WO2004108920A1 - 抗肥満薬のスクリーニング方法及び肥満モデル動物 - Google Patents
抗肥満薬のスクリーニング方法及び肥満モデル動物 Download PDFInfo
- Publication number
- WO2004108920A1 WO2004108920A1 PCT/JP2004/007692 JP2004007692W WO2004108920A1 WO 2004108920 A1 WO2004108920 A1 WO 2004108920A1 JP 2004007692 W JP2004007692 W JP 2004007692W WO 2004108920 A1 WO2004108920 A1 WO 2004108920A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- agf
- seq
- mice
- dna
- mouse
- Prior art date
Links
- 241001465754 Metazoa Species 0.000 title claims abstract description 76
- 238000000034 method Methods 0.000 title claims abstract description 76
- 239000003814 drug Substances 0.000 title claims abstract description 41
- 238000012216 screening Methods 0.000 title claims abstract description 27
- 208000008589 Obesity Diseases 0.000 title abstract description 46
- 235000020824 obesity Nutrition 0.000 title abstract description 46
- 101710085819 Angiopoietin-related protein 6 Proteins 0.000 claims abstract description 220
- 102100034599 Angiopoietin-related protein 6 Human genes 0.000 claims abstract description 209
- 206010012601 diabetes mellitus Diseases 0.000 claims abstract description 42
- 230000009261 transgenic effect Effects 0.000 claims abstract description 32
- 239000013598 vector Substances 0.000 claims abstract description 31
- 229940124597 therapeutic agent Drugs 0.000 claims abstract description 12
- 230000000694 effects Effects 0.000 claims description 104
- 210000004027 cell Anatomy 0.000 claims description 80
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 59
- 230000014509 gene expression Effects 0.000 claims description 57
- 241000282414 Homo sapiens Species 0.000 claims description 56
- 239000002773 nucleotide Substances 0.000 claims description 56
- 125000003729 nucleotide group Chemical group 0.000 claims description 56
- 108090000623 proteins and genes Proteins 0.000 claims description 51
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 49
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 49
- 150000001413 amino acids Chemical class 0.000 claims description 47
- 229920001184 polypeptide Polymers 0.000 claims description 45
- 239000000126 substance Substances 0.000 claims description 33
- 108700008625 Reporter Genes Proteins 0.000 claims description 30
- 208000021017 Weight Gain Diseases 0.000 claims description 25
- 230000004584 weight gain Effects 0.000 claims description 25
- 235000019786 weight gain Nutrition 0.000 claims description 25
- 230000008859 change Effects 0.000 claims description 17
- 102000040430 polynucleotide Human genes 0.000 claims description 15
- 108091033319 polynucleotide Proteins 0.000 claims description 15
- 239000002157 polynucleotide Substances 0.000 claims description 15
- 238000012360 testing method Methods 0.000 claims description 15
- 239000000883 anti-obesity agent Substances 0.000 claims description 14
- 229940125710 antiobesity agent Drugs 0.000 claims description 10
- 230000002401 inhibitory effect Effects 0.000 claims description 9
- 230000002950 deficient Effects 0.000 claims description 7
- 210000000349 chromosome Anatomy 0.000 claims description 6
- 125000000539 amino acid group Chemical group 0.000 claims description 5
- 230000001419 dependent effect Effects 0.000 claims description 5
- 210000001082 somatic cell Anatomy 0.000 claims description 2
- 239000002547 new drug Substances 0.000 abstract description 2
- 201000005577 familial hyperlipidemia Diseases 0.000 abstract 2
- 239000003529 anticholesteremic agent Substances 0.000 abstract 1
- 229940127226 anticholesterol agent Drugs 0.000 abstract 1
- 238000009509 drug development Methods 0.000 abstract 1
- 241000699670 Mus sp. Species 0.000 description 145
- 241000699666 Mus <mouse, genus> Species 0.000 description 114
- 108020004414 DNA Proteins 0.000 description 104
- 239000013612 plasmid Substances 0.000 description 42
- 210000001519 tissue Anatomy 0.000 description 36
- 239000012634 fragment Substances 0.000 description 31
- 101100434901 Mus musculus Angptl6 gene Proteins 0.000 description 30
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 28
- 210000002027 skeletal muscle Anatomy 0.000 description 25
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 24
- 210000001789 adipocyte Anatomy 0.000 description 23
- 229940079593 drug Drugs 0.000 description 23
- 210000004369 blood Anatomy 0.000 description 21
- 239000008280 blood Substances 0.000 description 21
- 210000000593 adipose tissue white Anatomy 0.000 description 18
- 101000924549 Homo sapiens Angiopoietin-related protein 6 Proteins 0.000 description 17
- 108010076504 Protein Sorting Signals Proteins 0.000 description 17
- 210000003486 adipose tissue brown Anatomy 0.000 description 17
- 239000003472 antidiabetic agent Substances 0.000 description 17
- 230000037396 body weight Effects 0.000 description 17
- 108060001084 Luciferase Proteins 0.000 description 16
- 210000000577 adipose tissue Anatomy 0.000 description 15
- 230000003579 anti-obesity Effects 0.000 description 15
- 108091008146 restriction endonucleases Proteins 0.000 description 15
- 238000011830 transgenic mouse model Methods 0.000 description 15
- 238000011144 upstream manufacturing Methods 0.000 description 15
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 14
- 239000008103 glucose Substances 0.000 description 14
- 230000001965 increasing effect Effects 0.000 description 14
- 108700028369 Alleles Proteins 0.000 description 13
- 239000005089 Luciferase Substances 0.000 description 13
- 235000009200 high fat diet Nutrition 0.000 description 13
- 238000004519 manufacturing process Methods 0.000 description 13
- 230000036284 oxygen consumption Effects 0.000 description 13
- 102000004877 Insulin Human genes 0.000 description 12
- 108090001061 Insulin Proteins 0.000 description 12
- 241000699660 Mus musculus Species 0.000 description 12
- 239000002299 complementary DNA Substances 0.000 description 12
- 235000013601 eggs Nutrition 0.000 description 12
- 239000005090 green fluorescent protein Substances 0.000 description 12
- 229940125396 insulin Drugs 0.000 description 12
- 210000004185 liver Anatomy 0.000 description 12
- 108700039691 Genetic Promoter Regions Proteins 0.000 description 11
- 208000031226 Hyperlipidaemia Diseases 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 11
- 201000010099 disease Diseases 0.000 description 11
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 11
- 210000004392 genitalia Anatomy 0.000 description 11
- 102000055024 human ANGPTL6 Human genes 0.000 description 11
- 230000001315 anti-hyperlipaemic effect Effects 0.000 description 10
- 238000010367 cloning Methods 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 230000002441 reversible effect Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 235000000891 standard diet Nutrition 0.000 description 9
- 238000013518 transcription Methods 0.000 description 9
- 230000035897 transcription Effects 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 210000005228 liver tissue Anatomy 0.000 description 8
- 210000002966 serum Anatomy 0.000 description 8
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 7
- 230000003178 anti-diabetic effect Effects 0.000 description 7
- 238000012258 culturing Methods 0.000 description 7
- 210000001339 epidermal cell Anatomy 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 238000011813 knockout mouse model Methods 0.000 description 7
- 108020004999 messenger RNA Proteins 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- 238000006467 substitution reaction Methods 0.000 description 7
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 7
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 6
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 6
- 206010020880 Hypertrophy Diseases 0.000 description 6
- 229930193140 Neomycin Natural products 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 6
- 238000009825 accumulation Methods 0.000 description 6
- 229940127003 anti-diabetic drug Drugs 0.000 description 6
- 239000003524 antilipemic agent Substances 0.000 description 6
- 210000002459 blastocyst Anatomy 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Natural products O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 6
- 210000002216 heart Anatomy 0.000 description 6
- 229960004927 neomycin Drugs 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 238000003753 real-time PCR Methods 0.000 description 6
- 210000000952 spleen Anatomy 0.000 description 6
- 210000004291 uterus Anatomy 0.000 description 6
- 108010053770 Deoxyribonucleases Proteins 0.000 description 5
- 102000016911 Deoxyribonucleases Human genes 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 206010022489 Insulin Resistance Diseases 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 230000035508 accumulation Effects 0.000 description 5
- 210000004102 animal cell Anatomy 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 210000001612 chondrocyte Anatomy 0.000 description 5
- 230000000875 corresponding effect Effects 0.000 description 5
- 238000007876 drug discovery Methods 0.000 description 5
- 210000003734 kidney Anatomy 0.000 description 5
- 210000000056 organ Anatomy 0.000 description 5
- 230000008685 targeting Effects 0.000 description 5
- 229940126585 therapeutic drug Drugs 0.000 description 5
- 238000001262 western blot Methods 0.000 description 5
- 241000287828 Gallus gallus Species 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 102000016267 Leptin Human genes 0.000 description 4
- 108010092277 Leptin Proteins 0.000 description 4
- 102000018653 Long-Chain Acyl-CoA Dehydrogenase Human genes 0.000 description 4
- 108010027062 Long-Chain Acyl-CoA Dehydrogenase Proteins 0.000 description 4
- 102000015494 Mitochondrial Uncoupling Proteins Human genes 0.000 description 4
- 108010050258 Mitochondrial Uncoupling Proteins Proteins 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 108700026244 Open Reading Frames Proteins 0.000 description 4
- 238000002105 Southern blotting Methods 0.000 description 4
- 102000008200 Uncoupling Protein 3 Human genes 0.000 description 4
- 108010021098 Uncoupling Protein 3 Proteins 0.000 description 4
- 238000007792 addition Methods 0.000 description 4
- 230000033115 angiogenesis Effects 0.000 description 4
- 229940125708 antidiabetic agent Drugs 0.000 description 4
- 239000013599 cloning vector Substances 0.000 description 4
- 238000012217 deletion Methods 0.000 description 4
- 230000037430 deletion Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 239000003623 enhancer Substances 0.000 description 4
- 238000007446 glucose tolerance test Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 229940039781 leptin Drugs 0.000 description 4
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 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 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- 241000725101 Clea Species 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102000003728 Peroxisome Proliferator-Activated Receptors Human genes 0.000 description 3
- 108090000029 Peroxisome Proliferator-Activated Receptors Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 238000003556 assay Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- WQZGKKKJIJFFOK-FPRJBGLDSA-N beta-D-galactose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-FPRJBGLDSA-N 0.000 description 3
- 108010005774 beta-Galactosidase Proteins 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 235000005911 diet Nutrition 0.000 description 3
- 230000037213 diet Effects 0.000 description 3
- 238000005265 energy consumption Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000013613 expression plasmid Substances 0.000 description 3
- 230000001605 fetal effect Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 102000039446 nucleic acids Human genes 0.000 description 3
- 108020004707 nucleic acids Proteins 0.000 description 3
- 150000007523 nucleic acids Chemical class 0.000 description 3
- -1 peptides) Chemical class 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000002062 proliferating effect Effects 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- OGYGFUAIIOPWQD-UHFFFAOYSA-N 1,3-thiazolidine Chemical compound C1CSCN1 OGYGFUAIIOPWQD-UHFFFAOYSA-N 0.000 description 2
- 108020004463 18S ribosomal RNA Proteins 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 241000972773 Aulopiformes Species 0.000 description 2
- 108010035563 Chloramphenicol O-acetyltransferase Proteins 0.000 description 2
- 102000001493 Cyclophilins Human genes 0.000 description 2
- 108010068682 Cyclophilins Proteins 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 description 2
- 101000834253 Gallus gallus Actin, cytoplasmic 1 Proteins 0.000 description 2
- 206010060378 Hyperinsulinaemia Diseases 0.000 description 2
- 206010020772 Hypertension Diseases 0.000 description 2
- 206010021033 Hypomenorrhoea Diseases 0.000 description 2
- 241000254158 Lampyridae Species 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 239000012807 PCR reagent Substances 0.000 description 2
- 102100038824 Peroxisome proliferator-activated receptor delta Human genes 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 239000012722 SDS sample buffer Substances 0.000 description 2
- 241000242583 Scyphozoa Species 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 229930189065 blasticidin Natural products 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 108091006047 fluorescent proteins Proteins 0.000 description 2
- 102000034287 fluorescent proteins Human genes 0.000 description 2
- 238000010230 functional analysis Methods 0.000 description 2
- 238000010363 gene targeting Methods 0.000 description 2
- 238000003505 heat denaturation Methods 0.000 description 2
- 125000005842 heteroatom Chemical group 0.000 description 2
- 230000003451 hyperinsulinaemic effect Effects 0.000 description 2
- 201000008980 hyperinsulinism Diseases 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 210000001596 intra-abdominal fat Anatomy 0.000 description 2
- 230000037356 lipid metabolism Effects 0.000 description 2
- 230000001000 lipidemic effect Effects 0.000 description 2
- 239000012139 lysis buffer Substances 0.000 description 2
- 230000036210 malignancy Effects 0.000 description 2
- 230000013011 mating Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 238000010172 mouse model Methods 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 108091008765 peroxisome proliferator-activated receptors β/δ Proteins 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 230000035790 physiological processes and functions Effects 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 230000014493 regulation of gene expression Effects 0.000 description 2
- 235000019515 salmon Nutrition 0.000 description 2
- 238000010845 search algorithm Methods 0.000 description 2
- 210000002363 skeletal muscle cell Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- QAPSNMNOIOSXSQ-YNEHKIRRSA-N 1-[(2r,4s,5r)-4-[tert-butyl(dimethyl)silyl]oxy-5-(hydroxymethyl)oxolan-2-yl]-5-methylpyrimidine-2,4-dione Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O[Si](C)(C)C(C)(C)C)C1 QAPSNMNOIOSXSQ-YNEHKIRRSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- YXQXLHSRJUFMFD-UHFFFAOYSA-N 3-[(3,4-dihydroxyphenyl)methyl]-2h-chromene-7,8-diol Chemical group C1=C(O)C(O)=CC=C1CC1=CC2=CC=C(O)C(O)=C2OC1 YXQXLHSRJUFMFD-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000006501 Angiopoietin-like Proteins Human genes 0.000 description 1
- 108010019425 Angiopoietin-like Proteins Proteins 0.000 description 1
- 101710085850 Angiopoietin-related protein 5 Proteins 0.000 description 1
- 102000009840 Angiopoietins Human genes 0.000 description 1
- 108010009906 Angiopoietins Proteins 0.000 description 1
- 241000272517 Anseriformes Species 0.000 description 1
- 241000272875 Ardeidae Species 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- 208000008035 Back Pain Diseases 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 108010051219 Cre recombinase Proteins 0.000 description 1
- 101000622007 Crotalus adamanteus Snake venom metalloproteinase adamalysin-2 Proteins 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108060002716 Exonuclease Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 206010018429 Glucose tolerance impaired Diseases 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 101001123331 Homo sapiens Peroxisome proliferator-activated receptor gamma coactivator 1-alpha Proteins 0.000 description 1
- 208000013016 Hypoglycemia Diseases 0.000 description 1
- 101150098499 III gene Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 229940122355 Insulin sensitizer Drugs 0.000 description 1
- 208000003947 Knee Osteoarthritis Diseases 0.000 description 1
- 208000008930 Low Back Pain Diseases 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- ZPXSCAKFGYXMGA-UHFFFAOYSA-N Mazindol Chemical compound N12CCN=C2C2=CC=CC=C2C1(O)C1=CC=C(Cl)C=C1 ZPXSCAKFGYXMGA-UHFFFAOYSA-N 0.000 description 1
- 206010027476 Metastases Diseases 0.000 description 1
- 102100029820 Mitochondrial brown fat uncoupling protein 1 Human genes 0.000 description 1
- 108050002686 Mitochondrial brown fat uncoupling protein 1 Proteins 0.000 description 1
- 102100040216 Mitochondrial uncoupling protein 3 Human genes 0.000 description 1
- 101710112412 Mitochondrial uncoupling protein 3 Proteins 0.000 description 1
- 101000800132 Mus musculus Thyroglobulin Proteins 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 102000017946 PGC-1 Human genes 0.000 description 1
- 108700038399 PGC-1 Proteins 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 102100038831 Peroxisome proliferator-activated receptor alpha Human genes 0.000 description 1
- 102100028960 Peroxisome proliferator-activated receptor gamma coactivator 1-alpha Human genes 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 101100218590 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) BDF2 gene Proteins 0.000 description 1
- 241001515850 Satellite Nucleic Acids Species 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 102000004338 Transferrin Human genes 0.000 description 1
- 108090000901 Transferrin Proteins 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000002491 angiogenic effect Effects 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 230000003276 anti-hypertensive effect Effects 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000003143 atherosclerotic effect Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000013592 cell lysate Substances 0.000 description 1
- 210000001638 cerebellum Anatomy 0.000 description 1
- 210000004720 cerebrum Anatomy 0.000 description 1
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 1
- 238000000975 co-precipitation Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013118 diabetic mouse model Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005553 drilling Methods 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 210000002889 endothelial cell Anatomy 0.000 description 1
- 238000009207 exercise therapy Methods 0.000 description 1
- 102000013165 exonuclease Human genes 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 235000020937 fasting conditions Nutrition 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 229940012952 fibrinogen Drugs 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 210000004602 germ cell Anatomy 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000009931 harmful effect Effects 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 230000001969 hypertrophic effect Effects 0.000 description 1
- 230000002218 hypoglycaemic effect Effects 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 208000024765 knee pain Diseases 0.000 description 1
- 102000005861 leptin receptors Human genes 0.000 description 1
- 108010019813 leptin receptors Proteins 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229960000299 mazindol Drugs 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000009401 metastasis Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 210000004088 microvessel Anatomy 0.000 description 1
- 244000309715 mini pig Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 210000000663 muscle cell Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- AHLBNYSZXLDEJQ-FWEHEUNISA-N orlistat Chemical compound CCCCCCCCCCC[C@H](OC(=O)[C@H](CC(C)C)NC=O)C[C@@H]1OC(=O)[C@H]1CCCCCC AHLBNYSZXLDEJQ-FWEHEUNISA-N 0.000 description 1
- 229960001243 orlistat Drugs 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 210000002824 peroxisome Anatomy 0.000 description 1
- 108091008725 peroxisome proliferator-activated receptors alpha Proteins 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000002708 random mutagenesis Methods 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 238000009781 safety test method Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229960004425 sibutramine Drugs 0.000 description 1
- UNAANXDKBXWMLN-UHFFFAOYSA-N sibutramine Chemical compound C=1C=C(Cl)C=CC=1C1(C(N(C)C)CC(C)C)CCC1 UNAANXDKBXWMLN-UHFFFAOYSA-N 0.000 description 1
- 230000003584 silencer Effects 0.000 description 1
- 238000002741 site-directed mutagenesis Methods 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000003868 tissue accumulation Effects 0.000 description 1
- 230000017423 tissue regeneration Effects 0.000 description 1
- 231100000041 toxicology testing Toxicity 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 239000012581 transferrin Substances 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- 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
- C07K14/475—Growth factors; Growth regulators
- C07K14/515—Angiogenesic factors; Angiogenin
-
- 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
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
- A01K2267/0306—Animal model for genetic diseases
-
- 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
- A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/30—Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
- C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
-
- 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
- C12N2840/00—Vectors comprising a special translation-regulating system
- C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
- C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES
Definitions
- the present invention relates to a method for screening an antiobesity drug, an obesity model animal, and a promoter of an angiopoietin-related growth factor (hereinafter, referred to as AGF).
- AGF angiopoietin-related growth factor
- Non-Patent Document 3 thiazolidine drugs have the side effect of hepatopathy 'edema' heart failure, and SU drugs are feared as a side effect that promotes obesity.Therefore, there is a strong demand for safe, non-weight-enhancing insulin sensitizers.
- Hypertrophic adipocytes which are abundant in visceral fat of obese diabetic patients, produce and secrete adipocytic dysins that induce insulin resistance, and adipocyte and / or muscle cells nearby. It is thought that it acts on cells to cause insulin resistance. Thus, adipose tissue changes into a tissue involved in the direction of enhancing insulin resistance in a diabetic condition in which fat cells are enlarged (Non-patent Documents 4 and 5).
- Lebutin is well known as a factor involved in the accumulation of adipose tissue that causes obesity and diabetes.
- Leptin is a weight gain inhibitory hormone, and it is known that deficiency of leptin increases appetite, reduces energy consumption, and leads to obesity.
- Such discovery of factors involved in adipose tissue accumulation and adipocyte hypertrophy is extremely useful for developing therapeutics for diseases such as obesity, diabetes mellitus, or hyperlipidemia (Non-patented). Reference 6).
- KKA y mice are widely used as obesity type II diabetes model mice. KKA y mice are associated with reduced insulin sensitivity and obesity and develop hyperglycemia, and are therefore being used in the development of antidiabetic drugs.
- the genes responsible for these phenotypes in KKA y mice have not been identified (Non-Patent Document 8). It is thought that many unknown genes are involved in obesity and diabetes (Non-Patent Document 9). Therefore, it has been desired to find a disease-related gene, generate a genetically modified mouse, and perform phenotypic analysis to develop a medicament for obesity or diabetes and further elucidate the pathological condition.
- Angiopoietin-related growth factor has a coiled-coil ) Domain is a secreted protein having a fibrinogen-like domain on the C-terminal side.
- AGF is the same as NL8 reported in Patent Document 1, and it has been reported that when NL8 is stably expressed in CHO cells and implanted subcutaneously in nude mice, CHO becomes capable of forming tumors.
- Patent Document 216 a sequence identical or similar to AGF is described in Patent Document 216.
- Patent Documents 4 and 5 include expression distribution (Patent Documents 4 and 5), vascular endothelial growth factor (VEGF) -stimulated growth inhibitory activity (Patent Document 5), or growth factor-treated human umbilical vein endothelial cells (HUVEC).
- VEGF vascular endothelial growth factor
- Patent Document 5 vascular endothelial growth factor
- HAVEC growth factor-treated human umbilical vein endothelial cells
- Detection of overexpression has been shown, and amino acids identical or similar to AGF are considered based on expression in vascular tissues, tumorigenicity, and homology with Z or one family molecule. It is described that a polypeptide consisting of a sequence is involved in angiogenesis.
- Non-Patent Document 10 excessive angiogenesis occurs in transgenic mice in which AGF is forcibly expressed in epidermal cells using the K14 promoter, and microvessels in the skin increase calories and activate the proliferation of keratinocytes. Is shown.
- Non-Patent Document 11 describes that AGF has epidermal cell proliferation activity.
- Non-patent document 2 Japanese clinical practice", 2003, vol. 61, extra number 6, obesity, p.649-654
- Non-patent document 3 Japanese clinical practice", 2002, vol. 60, extra number 9, new era Diabetes 3, p.310 -331
- Non-Patent Document 10 "Circulation”, (USA), November 5, 2002, 10
- Patent Document 4 WO 00/32221 pamphlet
- Patent Document 7 International Publication No. 02/08284 pamphlet
- Patent Document 8 US Patent Application Publication No. 2003/0105011
- Patent Document 13 US Pat. No. 6,350,450
- Patent Document 14 U.S. Patent No.6413770
- An object of the present invention is to provide a method for screening an antiobesity drug and an obesity model animal.
- nucleotides 2705 to 3001 in the nucleotide sequence represented by SEQ ID NO: 1 110 nucleotides are substituted, deleted, added, and / or inserted.
- DNA comprising a sequence and having angiopoietin-related growth factor promoter activity, or
- a method for screening an antiobesity agent, a therapeutic agent for diabetes, and / or an antihyperlipidemic agent comprising:
- a non-human animal or its progeny obtained by ontogenizing a totipotent cell into which a polynucleotide encoding a polypeptide selected together with a CAG promoter has been introduced, and the polynucleotide encoding the selected polypeptide.
- a non-human transgenic animal carrying nucleotides and expressing said polypeptide in somatic cells
- a step of introducing a DNA construct in which a CAG promoter is linked upstream of AGF cDNA into ES (Embryonic Stem) cells a step of selecting a clone into which AGF has been introduced, and a step of introducing the selected clone into a blastocyst.
- the present invention also includes a method for producing a non-human transgenic animal (preferably a mouse), which comprises a step of selecting an individual having the obtained DNA in the genome.
- Patent Document 1-116 A polypeptide having the same or homology as human or mouse AGF having the amino acid sequence described in 5 is described in Patent Document 1-116.
- Patent Documents 419 to 19 include diabetes as a disease that has been listed in large numbers, but this is an unfounded description without any concrete support.
- Non-Patent Document 12 Non-Patent Document 12
- the tissue on which the endogenous AGF acts is unknown, and there was no knowledge of any other physiological functions of AGF.
- the present inventors have produced AGF knockout mice and AGF transgenic mice that systemically overexpress AGF for the first time, and surprisingly, AGF knockout mice were obese, diabetic, and / or hyperlipidemic.
- AGF transgenic mice that overexpress AGF systemically are useful as a model animal for diabetes, and are useful for the identification of therapeutic drugs for obesity, diabetes, and / or hyperlipidemia, and drug discovery target molecules I found that.
- the DNA of the present invention includes, for example,
- DNA comprising the sequence and having angiopoietin-related growth factor promoter activity.
- angiopoietin-related growth factor (AGF) promoter activity means the promoter activity of the AGF gene, and more specifically, in the base sequence represented by SEQ ID NO: 1. It means the promoter activity of DNA that has both base strength and sequence strength of Nos. 2705 to 3001.
- the method for determining whether or not a certain DNA has “AGF promoter activity” is not particularly limited, but may be a known ordinary method, for example, an appropriate reporter gene 3 ′ downstream of the DNA.
- DNA can be ligated, introduced into a nucleated cell (preferably an animal cell line), cultured, and confirmed by measuring the expression level of a reporter gene in the cell. More specifically, it can be confirmed by the method described in Example 12.
- the DNA of the present invention can be produced by the following methods.
- the present invention is not limited to these methods.
- a primer set consisting of the nucleotide sequence represented by SEQ ID NO: 47 and SEQ ID NO: 46 is synthesized, PCR is performed using these primers and human genomic DNA, and the nucleotide sequence represented by SEQ ID NO: 1 DNA consisting of a sequence consisting of nucleotides 2705 to 3001 in the above can be produced.
- Human genomic DNA can be prepared from an appropriate human tissue by a commonly used method.
- the DNA of the present invention can be produced more specifically by the method described in Example 11, but is not limited to this method.
- the primer used is a restriction enzyme different from SEQ ID NO: 47 and SEQ ID NO: 46. Recognition sites can be added, and objects of different lengths can be used.
- a person skilled in the art would be able to modify the natural promoter DNA by modifying a part of the base sequence of the promoter sequence present in the natural form by substitution of another base, deletion of the base, and / or addition. It is possible to prepare a DNA having a promoter activity equivalent to the above.
- the DNA having the substitution, deletion, addition, Z or insertion base sequence in the natural base sequence and having the same promoter activity as the natural promoter DNA is also a DNA of the present invention.
- Modification of a base can be performed, for example, by introducing a deletion using a restriction enzyme or DNA exonuclease, or introducing a mutation using a site-directed mutagenesis method [
- Whether or not the DNA produced as described above has promoter activity is determined by a known ordinary method, for example, by linking an appropriate reporter gene DNA 3 'downstream of the DNA, and ligating the DNA to a nucleated cell (preferably Can be confirmed by introducing the cells into an animal cell line and culturing the cells, and measuring the expression level of the reporter gene in the cells. More specifically, it can be confirmed by the method described in Example 12.
- the recombinant vector of the present invention can be produced by incorporating the DNA of the present invention into a vector appropriately selected according to the purpose.
- a DNA containing a structural gene to be expressed can be inserted 3 'downstream of the DNA of the present invention.
- the structural gene is not particularly limited as long as it is a DNA encoding a protein, and may be all or part of ORF ( ⁇ pen Reading Frame).
- a DNA of the present invention that is, a DNA having AGF promoter activity
- a reporter gene such as luciferase has been incorporated. It is preferable to manufacture it.
- the “reporter gene” is not particularly limited as long as it is generally used, but is preferably an enzyme gene or the like, which can be easily measured quantitatively. Examples thereof include a chloramphenicol acetyltransferase gene (CAT) derived from a bacterial transposon, a luciferase gene (Luc) derived from a firefly, and a green fluorescent protein gene (GFP) derived from a jellyfish. Preferably, it can be produced by the method described in Example 11. Whether or not the produced recombinant vector has AGF promoter activity can be confirmed according to a known ordinary method. Specifically, the recombinant vector is transferred to a nucleated cell (preferably an animal cell line). It can be confirmed by introducing, culturing, and measuring the expression level of the reporter gene in the cells. More specifically, it can be confirmed by the method described in Example 12.
- CAT chloramphenicol acetyltransferase gene
- Luc luciferase gene
- the transformant of the present invention can be produced by introducing a recombinant vector containing the DNA of the present invention into a host cell appropriately selected according to the purpose.
- a host cell appropriately selected according to the purpose.
- cells derived from mammals eg, human, mouse, rat, etc.
- any cell having a transcription control factor or the like present in a normal cell can be used, and more specifically, for example, commercially available 293EBNA, HT-1080, or HepG2 can be used. .
- the method of introduction into a host cell is not limited to the following.
- the DEAE-dextran method [Luthman, H. and Magnusson, G. (1983) Nucleic Acids Res., 11,
- Whether or not the produced transformant has AGF promoter activity can be confirmed by measuring the expression level of a reporter gene in cells. More specifically, it can be confirmed by the method described in Example 12. By comparison with the case where the recombinant vector not containing the DNA of the present invention was introduced, the trait having the AGF promoter activity was determined. Transformants can be selected.
- AGF has an antiobesity, antidiabetic, and / or antihyperlipidemic effect. Therefore, by screening a substance that promotes AGF promoter activity, an antiobesity drug, an antidiabetic drug, and a substance effective for Z or antihyperlipidemia can be obtained.
- a step of bringing a transformant of the present invention into contact with a test substance and ii) measuring hAGF promoter activity and analyzing (detecting or measuring) a change in the activity dependent on the test substance.
- a screening method for an antiobesity agent, a therapeutic agent for diabetes, and a therapeutic agent for Z or antihyperlipidemia by the screening method of the present invention.
- an hAGF promoter region having a sequence strength of 2705 to 3001 in the nucleotide sequence represented by SEQ ID NO: 1 and a test substance contacted with a cell transformed with a reporter gene fused thereto.
- Antiobesity agents, antidiabetic agents, and / or antihyperlipidemic agents can be screened.
- the reporter gene Atsusei (Tamura et al., Transcription Factor Research, Yodosha, 1993) is a method for analyzing (detecting or measuring) the regulation of gene expression using the expression of a reporter gene as a marker.
- the regulation of gene expression is controlled by a part called the promoter region located in the 5 'upstream region, and the gene expression level at the transcription stage can be estimated by measuring the activity of this promoter.
- the test substance activates the promoter, it activates the transcription of a reporter gene located downstream of the promoter region.
- the promoter activating effect that is, the expression enhancing effect can be detected by replacing the expression with the reporter gene expression.
- DNA of the present invention preferably Is a sequence consisting of nucleotides 2705 to 3001 in the nucleotide sequence represented by SEQ ID NO: 1.
- the “reporter gene” fused to the hAGF promoter region is not particularly limited as long as it is commonly used. However, an enzyme gene or the like which is easy to quantitatively measure is preferable.
- Examples include a chloramphenicol acetyltransferase gene (CAT) derived from a bacterial transposon, a luciferase gene (Luc) derived from a firefly, and a green fluorescent protein gene (GFP) derived from a jellyfish.
- the reporter gene may be functionally fused with the DNA of the present invention (preferably, the promoter region of hAGF consisting of the nucleotide sequence represented by positions 2705 to 3001 of SEQ ID NO: 1).
- the reporter gene fused with the DNA of the present invention preferably the promoter region of hAGF
- is stably or transiently expressed in cells eg, animal cells or yeast).
- analyze the test substance-dependent change in promoter activity by comparing the expression level of the reporter gene when the transformed cell is contacted with the test substance in the culture medium and not. Can be.
- the method described in the above [1] can be used.
- the method of analyzing the expression level of the reporter gene is appropriately selected depending on the protein encoded by the reporter gene.
- the reporter gene encodes a fluorescent protein (for example, luciferase)
- the transfected cells are lysed by an appropriate method, luciferin as a substrate is added to the supernatant of the cell lysate, and then the appropriate fluorescent Fluorescence can be measured using a detector (eg, ML3000; Dynatech Laboratories, etc.) to determine the expression level of the reporter gene.
- a detector eg, ML3000; Dynatech Laboratories, etc.
- This reaction can also be carried out using a suitable commercially available detection kit, specifically, for example, a luciferase atsey system (Promega).
- a substance that enhances the expression of AGF ie, an antiobesity agent, a therapeutic agent for diabetes, and a therapeutic agent for Z or antihyperlipidemia.
- the substance promoting promoter activity which is preferably used in the method described in Example 12, a substance having a promoter monoactivation ability 1.5 times or more that when no compound is added is selected. Is preferred.
- the test substance used in the screening method of the present invention is not particularly limited.
- commercially available compounds including peptides
- various known compounds including peptides registered in a chemical file
- combinatorial chemistry technology Tetrahedron, Vol. 51, No. 8135-8173 (1995)
- a culture supernatant of a microorganism a natural component derived from a plant or marine organism, an animal tissue extract, and a compound selected by the screening method of the present invention.
- Compounds (including peptides) obtained by chemically or biologically modifying including peptides
- the non-human knockout animal of the present invention is not particularly limited as long as the gene function of the polynucleotide encoding AGF is deficient on the chromosome, and is generally used in the production of knockout animals.
- a sequence corresponding to an animal (species) to be prepared can be used.
- a sequence of GeneBank accession number AC073775.2 can be used.
- an AGF knockout animal can be obtained by selecting a mouse deficient in AGF gene function from a mouse produced by a random mutagenesis method (see, for example, Nature, 392, 608-611, 1998). it can.
- the non-human knockout animal of the present invention can be produced, for example, according to the method described in Example 1. That is, a DNA construct in which a part of the genomic sequence containing the ORF of the AGF gene was replaced with a drug resistance gene (for example, a neomycin resistance gene) was prepared, and the obtained DNA construct was introduced into ES cells. A resistant strain is obtained by culturing in the presence of an appropriate drug (eg, G418).
- a drug resistance gene for example, a neomycin resistance gene
- a clone in which the desired homologous recombination has occurred is selected by Southern blot analysis, microinjected into a blastocyst, and the engineered egg is transplanted into the uterus to obtain a chimeric mouse.
- a heterozygous mouse is obtained.
- homozygous mice can be obtained according to Mendel's law.
- the non-human knockout animal of the present invention (or the non-human transgenic animal of the present invention described below) can be prepared using any vertebrate other than human. Specifically, for example, in vertebrates such as mice, rats, egrets, miniature pigs, goats, sheep, and geese, knockout animals in which various gene introductions and expression levels have been modified have been created, The non-human knockout animals of the present invention also include these species. As the non-human knockout animal of the present invention (or the non-human transgenic animal of the present invention described below), a mouse, particularly a rodent-preferred animal, is preferable.
- the non-human knockout animal of the present invention showed remarkable obesity as shown in the Examples (Examples).
- the non-human knockout animals of the present invention may be obese, diabetic, and / or hyperlipidemic.
- Useful as a model animal That is, the non-human knockout animal of the present invention was screened for drugs for treating or preventing obesity, diabetes, and / or hyperlipidemia, etc., as well as elucidating the mechanism of these diseases and screening. It can also be used for drug safety testing.
- Use of an AGF non-human knockout animal as an animal model for obesity, diabetes, and Z or hyperlipidemia is also included in the present invention.
- the non-human transgenic animal of the present invention comprises the following transgenes (a) to (d):
- a polynucleotide encoding a polypeptide selected from the group consisting of (hereinafter, referred to as a polypeptide for producing a transgenic animal) is used as a modified chicken beta-actin promoter with CMV—IE enhancer (CAG) promoter 1 ⁇ [uENE ,
- the polypeptide for producing a transgenic animal is human AGF consisting of a sequence consisting of amino acids 1 to 450 in the amino acid sequence represented by SEQ ID NO: 3, or represented by SEQ ID NO: 5.
- Mouse AGF consisting of a sequence consisting of amino acids 1 to 433 in the amino acid sequence is preferred.
- the amino acid sequence represented by SEQ ID NO: 3 is the amino acid sequence of human AGF precursor.
- Human AGF has a signal sequence (120-1) at the N-terminus, and its signal sequence is cleaved when secreted and expressed extracellularly.
- Human mature AGF consisting of a sequence consisting of amino acids 1 to 450 in the amino acid sequence represented by SEQ ID NO: 3 from which the signal sequence has been cleaved.
- amino acid sequence represented by SEQ ID NO: 5 is the amino acid sequence of mouse AGF precursor It is.
- Mouse AGF has a signal sequence (1- 24--1) at the N-terminus, and the signal sequence is cleaved when secreted and expressed extracellularly.
- Mouse mature AGF consisting of a sequence consisting of amino acids 1 to 433 in the amino acid sequence represented by SEQ ID NO: 5 from which the signal sequence has been cleaved.
- polypeptide (b) that can be used as a polypeptide for producing a transgenic animal that is, the amino acid sequence No. 1 to No. 450 in the amino acid sequence represented by SEQ ID NO: 3 Or, in the sequence consisting of amino acids 1 to 433 in the amino acid sequence represented by SEQ ID NO: 5, 110 (for example, 11 or more) amino acid residues are substituted, deleted, and / or As a polypeptide comprising an inserted amino acid sequence and having a weight-gain suppressing activity, in each of the above-mentioned sequences, preferably 117, more preferably 115 amino acid residues are substituted, deleted, And / or a polypeptide comprising an inserted amino acid sequence and having an activity of suppressing weight gain.
- the amino acid to be substituted is preferably an amino acid having properties similar to the amino acid before substitution.
- the amino acids belonging to each group as shown below are amino acids having properties similar to each other within the group. Substitution of these amino acids for other amino acids in the group often does not impair the essential function of the protein. Such amino acid substitution is called a conservative substitution and is known as a technique for converting an amino acid sequence while maintaining the function of a polypeptide.
- Non-polar amino acids Ala, Val, Leu, Ile, Pro, Met, Phe, and T ⁇
- Uncharged amino acids Gly, Ser, Thr, Cys, Tyr, Asn, and Gin
- Acidic amino acids Asp and Glu
- the method for determining whether or not a certain polypeptide has a weight-gain-inhibiting activity is not particularly limited. For example, it can be confirmed by the method described in Example 4. That is, a transgenic animal produced using the gene encoding the polypeptide can be bred on a standard diet or a high-fat diet, and the change in body weight can be confirmed by comparing with a wild-type animal.
- the above-described polypeptide which can be used as a polypeptide for producing a transgenic animal.
- the conditions for hybridization are “5xSSPE, 5x Denhard's solution, 0.5% sodium dodecyl sulfate (SDS), 40% honoleamide. , 200 / ig / mL salmon sperm DNA, overnight at 37 ° C. More severe conditions include 5xSSPE, 5x Denhardt's solution, 0.5% SDS, 50% honolemamide, 200 ⁇ g ZmL salmon sperm. DNA, 42. C overnight.
- a loose condition is “5xSSC, 1% SDS, 42 ° C”
- a normal condition is “0.5xSSC, 0.1./.SDS, 42 ° C”.
- the conditions are more severe, such as “0.2xSSC, 0.1% SDS, 65 ° C”.
- the composition of 5XSSPE is 50 mmol / L sodium phosphate (pH 7.4), 0.75 mol / L NaCl, and 5 mmol / L EDTA, and the composition of 5XSSC is 0.75 molZL NaCl and 75 mmol / L Sodium (pH 7.0).
- the homology of the polypeptide (d), which can be used as a polypeptide for producing a transgenic animal, is at least 95% or more, and preferably 97% or more.
- the amino acid sequence homology can be determined using a BLAST search algorithm. Specifically, the BLAST package (sgi32bit version, version 2.0.12, available on 8 beams)) 1236 (1 program [below & & 1 ⁇ A. Tatusova,
- Gap insertion cost value is 0
- Gap extension cost value is 0 pair-wise alignment parameters
- SEG as query type filter
- BLOSUM62 is matrix.
- polypeptide for producing a transgenic animal those containing a signal sequence on the N-terminal side of the polypeptides (a) to (d) are preferable.
- the signal sequence is not limited as long as it is a sequence that leads the polypeptide through the membrane.
- the signal sequence described in Biochemistry, 28 (3), 923-930, 1989 can be used.
- the signal sequence (120-1) in the amino acid sequence represented by SEQ ID NO: 3 or the amino acid sequence represented by SEQ ID NO: 5 is the most preferred, including those containing the signal sequence (-24-1-1).
- the origin of the polypeptide for producing a transgenic animal is not limited to humans or mice.
- polypeptides derived from organisms other than humans or mice may also be used as long as they correspond to any one of the polypeptides (a) to (d).
- Polypeptides genetically engineered based on the sequence IJ consisting of amino acid No. 450 or the sequence consisting of amino acids No. 1-433 in the amino acid sequence represented by SEQ ID NO: 5 Can be used as a polypeptide for producing transgenic animals.
- Polynucleotides encoding the polypeptides (a) to (d), that is, polynucleotides that can be used for producing the non-human transgenic animal of the present invention include DNA and RNA. It's preferable that there is.
- the polynucleotide for example, the sequence IJ consisting of bases 61 to 1410 in the base sequence represented by SEQ ID NO: 2, or from the base 73 to 1371 in the base sequence represented by SEQ ID NO: 4 And a polynucleotide encoding a polypeptide having the activity of suppressing weight gain.
- a polynucleotide consisting of the nucleotide sequence represented by SEQ ID NO: 2 or the nucleotide sequence represented by SEQ ID NO: 4, or the nucleotide sequence consisting of nucleotides 61 to 1410 in the nucleotide sequence represented by SEQ ID NO: 2 It is a polynucleotide comprising a sequence consisting of bases 73 to 1371 in the base sequence represented by SEQ ID NO: 4 or SEQ ID NO: 4.
- the non-human transgenic animal of the present invention can be specifically produced, for example, according to the method described in Example 2. That is, a DNA construct in which a CAG promoter is ligated upstream of AGF cDNA is prepared, the resulting DNA construct is introduced into ES cells, and a clone expressing AGF is selected by Western blot analysis, for example.
- the chimeric mice can be delivered by microinjecting them into blastocysts and transplanting the engineered eggs into the uterus.
- a transgenic mouse can be obtained by crossing the chimeric mouse with a normal mouse.
- the non-human transgenic animal of the present invention can be used for identifying a therapeutic drug for obesity, diabetes, and Z or hyperlipidemia, and identifying Z or a drug discovery target molecule. More specifically, by crossing an animal (for example, a mouse) in which an arbitrary gene X is modified with a non-human transgenic animal of the present invention (for example, an AGF transgenic mouse), The offspring of which the genes have been modified can be produced, and the phenotype of the offspring can be analyzed.
- an animal for example, a mouse
- a non-human transgenic animal of the present invention for example, an AGF transgenic mouse
- genes X and Z or a function promoter for gene X can be used as a therapeutic agent for obesity, diabetes, and / or hyperlipidemia.
- the gene whose expression is fluctuated in the non-human transgenic animal of the present invention is a drug discovery target molecule for obesity, diabetes, and / or hyperlipidemia, and the gene or its agonist or antagonist is , Anti-obesity drugs, anti-diabetic drugs, and Z or anti-hyperlipidemic drugs.
- the non-human transgenic animal of the present invention is useful as a source of a material (eg, tissue or blood) for identifying a new drug target molecule.
- a targeting vector having an HSV-tk gene in order were prepared by the following method.
- mouse genomic library (Mouse Mouse) was used according to the attached manual, using as a probe a cDNA corresponding to the entire length of the protein coding region of mouse AGF prepared in the same manner as in Example 1 of WO03 / 083114.
- Genomic, 129 SVJ Library (Stratagene) was screened, and a phage clone with a sequence of approximately 17.9 kbp (90644-108544 of the mouse-pub-genome sequence AC073775.2 containing the AGF gene) was isolated. And sub-cooled to plasmid pBluescript (Stratagene).
- the plasmid clone (P BN2) is cleaved with restriction enzymes Sail and Mf el, and 5 to about 6 ⁇ 2 kbp, long arm (mouse-pub-genome sequence
- PCR was carried out using a primer set consisting of the nucleotide sequence represented by the following formula.
- Okbp including 105903 to 107914 of the mouse-pub-genome sequence AC073775.2 was obtained.
- the 5 'long arm and the 3' short arm were inserted into the Xhol and Xbal sites of plasmid pPNT (Cell, 1991, 65 (7), 1153-1163), respectively, to complete the targeting vector.
- the obtained targeting vector was digested with restriction enzyme Notl, and ES cell line R1 (Proceedings
- ES cells were cultured in a medium containing G418 to obtain a resistant strain. DNA was extracted from ES cells, and clones in which only homologous recombination had occurred were identified by Southern blot analysis. Specifically, SEQ ID NO: 8 (including AGF exon 4 and exon 5)
- the obtained ES cell line was microinjected into a blastocyst obtained from a BDF2 mouse, which was the second generation of C57BL / 6 mouse and DBA / 2 mouse hybrid, and the engineered egg was implanted into the uterus.
- Procedure Chimera mice were delivered from mice that had received eggs and were pregnant.
- a heterozygous mouse hereinafter, referred to as an AGF heterozygous KO mouse
- AGF heterozygous KO mouse a heterozygous mouse having a mutant allele lacking the initiation codon of AGF was obtained.
- Homozygous mice hereinafter referred to as AGF homo KO mice
- AGF homo KO mice Homozygous mice
- Reverse primer Rooster S system IJ number 12 (5'-ctacctacatccactcctac-3, mouse)
- PCR was performed on the obtained genomic DNA derived from mouse pups using the primers described above. PCR was performed using DNA polymerase (ExTaq; Takara) at 95 ° C (5 minutes) followed by heat denaturation at 95 ° C (1 minute), 60 ° C (1 minute), and 72 ° C (1 minute). (1 minute) for 30 cycles, followed by an extension reaction at 72 ° C. (7 minutes) to check the size of the amplified fragment.
- DNA polymerase ExTaq; Takara
- the genotype of the mouse was determined.
- the band of the mutant allele was detected and the band of the wild type allele was not detected
- the AGF heterozygous KO mouse both the band of the mutant allele and the band of the wild type allele were detected.
- no band of the mutant allele was detected, and a band of the wild-type allele was detected.
- CAG-AGF Tg mice AGF transgenic mice in which mouse AGF was forcibly expressed throughout the body under the control of a promoter [CMV-IE enhancer) promoter [GENE, 108 (1991) 193-200] were prepared.
- CMV-IE enhancer a promoter [CMV-IE enhancer] promoter
- mouse AGF full-length cDNA prepared in the same manner as in WO03 / 083114
- the above-described linear DNA fragment was introduced by electroporation ( 0.8 V, 3 / iF).
- ES cells were cultured in the presence of 0.4 Mg / mL blasticidin, and the gene was introduced. Cells were selected and 20 clones were established.
- the CAG_Cre vector (Blood,
- ES cells in which AGF is constantly expressed under the control of AGF Using the ES cells of each clone in which the 1 ⁇ 71– ⁇ interval was not removed before introducing the CAG-Cre vector as a negative control, it was confirmed that AGF was expressed in the selected 15 clones of ES cells in Example 1 ( It was confirmed by Western blotting using the same anti-AGF antibody as in 3). That is, Western blotting was performed using ES cells that had been diluted twice with a lysis buffer and a 2 ⁇ SDS sample buffer as a sample, and it was confirmed that AGF was expressed.
- Reverse primer Rooster 5 system IJ number 18 (5'_agccgggtcaacataacagc-3, mouse)
- PCR When PCR is performed using these primers, a 325 bp fragment is amplified from the transgene in PCR to detect AGF cDNA, a 320 bp fragment is amplified in PCR to detect LacZ, and these sizes are amplified from mouse genomic DNA. Should not be amplified.
- PCR was performed on the resulting genomic DNA derived from mouse pups. PCR was performed using DNA polymerase (ExTaq; Takara) and heat denaturation at 94 ° C (5 minutes), followed by 94 ° C (1 minute), 62 ° C (1 minute 30 seconds), and 72 ° C (1 minute 30 seconds), 28 cycles were performed, followed by extension reaction at 72 ° C (7 minutes), and the size of the amplified fragment was examined.
- CAG-AGF Tg mouse In the CAG-AGF Tg mouse prepared in Example 2, it was examined how much the AGF gene was expressed.
- CAG White fat (WAT), brown fat (BAT), cerebrum, cerebellum, hypothalamus, heart, liver, kidney, spleen, skeletal muscle, and AGF Tg mice and littermate wild-type mice (Ritamate WT mice)
- Total RNA was prepared from each tissue of the spleen using Trizol reagent (Invitrogen). Total RNA was treated with DNase and cleaned up using a commercially available RNA purification reagent (RNeasy; Qiagen) and DNase (Qiagen). 0.5 ⁇ g of the total RNA treated with DNase was transferred to Superscript 'First Strand System (for RT-PCR) (LIFE
- the measurement was performed by measuring the amount of fluorescence in real time using a PRISM 7900HT Sequence Detection System (Applied Biosystems). ⁇ cDNA as type ⁇ , primer designed for each gene as primer and Taqman probe (Taq
- PCR was performed using Universal PCR Master Mix (Applied Biosystems).
- the PCR reaction is carried out by performing an initial denaturation reaction at 95 ° C (10 minutes) and then repeating a cycle reaction consisting of 94 ° C (15 seconds) and 60 ° C (60 seconds) 45 times. Carried out.
- a cycle reaction consisting of 94 ° C (15 seconds) and 60 ° C (60 seconds) 45 times. Carried out.
- the same reaction was carried out using the above-mentioned cDNA or mouse genomic DNA as type III, and the gene expression level was calculated as a relative value between samples.
- the expression level of the AGF gene was increased in the tissue of the CAG-AGF Tg mouse compared to the tissue of the WT mouse.
- the expression was significantly increased in skeletal muscle, BAT, and heart.
- CAG-AGF Tg mice were backcrossed to C57BL / 6 for two generations to obtain F2 CAG-AGF Tg mice.
- CAG-AGF Tg mice and Rita-Mate WT mice were normally bred on a standard diet (CE-2; CLEA Japan).
- CE-2 CLEA Japan
- body weights of 6-week-old female mice were compared, CAG-AGF Tg mice were up to 15.7 g ⁇ 0.8 g (SD), and WT mice were up to 17.8 g ⁇ 0.5 g (SD).
- CAG-AGF Tg mice were 19.5 g ⁇ 0.7 g (SD)
- WT mice were 23.5 g ⁇ 2.5 g (SD)
- CAG-AGF Tg The mice were found to have lower body weight.
- AGF homo KO mice, AGF heterozygous K ⁇ mice, and Ritamate WT mice were bred normally with a standard diet. The weight of each mouse was measured every week until the age of 24 weeks. The result is shown in figure 2. As shown in FIG. 2, it was found that AGF homozygous mice began to gain weight at about 12 weeks of age as compared to WT mice, and thereafter continued to gain weight, resulting in marked obesity. The AGF heterozygous KO mouse had an intermediate phenotype between the AGF homozygous KO mouse and the Ritamate WT mouse.
- Tg mice had lower tissue weight per body weight than WT mice. That is, the weight loss of CAG-AGF Tg mice was suppressed by the weight of white adipose tissue. It was shown that the increase was suppressed. In other words, it was found that AGF had no effect on the weight of other organs and had an effect of suppressing an increase in adipose tissue weight due to obesity and the like.
- the results for white adipose tissue are shown in FIG. 3 (standard diet) and FIG. 4 (high fat diet load).
- a high fat diet load increases the weight of white adipose tissue and further enlarges fat cells. It is known that hypertrophy of fat cells is associated with malignant malignancy of diabetes (Ayumi Kagaku, 192, 513-518, 2000; Ayumi Kagaku, 192, 541-54 5, 2000).
- -The morphology of adipocytes of AGF Tg mice was examined. Genital adipose tissue was collected from CAG-AGF Tg mice and Rita-mate WT mice reared on a high fat diet for 12 weeks at 12 weeks and 24 weeks of age.
- FIG. 8 AGF homozygous K ⁇ mouse
- FIG. 9 Renid WT mouse
- the RAT-mate WT mouse WAT had normal size fat cells, whereas the AGF homo KO mouse WAT had fat cells hypertrophy.
- fat accumulation in BAT was observed in AGF homozygous KO mice compared to Rita-mate WT mice.
- CAG-AGF Tg mice (Tg) and Rita-mate WT mice (WT) were bred for 1 month and 3 months on a high fat diet (HFD-32; CLEA Japan), and skeletal muscle and liver tissue of each mouse TG in the tissue was extracted from a sample using a solution of formaldehyde in methanol (Chemical Experiment Lecture 3, Lipid Chemistry, Tokyo Kagaku Dojin). The concentration of the extracted TG was measured using a kit (Triglyceride E Test Co., Ltd .; Wako Pure Chemical Industries, Ltd.), and the TG content in the tissue was determined. The results are shown in FIG. 10 (liver) and FIG. 11 (skeletal muscle).
- CAG-AGF Tg mice in both skeletal muscle and liver showed lower tissue TG content than WT mice. I got it. From these results, it was revealed that AGF has an activity of decreasing TG content in skeletal muscle and liver, which is known to increase due to obesity.
- TG content in skeletal muscle (gastrocnemius muscle) and liver tissue of AGF KO mice was examined.
- TG in tissues was extracted from skeletal muscle and liver tissues of AGF KO mice and Rita-Mate WT mice by the method (1) described above.
- the concentration of the extracted TG was measured using a kit (Triglyceride E Test Co., Ltd .; Wako Pure Chemical Industries, Ltd.) to determine the TG content in the tissue.
- the results are shown in FIG.
- the AGF KO mice were found to have significantly increased TG content in skeletal muscle and liver tissues compared to WT mice.
- the AGF KO mice were found to have the opposite phenotype as the CAG-AGF Tg mice, and it was revealed that the physiological activity of AGF was to reduce the TG content in skeletal muscle and liver.
- a glucose tolerance test was performed on AGF homo KO mice and Rita-Mate WT mice, and blood glucose levels and blood insulin levels were examined. After the mice were fasted for 16 hours, lg / kg of D-gunolose was administered by intraperitoneal injection (ip), and blood was collected from the ocular vein before administration and at 15, 30, 60, and 120 minutes after administration. The blood glucose level was measured using Daltest Ace (Sanwa Chemical Laboratory), and the blood insulin concentration was measured using the RIA2 antibody method (SRL). The results are shown in FIG. 13 (blood glucose level) and FIG. 14 (serum insulin).
- the oxygen consumption of CAG-AGF Tg mice and Ritamate WT mice was measured. oxygen Using a consumption meter (OXYMAX; Columbus Instruments), the oxygen consumption of the mice was measured over a 24-hour period under fasting conditions. The measurement method followed the instruction manual attached to the oxygen consumption measuring device. The oxygen consumption of each of 14 CAG-AGF Tg mice and Rita-Mate WT mice was measured, and the light period was 12 hours (7:00 19:00), and the 12th period was 19 hours (19:00 7:00) , And 24-hour oxygen consumption were determined and compared between CAG-AGF Tg mice and WT mice. The results are shown in FIG. It was found that the CAG-AGF Tg mice had a higher oxygen consumption (VO 2) than the WT mice at any time. As a result, AGF
- UCP1 was induced in BAT of CAG-AGF Tg mice, and the expression of UCP3, PPAR_a, and PPAR-5 was induced in skeletal muscle of CAG-AGF Tg mice.
- AGF induced the expression of UCP1 in BAT, and induced the expression of UCP3, PPAR-H, and PPAR-5 in skeletal muscle.
- UCP which enhances heat consumption by AGF
- PPAR which enhances heat consumption and lipid metabolism.Increases oxygen consumption, suppresses weight gain, suppresses fat tissue weight increase by AGF, etc. It is clear that this is one of the mechanisms of action.
- Human AGF and mouse AGF were expressed and purified according to the method described in WO03 / 083114 (Example 19). That is, an expression plasmid obtained by introducing a DNA fragment of about 1.4 kbp (human) or about 1.3 kbp (mouse) obtained by PCR into plasmid pcDNA_Signal_FLAG was introduced into HEK293 cells. The culture supernatant of the human AGF and mouse AGF expression cell lines was purified using an anti-FLAG-M2 monoclonal antibody agarose affinity gel (ANTI-FLAG).
- ANTI-FLAG anti-FLAG-M2 monoclonal antibody agarose affinity gel
- Affinity purification was performed using M2 Monoclonal Antibody Agarose Affinity Gel (Sigma) to obtain recombinant human and mouse AGF proteins.
- C2C12 cells obtained from ATCC to confluence, replace the culture medium from DMEM containing 10% fetal serum (FCS) with DMEM containing 2.5% fetal serum and continue culturing for 8 days Then, the cells were differentiated into skeletal muscle cells. After staring for 12 hours, C / C12 cells differentiated from skeletal muscle were added with or without human / mouse AGF recombinant protein at 3 / ig / mL, and then added for 4, 8, 12, and 24 hours after addition. Reacted. Without AGF Total RNA was prepared from stimulated and AGF-stimulated cells, treated with DNase, and then subjected to cDNA synthesis. PPAR— ⁇ , PG Shiichi 1 (Peroxisome Proliferat or- Act ivat ed
- Receptor-coactivator 1 alpha and CYP (cyclophilin) gene expression levels were measured by performing quantitative PCR according to the method described in Example 10 above.
- the primers shown in Tables 1 and 2 were used.
- the primers shown in Tables 1 and 2 were used.
- a commercially available PCR reagent as a commercially available PCR reagent,
- LCAD long-chain acyl-CoA dehydrogenase
- AGF induced the expression of PPAR-hi, LCAD, and PGC-l, supporting the anti-obesity effect of AGF.
- the primer sets include (SEQ ID NO: 45, SEQ ID NO: 46), (SEQ ID NO: 47, SEQ ID NO: 46), (SEQ ID NO: 48, SEQ ID NO: 46), (SEQ ID NO: 49, SEQ ID NO: 46), and (SEQ ID NO: 50).
- primer sets consisting of the nucleotide sequence represented by SEQ ID NO: 46
- fragments of about 200, about 300, about 400, about 600, and about 800 bp obtained were each subcloned into a cloning vector (PCR2.1-TOPO; Invitrogen).
- Each subclone obtained here is treated with restriction enzymes Kpnl and Nhel, and bases 2790 to 3001, 2705 to 3001, and 2604 of base ⁇ 's system' J represented by IJ number 1
- a fragment containing the base sequence J 'represented by No. 3001, No. 2406-3001, and No. 2206-3001 was treated with restriction enzymes Kpnl and Nehl for the luciferase atssay system vector pGV_B2 (PicaGene
- Vector 2 basic vector; Toyo Ink to obtain pGV_hAGFpro200 (N6), pGV-hAGFpro300 (N6), pGV-hAGFpro400 (N6), pGV-hAGFpro600 (N6), and pGV-hAGFpro800 (N6).
- Cloning of about lk and about 1.3 k upstream of the hAGF mRNA transcription region was performed using PCR.
- a DNA consisting of the base sequence represented by SEQ ID NO: 51 was used as the forward primer
- a DNA consisting of the base sequence represented by SEQ ID NO: 52 was used as the reverse primer.
- a DNA consisting of the nucleotide sequence of SEQ ID NO: 53 was used as a forward primer
- 'A DNA consisting of the base sequence represented by SEQ ID NO: 52 was used as one.
- the human genome (Genomic DNA; Clontech) was converted into type III, and these primer sets and DNA polymerase (TaKaRa LA taq TM; Takara Shuzo) were used. After 95 ° C for 2 minutes, 94 ° C for 30 seconds, 60 ° C30 PCR was performed 45 times for 2 seconds and at 3 minutes at 72 ° C, followed by 5 minutes at 72 ° C. The obtained fragments of about lkbp and 1.3 kbp were subcloned into a cloning vector (pCR-XL-T ⁇ PO; Invitrogen).
- Each subclone was treated with Kpn I and Nhel, and the resulting fragment (ie, containing the nucleotide sequences represented by nucleotides 202 1-3028 and 1640-3028 of the nucleotide sequence represented by SEQ ID NO: 1) The fragment) was introduced into pGV_B2 treated with Kpnl and Nhel to obtain pGV_hAGFprolk (N4) and pGV_hAGFprol.3k (N4).
- a human genome (Genomic DNA; Clontech) was used.
- a DNA polymerase (TaKaRa LA taq TM; Takara Shuzo Co., Ltd.) for 95 minutes at 95 ° C for 2 minutes, followed by 45 cycles of 94 ° C for 30 seconds, 63 ° C for 30 seconds, and 72 ° C for 2 minutes and 30 seconds.
- PCR was performed at 72 ° C. for 5 minutes.
- the obtained fragment of about 2 kb was subcloned into a cloning vector (pCR-XL-TOPO; Invitrogen).
- the subclone obtained here was treated with Kpnl and Xmal to obtain a fragment containing the nucleotide sequence represented by No. 1 to 1768 of the nucleotide sequence represented by SEQ ID NO: 1.
- 3k (N4) in pGV—hAGFprol. 3k (N4) has two Xmal restriction enzyme sites in the upstream sequence of the hAGF mRNA transcription region and in the cloning site of the pGV—B2 vector. 8.7 ⁇ g (pGV-hAGFprol.
- 3k (N4) was treated with 5 units of Xmal restriction enzyme for 110 minutes to prepare a plasmid in which Xmal was cut at 0, 1, or 2 places.
- the plasmid, which had been cut only at the site, was separated and extracted by electrophoresis, further treated with Kpnl, and obtained by electrophoresis to obtain a fragment separated to a size of 6. lkbp, thereby obtaining Xmal in the cloning site of the pGV-B2 vector.
- a plasmid was obtained in which the site was not cleaved and only the Xmal site in the sequence of the hAGF promoter region was cleaved, and the plasmid represented by SEQ ID NO: 1 obtained by the method described above (Kpnl and Xmal treatment) was used.
- a fragment containing each base sequence represented by Nos. 1 to 1768 of the base sequence was inserted to obtain pGV-hAGFpro3k (N4).
- pGV-hAGFpro800 (N6) was treated with SnaBI and Xbal to obtain a fragment (N6 fragment) of about 2 kbp.
- pGV-hAGFprolk N2
- pGV-hAGFpro1.3k N4
- pGV-hAGFpro3k N4
- SnaBI and Xbal SnaBI and Xbal
- the upstream of the hAGF mRNA transcription region which is the nucleotide sequence represented by nucleotides 2021 to 3001, 1640 to 3101, and 1 to 3001 of the nucleotide sequence represented by SEQ ID NO: 1
- the regions of about lkbp, 1.3 kbp and about 3 kbp were cloned to prepare a plasmid whose promoter activity could be measured.
- pGV-B2 empty vector
- plasmids obtained in Example 11 pGV-hAGFpro200 (N6), pGV-hAGFpro300 (N6), pGV-hAGFpro400 (N6), pGV-hAGFpro600 (N6), pGV -hAGFpro800 (N6), pGV-hAGFprolk (N6), pGV-hAGFpro1.3k (N6), and pGV_hAGFpro3k (N6), and ii) a j3-gal expression plasmid (pCHHO; Amersham Pharmacia Biotech) Transfection reagent (
- 293EBNA cells (FuGene-6; Japan Roche) cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal serum, 100 / ig / mL penicillin, and 100 ⁇ g / mL streptomycin (Invitrogen).
- DMEM Dulbecco's modified Eagle medium
- Luciferase activity after culturing for 48 hours under normal culture conditions was measured using a commercially available measurement kit (PicaGene color development kit; Toyo Ink). At this time, the measured value was corrected with the activity value of / 3-gal expressed from the co-introduced / 3-gal expression plasmid.
- ⁇ -gal activity can be measured using a commercially available assay kit (Galacto-Light
- a plasmid containing DNA of about 400 bp and about 3 kbp pGV-hAGFpro400 (N6), pGV-hAGFpro600 (N6), pGV-hAGFpro800 (N6), pGV-hAGFprolk (N6), pGV-hAGFprol.3k (N6 ), And pGV_hA No increase in luciferase activity was observed in cells into which GFpro3k (N6)] was introduced.
- a reporter assay using a DNA sequence of about 300 bp represented by a sequence consisting of nucleotides 2705 to 3001 in the nucleotide sequence represented by SEQ ID NO: 1 can be constructed. Activity was found to be included.
- the test compound is preferably added to the cells after the introduction of pGV-hAGFpro300 (N6), and the change in luciferase activity is analyzed to promote the AGF gene promoter activity and enhance AGF expression.
- Anti-obesity drugs, anti-diabetic drugs, and / or anti-hyperlipidemic drugs can be screened.
- an oligonucleotide consisting of the base sequence IJ represented by SEQ ID NO: 56 was used as the forward primer
- an oligonucleotide consisting of the base sequence represented by SEQ ID NO: 57 was used as the reverse primer.
- PCR use Pyrobest DNA polymerase (Takara Shuzo), 98 ° C (20 seconds) / 64 ° C (30 seconds) / 74 ° C (3 minutes) in the presence of 5% formamide ) Cycle was repeated 35 times. As a result, a DNA fragment of about 1.5 kbp was amplified.
- This fragment was cloned using pCR2.1 plasmid (Invitrogen) to obtain plasmid pCR2.1.m_New.
- the nucleotide sequence of the clone obtained was determined by the dideoxy termination method using the DNA sequencer (ABI377).
- SEQ ID NO: 58 This sequence has an open reading frame of 1374 bases. It has column number 58 No. 1-1374).
- the amino acid sequence (457 amino acids) predicted from the open reading frame is shown in SEQ ID NO: 59.
- Mouse AGF has a signal sequence (124-1-) at the N-terminus, and its signal sequence is cleaved when secreted and expressed extracellularly. It has mouse mature AGF force S consisting of amino acids 1 to 433 in the amino acid sequence represented by SEQ ID NO: 59 from which the signal sequence has been cleaved, and has biological activity.
- amino acid sequence represented by SEQ ID NO: 59 the sequence consisting of amino acids 1 to 433 (mouse) is human NL8ZNEW, angiopoietin-like 6 or angiopoietin-related protein 5 (angiopoietin-like protein). related protein
- the plasmid pCR2.l_mNew prepared in Reference Example 3 was digested with restriction enzymes Xbal and Spel to obtain a fragment containing a 1.4 kb mouse AGF gene. This fragment was introduced into pEF-BOS-neo [Mizushima, S., & Nagata, S. Nucleic Acids Res. 18: 5322 (1990)] which had been digested with Xbal and treated with BAP to express mouse AGF. Create the vector pEF_BOS_mAGF Was. Fuyu 1 ⁇ ji 1 ⁇ on 6 (Fugene6) (Roche
- transfection of pEF-BOS-mAGF The transfected cells were cultured in the presence of 300 ⁇ g / mL Genetics (Geneticin; Roche Diagnostics) to obtain a mouse AGF stable expression strain (OP9ZAGF).
- OP9ZAGF mouse AGF stable expression strain
- an OP9 cell line ( ⁇ P9 / vector) transfected with only the pEF-BOS_neo vector containing no mouse AGF gene was obtained for negative control.
- a green fluorescent protein gene (green) that can be used in a retrovirus expression system
- GFP fluorescent protein
- inflation Ekushi Yong cells were ATDC5 culture medium [DMEM / F_12 (Lifetechnologies Inc.), 5% FCS, 5 ⁇ g / mL insulin, 5 ⁇ g / mL transferrin, 3 X 10- 8 mol / L selenite Sodium], and before being confluent, the cells were dispersed by removing from the culture plate by trypsin treatment. The dispersed cells were applied to a cell separator (FACS vantage; Becton Dickinson) to separate and collect ATDC5 having GFP fluorescence to obtain ATDC5 expressing GFP.
- FACS vantage Becton Dickinson
- the AGF promoter or screening method of the present invention and the non-human knockout animal of the present invention can be applied to the use of screening for antiobesity drugs, antidiabetic drugs, and / or antihyperlipidemic drugs. Further, the non-human transgenic animal of the present invention can be applied to the use of developing antiobesity drugs, antidiabetic drugs, and / or antihyperlipidemic drugs.
- the present invention has been described in connection with the specific embodiments, but modifications and improvements obvious to those skilled in the art are included in the scope of the present invention.
- FIG. 1 is a drawing schematically showing the structure of plasmid pBS-loxP-1 ⁇ 71-mAGF-iS geo.
- the symbols “pro.” And “pri.” Mean the promoter and primer, respectively.
- FIG. 2 is a graph showing changes in body weight of AGF KO mice.
- the horizontal axis indicates the age (week), and the vertical axis indicates the body weight (g).
- the symbols “WT”, “HTR”, and “HM” mean WT mouse, heterozygous KO mouse, and homozygous mouse, respectively.
- FIG. 3 is a graph showing changes in the weight of genital fat (white adipose tissue) in CAG-AGF Tg mice (standard diet).
- the vertical axis indicates white adipose tissue weight (g) / body weight (g).
- the symbols “WT” and “Tg” mean WT mouse and Tg mouse, respectively.
- FIG. 4 is a graph showing changes in genital fat (white adipose tissue) weight of CAG-AGF Tg mice (high fat diet load).
- the vertical axis indicates white adipose tissue weight (g) and Z body weight (g).
- the numbers "WT” and “Tg” mean WT and Tg mice, respectively.
- FIG. 5 is a graph showing changes in genital fat (white adipose tissue) weight (white adipose tissue weight) of AGF KO mice.
- the vertical axis indicates white adipose tissue weight (g).
- the symbols “WT”, “HTR”, and “HM” mean WT mice, AGF heterozygous KO mice, and AGF homozygous KO mice, respectively.
- FIG. 6 is a graph showing changes in genital fat (white adipose tissue) weight (white adipose tissue weight / body weight) of AGF KO mice.
- the vertical axis indicates white adipose tissue weight (g) and Z body weight (g).
- the symbols “WT”, “HTR”, and “HM” mean a WT mouse, an AGF heterozygous KO mouse, and an AGF homozygous KO mouse, respectively.
- FIG. 9 shows the results of microscopic observation of the morphology of adipocytes in Rita-Mate WT mice.
- FIG. 10 is a graph showing the TG content in the tissue (liver) of CAG—AGF Tg mice.
- the horizontal axis shows the high fat diet load (for months), and the vertical axis shows the TG content (mg / g tissue).
- the symbols “WT” and “Tg” mean WT mouse and Tg mouse, respectively.
- CAG-AGF is a graph showing TG content in tissues (skeletal muscle) of Tg mice.
- the horizontal axis shows the high fat diet load (for months), and the vertical axis shows the TG content (mg / g tissue). Also, the symbol "W
- T and Tg mean WT and Tg mice, respectively.
- FIG. 12 is a graph showing the TG content in tissues of AGF KO mice. The vertical axis shows the TG content (mg
- the symbols “L” and “SM” on the horizontal axis mean liver and skeletal muscle, respectively, and the symbols “WT”, “HTR”, and “HM” mean WT mouse, hetero KO mouse, And homozygous K ⁇ mice.
- FIG. 13 is a graph showing the results of a glucose tolerance test (blood glucose level) of AGF KO mice.
- the horizontal axis indicates time (minutes), and the vertical axis indicates blood glucose level (mg / dL).
- the symbols “WT” and “HM” mean a WT mouse and a homo KO mouse, respectively.
- FIG. 14 is a graph showing the results of a glucose tolerance test (serum insulin) in AGF KO mice.
- the horizontal axis represents time (minutes), and the vertical axis represents serum insulin (ng / mL).
- the symbols “WT” and “HM” mean a WT mouse and a homo KO mouse, respectively.
- FIG. 15 is a graph showing oxygen consumption of CAG_AGF Tg mice.
- lane 1 is “light period”
- lane 2 is “period”
- lane 3 is “24 hours”
- the vertical axis is V ⁇ (mL /
- WT and Tg mean WT mouse and Tg mouse, respectively.
- FIG. 16 is a graph showing luciferase activity. The vertical axis indicates luciferase / ⁇ -gal.
- Fig. 17 is a graph showing luciferase activity. The vertical axis indicates luciferase / ⁇ -gal.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Environmental Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Biochemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Toxicology (AREA)
- Microbiology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Plant Pathology (AREA)
- Vascular Medicine (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Description
Claims
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US10/551,964 US20060156423A1 (en) | 2003-06-06 | 2004-06-03 | Method of screening antiobesity agents and animal model of obesity |
JP2005506774A JPWO2004108920A1 (ja) | 2003-06-06 | 2004-06-03 | 抗肥満薬のスクリーニング方法及び肥満モデル動物 |
CA002518627A CA2518627A1 (en) | 2003-06-06 | 2004-06-03 | Method of screening antiobesity agents and animal model of obesity |
EP04745548A EP1593740A4 (en) | 2003-06-06 | 2004-06-03 | PROCEDURE FOR SCREENING MEDICINE AGAINST OBESITY AND OBESITY ANIMAL MODEL |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2003163016 | 2003-06-06 | ||
JP2003-163016 | 2003-06-06 | ||
JP2004111500 | 2004-04-05 | ||
JP2004-111500 | 2004-04-05 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2004108920A1 true WO2004108920A1 (ja) | 2004-12-16 |
Family
ID=33513394
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2004/007692 WO2004108920A1 (ja) | 2003-06-06 | 2004-06-03 | 抗肥満薬のスクリーニング方法及び肥満モデル動物 |
Country Status (5)
Country | Link |
---|---|
US (1) | US20060156423A1 (ja) |
EP (1) | EP1593740A4 (ja) |
JP (1) | JPWO2004108920A1 (ja) |
CA (1) | CA2518627A1 (ja) |
WO (1) | WO2004108920A1 (ja) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005097171A1 (ja) * | 2004-04-05 | 2005-10-20 | Astellas Pharma Inc. | 抗肥満薬 |
WO2006059691A1 (ja) * | 2004-12-03 | 2006-06-08 | Astellas Pharma Inc. | 抗肥満薬のスクリーニング方法 |
WO2009139459A1 (ja) * | 2008-05-15 | 2009-11-19 | 国立大学法人 岡山大学 | Psgl-1阻害によるメタボリックシンドロームの予防及び治療法 |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
HU0700675D0 (en) * | 2007-10-15 | 2007-12-28 | Mta Tamogatott Kutatohelyek Ir | Method for monitoring stem cell differentiation |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999015653A2 (en) * | 1997-09-19 | 1999-04-01 | Genentech, Inc. | Tie ligand homologues |
JP2000300263A (ja) * | 1999-04-14 | 2000-10-31 | Herikkusu Kenkyusho:Kk | 血管新生に関連するタンパク質「410」および「new」、ならびに該タンパク質をコードする遺伝子 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6350450B1 (en) * | 1997-09-19 | 2002-02-26 | Genentech, Inc. | TIE ligand homologue antibody |
US5972338A (en) * | 1997-09-19 | 1999-10-26 | Genentech, Inc. | Tie ligands homologues |
US7265210B2 (en) * | 2000-09-15 | 2007-09-04 | Genentech, Inc. | Anti-PRO9821 antibodies |
US20020174484A1 (en) * | 2001-05-25 | 2002-11-28 | Gotfried Bradley L. | Combination equipment cover and sleeping device |
US7135334B2 (en) * | 2001-06-20 | 2006-11-14 | Genentech, Inc. | PRO20044 nucleic acids |
-
2004
- 2004-06-03 WO PCT/JP2004/007692 patent/WO2004108920A1/ja not_active Application Discontinuation
- 2004-06-03 EP EP04745548A patent/EP1593740A4/en not_active Withdrawn
- 2004-06-03 CA CA002518627A patent/CA2518627A1/en not_active Abandoned
- 2004-06-03 JP JP2005506774A patent/JPWO2004108920A1/ja not_active Withdrawn
- 2004-06-03 US US10/551,964 patent/US20060156423A1/en not_active Abandoned
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999015653A2 (en) * | 1997-09-19 | 1999-04-01 | Genentech, Inc. | Tie ligand homologues |
JP2000300263A (ja) * | 1999-04-14 | 2000-10-31 | Herikkusu Kenkyusho:Kk | 血管新生に関連するタンパク質「410」および「new」、ならびに該タンパク質をコードする遺伝子 |
Non-Patent Citations (2)
Title |
---|
OIKE Y. ET AL: "Angiopoietin-related growth factor (AGF) promotes epidermal proliferation, remodeling, and regeneration", PNAS USA, vol. 100, 2003, pages 9494 - 9499, XP002981337 * |
See also references of EP1593740A4 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005097171A1 (ja) * | 2004-04-05 | 2005-10-20 | Astellas Pharma Inc. | 抗肥満薬 |
WO2006059691A1 (ja) * | 2004-12-03 | 2006-06-08 | Astellas Pharma Inc. | 抗肥満薬のスクリーニング方法 |
WO2009139459A1 (ja) * | 2008-05-15 | 2009-11-19 | 国立大学法人 岡山大学 | Psgl-1阻害によるメタボリックシンドロームの予防及び治療法 |
Also Published As
Publication number | Publication date |
---|---|
EP1593740A1 (en) | 2005-11-09 |
CA2518627A1 (en) | 2004-12-16 |
EP1593740A4 (en) | 2006-12-06 |
JPWO2004108920A1 (ja) | 2006-07-20 |
US20060156423A1 (en) | 2006-07-13 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
RU2654565C2 (ru) | Грызуны с гуманизированным il-7 | |
McCright et al. | Generation of new Notch2 mutant alleles | |
EP1727560B1 (en) | Gastrointestinal proliferative factor and uses thereof | |
KR20040015711A (ko) | 약물학적 및 독성학적 연구용 트랜스제닉 비인간 동물 | |
AU2015312098B2 (en) | Animal model of longevity and related methods for increasing longevity and inhibiting tumorigenesis | |
AU2002303199A1 (en) | Gastrokines and derived peptides including inhibitors | |
EP1423409A2 (en) | Gastrokines and derived peptides including inhibitors | |
WO2004108920A1 (ja) | 抗肥満薬のスクリーニング方法及び肥満モデル動物 | |
WO2005097171A1 (ja) | 抗肥満薬 | |
US20030041341A1 (en) | Non-human transgenic animal whose germ cells and somatic cells contain a knockout mutation in DNA encoding 4E-BP1 | |
JP5804377B2 (ja) | 2型糖尿病モデル非ヒト動物 | |
JP2006314242A (ja) | 哺乳動物由来の変異sdhc遺伝子を有する遺伝子組み換え動物 | |
CA3155265A1 (en) | A rodent model of increased bone mineral density | |
JPWO2006059691A1 (ja) | 抗肥満薬のスクリーニング方法 | |
JP2008154489A (ja) | MafK/MafA遺伝子改変非ヒト動物及び該非ヒト動物の作成方法 | |
US20120079613A1 (en) | Homeobox Transcription Factor BSX and Uses Thereof for Treating Diseases, in Particular Obesity | |
WO2003021266A1 (en) | The great gene and protein | |
JP2005013151A (ja) | ロスモンド・トムソン症候群の特徴を示すマウス及びその作製方法 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
DPEN | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed from 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
WWE | Wipo information: entry into national phase |
Ref document number: 2005506774 Country of ref document: JP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2004745548 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2518627 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2006156423 Country of ref document: US Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 10551964 Country of ref document: US |
|
WWP | Wipo information: published in national office |
Ref document number: 2004745548 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 10551964 Country of ref document: US |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 2004745548 Country of ref document: EP |