법 및 조성물 | |
KR101917346B1 (ko) | Thrsp 과발현 형질전환 동물모델 및 이의 용도 | |
JP3483552B2 (ja) | 新規時計遺伝子プロモーター | |
Toperzer | CRISPR-Based Genome Editing of Caenorhabditis Elegans and Cultured Human Cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 12806075 Country of ref document: EP Kind code of ref document: A1 |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 12806075 Country of ref document: EP Kind code of ref document: A1 |