WO2001033956A1 - Melanocortin-4 receptor deficient cells, non-human transgenic animals and methods of selecting compounds which regulate body weight - Google Patents
Melanocortin-4 receptor deficient cells, non-human transgenic animals and methods of selecting compounds which regulate body weight Download PDFInfo
- Publication number
- WO2001033956A1 WO2001033956A1 PCT/US2000/031061 US0031061W WO0133956A1 WO 2001033956 A1 WO2001033956 A1 WO 2001033956A1 US 0031061 W US0031061 W US 0031061W WO 0133956 A1 WO0133956 A1 WO 0133956A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- compound
- mouse
- subunit
- transgenic
- Prior art date
Links
- 241001465754 Metazoa Species 0.000 title claims abstract description 118
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 97
- 238000000034 method Methods 0.000 title claims abstract description 72
- 230000037396 body weight Effects 0.000 title claims abstract description 46
- 108010021436 Type 4 Melanocortin Receptor Proteins 0.000 title claims description 387
- 102000001796 Melanocortin 4 receptors Human genes 0.000 title claims description 338
- 150000001875 compounds Chemical class 0.000 title claims description 79
- 230000002950 deficient Effects 0.000 title abstract description 32
- 230000000694 effects Effects 0.000 claims abstract description 81
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 74
- 238000012360 testing method Methods 0.000 claims abstract description 72
- 230000037323 metabolic rate Effects 0.000 claims abstract description 49
- 230000037406 food intake Effects 0.000 claims abstract description 32
- 235000012631 food intake Nutrition 0.000 claims abstract description 32
- 210000004027 cell Anatomy 0.000 claims description 200
- 230000014509 gene expression Effects 0.000 claims description 68
- 239000000126 substance Substances 0.000 claims description 60
- 239000013604 expression vector Substances 0.000 claims description 58
- 239000012528 membrane Substances 0.000 claims description 32
- 239000003446 ligand Substances 0.000 claims description 29
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 28
- 208000008589 Obesity Diseases 0.000 claims description 27
- 235000020824 obesity Nutrition 0.000 claims description 27
- 210000000287 oocyte Anatomy 0.000 claims description 26
- 102000004169 proteins and genes Human genes 0.000 claims description 26
- 208000035475 disorder Diseases 0.000 claims description 25
- 238000011830 transgenic mouse model Methods 0.000 claims description 24
- 230000001131 transforming effect Effects 0.000 claims description 18
- 230000006870 function Effects 0.000 claims description 17
- 108010000239 Aequorin Proteins 0.000 claims description 16
- 206010012601 diabetes mellitus Diseases 0.000 claims description 10
- 230000007812 deficiency Effects 0.000 claims description 8
- 208000002193 Pain Diseases 0.000 claims description 7
- 241000269370 Xenopus <genus> Species 0.000 claims description 7
- 210000004602 germ cell Anatomy 0.000 claims description 7
- 230000015654 memory Effects 0.000 claims description 7
- 230000036407 pain Effects 0.000 claims description 7
- 206010057671 Female sexual dysfunction Diseases 0.000 claims description 6
- 206010057672 Male sexual dysfunction Diseases 0.000 claims description 6
- 230000007423 decrease Effects 0.000 claims description 6
- 230000003993 interaction Effects 0.000 claims description 6
- 230000009689 neuronal regeneration Effects 0.000 claims description 6
- 230000007823 neuropathy Effects 0.000 claims description 6
- 201000001119 neuropathy Diseases 0.000 claims description 6
- 208000033808 peripheral neuropathy Diseases 0.000 claims description 6
- 208000037824 growth disorder Diseases 0.000 claims description 5
- 238000012544 monitoring process Methods 0.000 claims description 3
- 210000001082 somatic cell Anatomy 0.000 claims description 2
- 102000002067 Protein Subunits Human genes 0.000 claims 4
- 108010001267 Protein Subunits Proteins 0.000 claims 4
- 208000011580 syndromic disease Diseases 0.000 claims 4
- 101100286588 Mus musculus Igfl gene Proteins 0.000 claims 2
- 102000005962 receptors Human genes 0.000 abstract description 26
- 108020003175 receptors Proteins 0.000 abstract description 26
- 230000006583 body weight regulation Effects 0.000 abstract description 16
- 238000012216 screening Methods 0.000 abstract description 9
- 241000699670 Mus sp. Species 0.000 description 65
- 239000000556 agonist Substances 0.000 description 58
- 238000003556 assay Methods 0.000 description 41
- 108020004414 DNA Proteins 0.000 description 40
- 239000005557 antagonist Substances 0.000 description 38
- 238000011813 knockout mouse model Methods 0.000 description 37
- 239000013598 vector Substances 0.000 description 24
- 230000004913 activation Effects 0.000 description 20
- 238000011282 treatment Methods 0.000 description 20
- JDKLPDJLXHXHNV-MFVUMRCOSA-N (3s,6s,9r,12s,15s,23s)-15-[[(2s)-2-acetamidohexanoyl]amino]-9-benzyl-6-[3-(diaminomethylideneamino)propyl]-12-(1h-imidazol-5-ylmethyl)-3-(1h-indol-3-ylmethyl)-2,5,8,11,14,17-hexaoxo-1,4,7,10,13,18-hexazacyclotricosane-23-carboxamide Chemical compound C([C@@H]1C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCNC(=O)C[C@@H](C(N[C@@H](CC=2NC=NC=2)C(=O)N1)=O)NC(=O)[C@@H](NC(C)=O)CCCC)C(N)=O)C1=CC=CC=C1 JDKLPDJLXHXHNV-MFVUMRCOSA-N 0.000 description 19
- 101710201349 Metallothionein B Proteins 0.000 description 18
- 101710094505 Metallothionein-2 Proteins 0.000 description 18
- 102100031347 Metallothionein-2 Human genes 0.000 description 18
- 108700019146 Transgenes Proteins 0.000 description 18
- 108700028369 Alleles Proteins 0.000 description 16
- 241000699666 Mus <mouse, genus> Species 0.000 description 16
- 210000001671 embryonic stem cell Anatomy 0.000 description 16
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 15
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 15
- 102000014384 Type C Phospholipases Human genes 0.000 description 15
- 108010079194 Type C Phospholipases Proteins 0.000 description 15
- 239000011575 calcium Substances 0.000 description 15
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 14
- 108091006027 G proteins Proteins 0.000 description 14
- 102000030782 GTP binding Human genes 0.000 description 14
- 108091000058 GTP-Binding Proteins 0.000 description 14
- 102000004378 Melanocortin Receptors Human genes 0.000 description 14
- 108090000950 Melanocortin Receptors Proteins 0.000 description 14
- 229910052791 calcium Inorganic materials 0.000 description 14
- 210000002257 embryonic structure Anatomy 0.000 description 14
- 238000012217 deletion Methods 0.000 description 13
- 230000037430 deletion Effects 0.000 description 13
- 239000012634 fragment Substances 0.000 description 13
- 102000006822 Agouti Signaling Protein Human genes 0.000 description 12
- 108010072151 Agouti Signaling Protein Proteins 0.000 description 12
- 241000699660 Mus musculus Species 0.000 description 12
- 230000029918 bioluminescence Effects 0.000 description 12
- 238000005415 bioluminescence Methods 0.000 description 12
- 238000001727 in vivo Methods 0.000 description 12
- 230000035772 mutation Effects 0.000 description 12
- 210000004556 brain Anatomy 0.000 description 11
- 239000002299 complementary DNA Substances 0.000 description 11
- 238000000338 in vitro Methods 0.000 description 11
- 230000003834 intracellular effect Effects 0.000 description 11
- 238000005259 measurement Methods 0.000 description 11
- 230000008685 targeting Effects 0.000 description 11
- 108091026890 Coding region Proteins 0.000 description 10
- 241000484025 Cuniculus Species 0.000 description 10
- 102000030621 adenylate cyclase Human genes 0.000 description 10
- 108060000200 adenylate cyclase Proteins 0.000 description 10
- 230000002068 genetic effect Effects 0.000 description 10
- 239000000203 mixture Substances 0.000 description 10
- 241000238631 Hexapoda Species 0.000 description 9
- 241000700159 Rattus Species 0.000 description 9
- 210000004102 animal cell Anatomy 0.000 description 9
- 210000000349 chromosome Anatomy 0.000 description 9
- 108020004999 messenger RNA Proteins 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 206010006895 Cachexia Diseases 0.000 description 8
- 102100034216 Melanocyte-stimulating hormone receptor Human genes 0.000 description 8
- 102000009493 Neurokinin receptors Human genes 0.000 description 8
- 108050000302 Neurokinin receptors Proteins 0.000 description 8
- 108700026244 Open Reading Frames Proteins 0.000 description 8
- YHIPILPTUVMWQT-UHFFFAOYSA-N Oplophorus luciferin Chemical compound C1=CC(O)=CC=C1CC(C(N1C=C(N2)C=3C=CC(O)=CC=3)=O)=NC1=C2CC1=CC=CC=C1 YHIPILPTUVMWQT-UHFFFAOYSA-N 0.000 description 8
- 229910019142 PO4 Inorganic materials 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 8
- 108010021428 Type 1 Melanocortin Receptor Proteins 0.000 description 8
- 230000001404 mediated effect Effects 0.000 description 8
- 239000002773 nucleotide Substances 0.000 description 8
- 125000003729 nucleotide group Chemical group 0.000 description 8
- 235000021317 phosphate Nutrition 0.000 description 8
- 239000013612 plasmid Substances 0.000 description 8
- 108090000765 processed proteins & peptides Proteins 0.000 description 8
- 230000009467 reduction Effects 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 239000003981 vehicle Substances 0.000 description 8
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 7
- 235000013305 food Nutrition 0.000 description 7
- 238000009396 hybridization Methods 0.000 description 7
- 238000002347 injection Methods 0.000 description 7
- 239000007924 injection Substances 0.000 description 7
- 229960000367 inositol Drugs 0.000 description 7
- -1 inositol phosphates Chemical class 0.000 description 7
- 230000004044 response Effects 0.000 description 7
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 7
- 102000034353 G alpha subunit Human genes 0.000 description 6
- 108091006099 G alpha subunit Proteins 0.000 description 6
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 208000022531 anorexia Diseases 0.000 description 6
- 206010061428 decreased appetite Diseases 0.000 description 6
- 230000001965 increasing effect Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 150000007523 nucleic acids Chemical group 0.000 description 6
- 238000003752 polymerase chain reaction Methods 0.000 description 6
- 238000001890 transfection Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 108091028043 Nucleic acid sequence Proteins 0.000 description 5
- 210000002459 blastocyst Anatomy 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 239000012636 effector Substances 0.000 description 5
- 235000013601 eggs Nutrition 0.000 description 5
- 230000002538 fungal effect Effects 0.000 description 5
- 238000002744 homologous recombination Methods 0.000 description 5
- 230000006801 homologous recombination Effects 0.000 description 5
- 238000007901 in situ hybridization Methods 0.000 description 5
- 238000003780 insertion Methods 0.000 description 5
- 230000037431 insertion Effects 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 238000000520 microinjection Methods 0.000 description 5
- 102000039446 nucleic acids Human genes 0.000 description 5
- 108020004707 nucleic acids Proteins 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
- 108091033319 polynucleotide Proteins 0.000 description 5
- 102000040430 polynucleotide Human genes 0.000 description 5
- 239000002157 polynucleotide Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 4
- 101000860415 Homo sapiens Galanin peptides Proteins 0.000 description 4
- 108010008364 Melanocortins Proteins 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 241000700605 Viruses Species 0.000 description 4
- UAHFGYDRQSXQEB-LEBBXHLNSA-N afamelanotide Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(N)=O)NC(=O)[C@H](CO)NC(C)=O)C1=CC=C(O)C=C1 UAHFGYDRQSXQEB-LEBBXHLNSA-N 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 230000000295 complement effect Effects 0.000 description 4
- 238000004520 electroporation Methods 0.000 description 4
- 210000003527 eukaryotic cell Anatomy 0.000 description 4
- 102000050963 human GAL Human genes 0.000 description 4
- 238000007912 intraperitoneal administration Methods 0.000 description 4
- PHTQWCKDNZKARW-UHFFFAOYSA-N isoamylol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 230000000699 topical effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- NCYCYZXNIZJOKI-IOUUIBBYSA-N 11-cis-retinal Chemical compound O=C/C=C(\C)/C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C NCYCYZXNIZJOKI-IOUUIBBYSA-N 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 3
- 241000282693 Cercopithecidae Species 0.000 description 3
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 3
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 3
- 108020005187 Oligonucleotide Probes Proteins 0.000 description 3
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 3
- 102100040756 Rhodopsin Human genes 0.000 description 3
- 108090000820 Rhodopsin Proteins 0.000 description 3
- 241000283984 Rodentia Species 0.000 description 3
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 3
- 239000013543 active substance Substances 0.000 description 3
- 238000010171 animal model Methods 0.000 description 3
- 230000000692 anti-sense effect Effects 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000007707 calorimetry Methods 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000008878 coupling Effects 0.000 description 3
- 238000010168 coupling process Methods 0.000 description 3
- 238000005859 coupling reaction Methods 0.000 description 3
- 235000005911 diet Nutrition 0.000 description 3
- 230000037213 diet Effects 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000009472 formulation Methods 0.000 description 3
- 238000002825 functional assay Methods 0.000 description 3
- 230000037308 hair color Effects 0.000 description 3
- 238000013537 high throughput screening Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 230000031146 intracellular signal transduction Effects 0.000 description 3
- 229910001629 magnesium chloride Inorganic materials 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000002865 melanocortin Substances 0.000 description 3
- 239000000336 melanocortin receptor agonist Substances 0.000 description 3
- 239000002751 oligonucleotide probe Substances 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000012552 review Methods 0.000 description 3
- 230000019491 signal transduction Effects 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 235000019786 weight gain Nutrition 0.000 description 3
- 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 2
- 108020005544 Antisense RNA Proteins 0.000 description 2
- 241000269350 Anura Species 0.000 description 2
- 241000255789 Bombyx mori Species 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 206010021033 Hypomenorrhoea Diseases 0.000 description 2
- 102000016267 Leptin Human genes 0.000 description 2
- 108010092277 Leptin Proteins 0.000 description 2
- 102100023724 Melanocortin receptor 4 Human genes 0.000 description 2
- 101710085775 Melanocortin receptor 4 Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 108010069820 Pro-Opiomelanocortin Proteins 0.000 description 2
- 239000000683 Pro-Opiomelanocortin Substances 0.000 description 2
- 102100027467 Pro-opiomelanocortin Human genes 0.000 description 2
- 101710137500 T7 RNA polymerase Proteins 0.000 description 2
- 102000003425 Tyrosinase Human genes 0.000 description 2
- 108060008724 Tyrosinase Proteins 0.000 description 2
- 241000269368 Xenopus laevis Species 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000012190 activator Substances 0.000 description 2
- 210000000577 adipose tissue Anatomy 0.000 description 2
- 230000008485 antagonism Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
- 230000002759 chromosomal effect Effects 0.000 description 2
- 238000007398 colorimetric assay Methods 0.000 description 2
- 239000003184 complementary RNA Substances 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000010432 diamond Substances 0.000 description 2
- 210000001840 diploid cell Anatomy 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000012869 ethanol precipitation Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000010363 gene targeting Methods 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 238000003306 harvesting Methods 0.000 description 2
- 210000001320 hippocampus Anatomy 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 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 2
- 238000000185 intracerebroventricular administration Methods 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- QWTDNUCVQCZILF-UHFFFAOYSA-N isopentane Chemical compound CCC(C)C QWTDNUCVQCZILF-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 229940039781 leptin Drugs 0.000 description 2
- 238000001638 lipofection Methods 0.000 description 2
- 230000002018 overexpression Effects 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 210000001938 protoplast Anatomy 0.000 description 2
- 230000009257 reactivity Effects 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000010076 replication Effects 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000011664 signaling Effects 0.000 description 2
- 210000003491 skin Anatomy 0.000 description 2
- 229940054269 sodium pyruvate Drugs 0.000 description 2
- ATHGHQPFGPMSJY-UHFFFAOYSA-N spermidine Chemical compound NCCCCNCCCN ATHGHQPFGPMSJY-UHFFFAOYSA-N 0.000 description 2
- 238000010561 standard procedure 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
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 210000002820 sympathetic nervous system Anatomy 0.000 description 2
- 230000035924 thermogenesis Effects 0.000 description 2
- 239000003104 tissue culture media Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 230000014621 translational initiation Effects 0.000 description 2
- RWBLWXCGQLZKLK-USVTTYPOSA-N (2s)-2-[(2-aminoacetyl)amino]-n-[(2s)-1-[[(2s)-1-[[(2s)-1-[[(2s)-1-[[2-[[(2s)-1-[[(2s)-1-[[(2s)-1-amino-4-methylsulfanyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1h-imidazol-5-yl)-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-methyl-1-oxob Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(N)=O)NC(=O)CN)C(C)C)C1=CN=CN1 RWBLWXCGQLZKLK-USVTTYPOSA-N 0.000 description 1
- GGYWLZFXFKFWKL-GLWNXGLNSA-N (3s,6s,9r,12s,15r,23s)-15-[[(2r)-2-acetamidohexanoyl]amino]-6-[3-(diaminomethylideneamino)propyl]-12-(1h-imidazol-5-ylmethyl)-3-(1h-indol-3-ylmethyl)-9-(naphthalen-2-ylmethyl)-2,5,8,11,14,17-hexaoxo-1,4,7,10,13,18-hexazacyclotricosane-23-carboxamide Chemical compound C([C@H]1C(=O)N[C@H](CC=2C=C3C=CC=CC3=CC=2)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCNC(=O)C[C@H](C(N1)=O)NC(=O)[C@H](NC(C)=O)CCCC)C(N)=O)C1=CNC=N1 GGYWLZFXFKFWKL-GLWNXGLNSA-N 0.000 description 1
- 101710139410 1-phosphatidylinositol phosphodiesterase Proteins 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
- 108020005065 3' Flanking Region Proteins 0.000 description 1
- 108700034262 4-Nle-7-Phe-alpha- MSH Proteins 0.000 description 1
- 108060003345 Adrenergic Receptor Proteins 0.000 description 1
- 102000017910 Adrenergic receptor Human genes 0.000 description 1
- 108020000948 Antisense Oligonucleotides Proteins 0.000 description 1
- 108010051479 Bombesin Proteins 0.000 description 1
- 102000013585 Bombesin Human genes 0.000 description 1
- 108010073466 Bombesin Receptors Proteins 0.000 description 1
- 102100028628 Bombesin receptor subtype-3 Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000700198 Cavia Species 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- 101100007328 Cocos nucifera COS-1 gene Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 102000029816 Collagenase Human genes 0.000 description 1
- 108060005980 Collagenase Proteins 0.000 description 1
- OMFXVFTZEKFJBZ-UHFFFAOYSA-N Corticosterone Natural products O=C1CCC2(C)C3C(O)CC(C)(C(CC4)C(=O)CO)C4C3CCC2=C1 OMFXVFTZEKFJBZ-UHFFFAOYSA-N 0.000 description 1
- 241000192700 Cyanobacteria Species 0.000 description 1
- 102000001189 Cyclic Peptides Human genes 0.000 description 1
- 108010069514 Cyclic Peptides Proteins 0.000 description 1
- 108010008286 DNA nucleotidylexotransferase Proteins 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 102100029764 DNA-directed DNA/RNA polymerase mu Human genes 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 101800002068 Galanin Proteins 0.000 description 1
- 102000019432 Galanin Human genes 0.000 description 1
- 102000011392 Galanin receptor Human genes 0.000 description 1
- 108050001605 Galanin receptor Proteins 0.000 description 1
- 241000287828 Gallus gallus Species 0.000 description 1
- 101000887162 Gallus gallus Gallinacin-5 Proteins 0.000 description 1
- 101000887167 Gallus gallus Gallinacin-6 Proteins 0.000 description 1
- 101000887166 Gallus gallus Gallinacin-7 Proteins 0.000 description 1
- 101000887235 Gallus gallus Gallinacin-9 Proteins 0.000 description 1
- 102000047481 Gastrin-releasing peptide receptors Human genes 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 108010016122 Ghrelin Receptors Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 102100039256 Growth hormone secretagogue receptor type 1 Human genes 0.000 description 1
- 101000996727 Homo sapiens Gonadotropin-releasing hormone receptor Proteins 0.000 description 1
- 101000617830 Homo sapiens Sterol O-acyltransferase 1 Proteins 0.000 description 1
- 206010060378 Hyperinsulinaemia Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000003746 Insulin Receptor Human genes 0.000 description 1
- 108010001127 Insulin Receptor Proteins 0.000 description 1
- 108010025815 Kanamycin Kinase Proteins 0.000 description 1
- 108091026898 Leader sequence (mRNA) Proteins 0.000 description 1
- 229940126661 MC4 antagonist Drugs 0.000 description 1
- 241000282560 Macaca mulatta Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 108010047068 Melanin-concentrating hormone receptor Proteins 0.000 description 1
- 102000029828 Melanin-concentrating hormone receptor Human genes 0.000 description 1
- 229940117029 Melanocortin receptor agonist Drugs 0.000 description 1
- 239000000637 Melanocyte-Stimulating Hormone Substances 0.000 description 1
- 102400000740 Melanocyte-stimulating hormone alpha Human genes 0.000 description 1
- 101710200814 Melanotropin alpha Proteins 0.000 description 1
- 102400000744 Melanotropin gamma Human genes 0.000 description 1
- 101800000520 Melanotropin gamma Proteins 0.000 description 1
- 241000158728 Meliaceae Species 0.000 description 1
- 102000057413 Motilin receptors Human genes 0.000 description 1
- 108700040483 Motilin receptors Proteins 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 101000608766 Mus musculus Galectin-6 Proteins 0.000 description 1
- 101100129532 Mus musculus Mc4r gene Proteins 0.000 description 1
- 102000030937 Neuromedin U receptor Human genes 0.000 description 1
- 108010002741 Neuromedin U receptor Proteins 0.000 description 1
- 102400000064 Neuropeptide Y Human genes 0.000 description 1
- 108091092724 Noncoding DNA Proteins 0.000 description 1
- 108050000742 Orexin Receptor Proteins 0.000 description 1
- 102000008834 Orexin receptor Human genes 0.000 description 1
- 102000016978 Orphan receptors Human genes 0.000 description 1
- 108070000031 Orphan receptors Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000010222 PCR analysis Methods 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 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
- 241000235648 Pichia Species 0.000 description 1
- 229920005372 Plexiglas® Polymers 0.000 description 1
- 102100037097 Protein disulfide-isomerase A3 Human genes 0.000 description 1
- 108091034057 RNA (poly(A)) Proteins 0.000 description 1
- 230000006819 RNA synthesis Effects 0.000 description 1
- 101000608768 Rattus norvegicus Galectin-5 Proteins 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108010021820 SHU 9119 Proteins 0.000 description 1
- 241000242583 Scyphozoa Species 0.000 description 1
- 201000001880 Sexual dysfunction Diseases 0.000 description 1
- 238000002105 Southern blotting Methods 0.000 description 1
- 241000256251 Spodoptera frugiperda Species 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 102100021993 Sterol O-acyltransferase 1 Human genes 0.000 description 1
- 101000697584 Streptomyces lavendulae Streptothricin acetyltransferase Proteins 0.000 description 1
- 108091036066 Three prime untranslated region Proteins 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 102000008318 Type 3 Melanocortin Receptor Human genes 0.000 description 1
- 108010021433 Type 3 Melanocortin Receptor Proteins 0.000 description 1
- 241000269360 Xenopus sp. Species 0.000 description 1
- FHHZHGZBHYYWTG-INFSMZHSSA-N [(2r,3s,4r,5r)-5-(2-amino-7-methyl-6-oxo-3h-purin-9-ium-9-yl)-3,4-dihydroxyoxolan-2-yl]methyl [[[(2r,3s,4r,5r)-5-(2-amino-6-oxo-3h-purin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl] phosphate Chemical compound N1C(N)=NC(=O)C2=C1[N+]([C@H]1[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=C(C(N=C(N)N4)=O)N=C3)O)O1)O)=CN2C FHHZHGZBHYYWTG-INFSMZHSSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 210000003486 adipose tissue brown Anatomy 0.000 description 1
- 230000010386 affect regulation Effects 0.000 description 1
- 230000008484 agonism Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 230000036528 appetite Effects 0.000 description 1
- 235000019789 appetite Nutrition 0.000 description 1
- 210000001106 artificial yeast chromosome Anatomy 0.000 description 1
- 239000012131 assay buffer Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 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 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- DNDCVAGJPBKION-DOPDSADYSA-N bombesin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(N)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC=1NC2=CC=CC=C2C=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H]1NC(=O)CC1)C(C)C)C1=CN=CN1 DNDCVAGJPBKION-DOPDSADYSA-N 0.000 description 1
- 108010063504 bombesin receptor subtype 3 Proteins 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000005013 brain tissue Anatomy 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000009084 cardiovascular function Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000000423 cell based assay Methods 0.000 description 1
- 210000004671 cell-free system Anatomy 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229940107161 cholesterol Drugs 0.000 description 1
- 238000011260 co-administration Methods 0.000 description 1
- 229960002424 collagenase Drugs 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- OMFXVFTZEKFJBZ-HJTSIMOOSA-N corticosterone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@H](CC4)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 OMFXVFTZEKFJBZ-HJTSIMOOSA-N 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 235000021316 daily nutritional intake Nutrition 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 239000007933 dermal patch Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229910003460 diamond Inorganic materials 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- AFABGHUZZDYHJO-UHFFFAOYSA-N dimethyl butane Natural products CCCC(C)C AFABGHUZZDYHJO-UHFFFAOYSA-N 0.000 description 1
- 208000037765 diseases and disorders Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 230000001158 estrous effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 230000004634 feeding behavior Effects 0.000 description 1
- 231100000502 fertility decrease Toxicity 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 231100000221 frame shift mutation induction Toxicity 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- YFHXZQPUBCBNIP-UHFFFAOYSA-N fura-2 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=3OC(=CC=3C=2)C=2OC(=CN=2)C(O)=O)N(CC(O)=O)CC(O)=O)=C1 YFHXZQPUBCBNIP-UHFFFAOYSA-N 0.000 description 1
- 238000012224 gene deletion Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 210000002980 germ line cell Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960001031 glucose Drugs 0.000 description 1
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000003780 hair follicle Anatomy 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 102000034345 heterotrimeric G proteins Human genes 0.000 description 1
- 108091006093 heterotrimeric G proteins Proteins 0.000 description 1
- 235000009200 high fat diet Nutrition 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 102000057996 human GNRHR Human genes 0.000 description 1
- 230000003451 hyperinsulinaemic effect Effects 0.000 description 1
- 201000008980 hyperinsulinism Diseases 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- PNDZEEPOYCVIIY-UHFFFAOYSA-N indo-1 Chemical compound CC1=CC=C(N(CC(O)=O)CC(O)=O)C(OCCOC=2C(=CC=C(C=2)C=2N=C3[CH]C(=CC=C3C=2)C(O)=O)N(CC(O)=O)CC(O)=O)=C1 PNDZEEPOYCVIIY-UHFFFAOYSA-N 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229940125425 inverse agonist Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 102000005861 leptin receptors Human genes 0.000 description 1
- 108010019813 leptin receptors Proteins 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- SLZIZIJTGAYEKK-CIJSCKBQSA-N molport-023-220-247 Chemical compound C([C@@H](C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(N)=O)NC(=O)[C@H]1N(CCC1)C(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)CN)[C@@H](C)O)C1=CNC=N1 SLZIZIJTGAYEKK-CIJSCKBQSA-N 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000010004 neural pathway Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000000424 optical density measurement Methods 0.000 description 1
- 239000006186 oral dosage form Substances 0.000 description 1
- 230000002611 ovarian Effects 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000003076 paracrine Effects 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 210000003899 penis Anatomy 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 230000006461 physiological response Effects 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 1
- 239000004926 polymethyl methacrylate Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 108010030587 rat melanocortin receptor type 4 Proteins 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000027425 release of sequestered calcium ion into cytosol Effects 0.000 description 1
- 230000003362 replicative effect Effects 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 210000001995 reticulocyte Anatomy 0.000 description 1
- 230000001177 retroviral effect Effects 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 231100000872 sexual dysfunction Toxicity 0.000 description 1
- 230000036299 sexual function Effects 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 229940063673 spermidine Drugs 0.000 description 1
- 210000000278 spinal cord Anatomy 0.000 description 1
- 238000012453 sprague-dawley rat model Methods 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 230000037317 transdermal delivery Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- FQZJYWMRQDKBQN-UHFFFAOYSA-N tricaine methanesulfonate Chemical compound CS([O-])(=O)=O.CCOC(=O)C1=CC=CC([NH3+])=C1 FQZJYWMRQDKBQN-UHFFFAOYSA-N 0.000 description 1
- 229940013180 tricaine methanesulfonate Drugs 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
- 230000001960 triggered effect Effects 0.000 description 1
- 241000701447 unidentified baculovirus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 241001430294 unidentified retrovirus Species 0.000 description 1
- 108700026220 vif Genes Proteins 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000004260 weight control Methods 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
- WHNFPRLDDSXQCL-UHFFFAOYSA-N α-melanotropin Chemical compound C=1N=CNC=1CC(C(=O)NC(CC=1C=CC=CC=1)C(=O)NC(CCCNC(N)=N)C(=O)NC(CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)NC(CCCCN)C(=O)N1C(CCC1)C(=O)NC(C(C)C)C(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(CCSC)NC(=O)C(CO)NC(=O)C(NC(=O)C(CO)NC(C)=O)CC1=CC=C(O)C=C1 WHNFPRLDDSXQCL-UHFFFAOYSA-N 0.000 description 1
- WHNFPRLDDSXQCL-UAZQEYIDSA-N α-msh Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C(C)C)C(N)=O)NC(=O)[C@H](CO)NC(C)=O)C1=CC=C(O)C=C1 WHNFPRLDDSXQCL-UAZQEYIDSA-N 0.000 description 1
- GZWUQPQBOGLSIM-VOOUCTBASA-N γ msh Chemical compound C([C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)NCC(O)=O)C(C)C)C1=CC=C(O)C=C1 GZWUQPQBOGLSIM-VOOUCTBASA-N 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
- C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
-
- 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
- A01K2217/00—Genetically modified animals
- A01K2217/20—Animal model comprising regulated expression system
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/50—Amphibians, e.g. Xenopus
-
- 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
- A01K2267/035—Animal model for multifactorial diseases
- A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
Definitions
- MELANOCORTIN-4 RECEPTOR DEFICIENT CELLS, NON-HUMAN TRANSGENIC ANIMALS AND METHODS OF SELECTING COMPOUNDS WHICH REGULATE BODY WEIGHT
- the present invention relates to cells and non-human transgenic animals that have been engineered to be deficient in the gene encoding the melcanocortin-4 receptor protein (MC-4R). It is shown herein that male MC-4R deficient transgenic animals of the present invention show increased fatmass and are obese, while female heterozygous MC-4R deficient transgenic animals have similar body weight to wild type mice.
- the MC-4R deficient transgenic animals of the present invention can be used to select for and test potential modulators (e.g., agonists or antagonists) of MC-4R which control food intake and metabolic rate.
- the present invention also relates to methods of screening for MC-4R modulators which effect body weight in general but more specifically impart effects on the control of food intake and metabolic rate, in conjunction with associated methods of treating various disorders associated with inappropriate regulation of body weight.
- GPCR G-protein coupled receptors
- melanocortin receptors belong to the rhodopsin sub-family of G-protein coupled receptors (GPCR's). Five different subtypes are known. These melanocortin receptors bind and are activated by peptides such as -, ⁇ , or ⁇ -melanocyte stimulating hormones ( ⁇ -, ⁇ -, ⁇ -MSH) derived from the pro-opiomelanocortin (POMC) gene. A wide range of physiological functions are believed to be mediated by melanocortin peptides and their receptors.
- peptides such as -, ⁇ , or ⁇ -melanocyte stimulating hormones ( ⁇ -, ⁇ -, ⁇ -MSH) derived from the pro-opiomelanocortin (POMC) gene.
- POMC pro-opiomelanocortin
- MC-IR agonism through its natural ligand, a-MSH raises cAMP and the expression of the enzyme tyrosinase.
- Low levels of tyrosinase which result from agouti antagonism of MC-IR, result in reduced conversion of the hair color pigment pheomelanin to eumelanin. As a result a light (agouti) rather than black hair color results.
- the obese phenotype of the agouti mouse was ascribed to the expression of agouti in the brain, where it antagonizes MC-4R and MC-4R receptors.
- MC-Rs are expressed in a variety of tissues including the central nervous system, where the MC-4R is believed to be expressed exclusively.
- MC-4R melanocortin 4 receptor
- Huszar et al., 1997, Cell 88: 141-141 see also U.S. Patent No. 5,942, 779, issued August 4, 1999 to Lee et al
- MC-4R knock out mice have a predisposition to obesity and hyperinsulinemia first manifested after about 8 weeks of age. Since the phenotype of MC-4R knock out mice recapitulates the obesity observed in the A vy mouse it is assumed that the effects of agouti on the MC-4R receptor mediate the obese phenotype of the A vy mouse (the effects of agouti on skin color are exerted through the MC-IR).
- MC-4R has also been implicated as a regulator of feeding behavior which regulates body weight through studies with peptide agonists and antagonists (Fan et al., 1997, Nature 485: 165-168).
- melanocortin receptor targets through which regulation of body weight is mediated by both metabolic rate and food intake.
- the present invention addresses and meets these needs by disclosing MC-4R-deficient animal cells, related non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are also MC-4R-deficient.
- the present invention also addresses and meets these needs by disclosing methods of screening for compounds which effect body weight comprising the screening and selection of compounds which modulate the MC-4R, specifically by mediating both metabolic rate and food intake.
- the present invention relates to animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are MC-4R deficient (MC-4R null) due to a disruption in the gene encoding MC-4R. More specifically, this portion of the invention relates to a transgenic animal, preferably a transgenic mouse, wherein homozygous or heterozygous male MC-4R deficient transgenic animals show increased fatmass and are obese, while heterozygous female MC-4R deficient transgenic animals have similar body weight to wild type mice. Transgenic female mice homozygous for alteration of the MC-4R gene become obese.
- the MC-4R deficient transgenic animals of the present invention can be used to select for and test potential modulators (e.g., agonists or antagonists) of MC-4R which control both food intake and metabolic rate.
- the present invention further relates to animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates heterozygous for a functional MC-4R gene native to that animal which show the male/female obesity phenotypic difference described herein.
- the preferable transgenic animal is a mouse.
- the present invention also relates to transgenic embryos, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are either homozygous or heterozygous for deletion of the MC-4R gene in combination with a homozygous or heterozygous deletion at separate alleles which encode at least one additional melanocortin receptor, especially a melanocortin receptor shown to be involved in body weight regulation, such as MC-4R, wherein such transgenic animals are derived from MC-4R transgenic animals, preferably mice, which show the male/female obesity phenotype described herein.
- the transgenic cells and animals of the present invention are useful in the study of the effect of modulators on the activity of the MC-4R gene and/or protein or the expression of the MC-4R gene and/or protein as concerning the regulation of body weight, including but not limited to disorders such as obesity, diabetes, anorexia and cachexia, as well as diseases and disorders such as cancer, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy.
- the present invention also relates to methods of selecting for modulators which regulate body weight by selecting such modulators which show an ability to effect both metabolic rate and food intake when interacting with the MC-4R.
- in vivo e.g., transgenic mice
- in vitro e.g., recombinant cell- or membrane-based assays measuring the modulation of the MC-4R
- preferred modulators of MC-4R that is, modulators of MC-4R which effect both metabolic rate and food intake.
- the term "functional” is used to describe a gene or protein that, when present in a cell or in vitro system, performs normally as if in a native or unaltered condition or environment. Therefore, a gene which is not functional (i.e., “non-functional”, “disrupted”, “altered”, or the like) will encode a protein which does not function as a wild type, native or non-altered protein, or encodes no protein at all.
- Such a non-functional gene such as a non-functional MC-4R gene, may be the product of a homologous recombination event as described herein, where a non-functional gene is targeted specifically to the region of the target chromosome which contains a functional form of the gene, resulting in a "knock-out" of the wild type or native gene.
- a “modulator” is a compound that causes a change in the expression or activity of MC-4R, or causes a change in the effect of the interaction of MC-4R with its ligand(s), or other protein(s).
- transgenes and “genes”.
- a transgene is a genetic construct including a gene.
- the transgene is integrated into one or more chromosomes in the cells in an animal by methods known in the art. Once integrated, the transgene is carried in at least one place in the chromosomes of a transgenic animal.
- a gene is a nucleotide sequence that encodes a protein.
- the gene and/or transgene may also include genetic regulatory elements and/or structural elements known in the art.
- transgenic animal is used herein to include all mammals, except that when referring to transgenic animals, the use of this term excludes humans. It also includes an individual animal in all stages of development, including embryonic and fetal stages.
- a "transgenic animal” is an animal containing one or more cells bearing genetic information received, directly or indirectly, by deliberate genetic manipulation at a subcellular level, such as by microinjection or infection with recombinant virus. This introduced DNA molecule can be integrated within a chromosome, or it can be extra- chromosomally replicating DNA.
- transgenic animal refers to a transgenic animal in which the genetic information was introduced into a germ line cell, thereby conferring the ability to transfer the information to offspring. If offspring in fact possess some or all of the genetic information, then they, too, are transgenic animals.
- the genetic information is typically provided in the form of a transgene carried by the transgenic animal.
- a “targeted gene” or “Knockout” (KO) is a DNA sequence introduced into the germline of a non-human animal by way of human intervention, including but not limited to, the methods described herein.
- the targeted genes of the invention include nucleic acid sequences which are designed to specifically alter cognate endogenous alleles, especially endogenous alleles which encode MC-4R.
- MC-IR refers to the melanocortin- 1 receptor.
- M-3R refers to the melanocortin-3 receptor.
- M-4R refers to the melanocortin-4 receptor.
- FIGURES Figure 1 shows the nucleotide sequence which encodes the human MC-4R (SEQ ID NO: 1
- Figure 2 shows the amino acid sequence of the human MC-4R (SEQ ID NO:2).
- Figure 3 shows a schematic description of the strategy utilized in construction of the targeting gene vector, pAJ7.
- Figure 4 shows the strategy utilizing the targeting gene vector, pAJ7, for homologous recombination with mouse genomic sequences encoding MC-4R.
- Figure 5A-B show the mean body weights of MC-4R knockout mice and control littermates.
- Figure 6A-D show a lack of MC-4R mRNA expression in the brain of the
- MC-4R knock out mice In situ hybridization was performed on 14 ⁇ m coronal brain sections from wild type control (A and B) and MC-4R knock out (C and D) mice. 33 P-labeled oligonucleotide probes were used for detecting the mRNA for MR-4R in the absence (A and C) and presence (B and D) of 100 fold molar excess of non- labeled oligonucleotide probes. Arrows indicate specific hybridization for MC-4R mRNA in the hippocampus of the wild type mouse brain.
- Figure 7 shows the metabolic rate measurements in wild type (+/+) and MC-4R knock out (-/-) mice. Data are the average metabolic rate (cal/hr) from 30 minutes to 4 hours post dosing, mean + SE of 8 animals/group. MT-II was administered at 20 mg/kg, ip. * P ⁇ 0.01 vs vehicle control group.
- Saline vehicle was injected to the mice for two days prior to MT-II treatment.
- the average daily food intake for the two days was used as vehicle baseline (open bar). **P ⁇ 0.01, * P ⁇ 0.05 (paired-comparisons T test).
- Figure 9 shows an aequorin bioluminescence assay in which the rat melanocortin 4 receptor (MC-4R), upon stimulation by the melanocortin agonist peptide ⁇ NDP-MSH (1 ⁇ M), can functionally couple to activation of phospholipase C (mobilization of intracellular calcium reported by aequorin bioluminescence) when expressed in Xenopus oocytes in the presence of the G ⁇ l5 subunit.
- M-4R rat melanocortin 4 receptor
- ⁇ NDP-MSH 1 ⁇ M
- Figure 10 shows an experiment similar to that described for Figure 9, using the G ⁇ q i 5 subunit.
- the human galanin receptor 1 hGALRl
- hGALRl human galanin receptor 1
- the present invention relates to animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates which are MC-4R deficient (MC-4R null) due to a disruption in the gene encoding MC-4R.
- the invention also relates to a transgenic animal, preferably a transgenic mouse, wherein the male mice homozygous or heterozygous for the altered MC-4R gene show increased fatmass and are obese, while females homozygous for the altered MC-4R gene become obese while females heterozygous for the altered MC-4R gene have body weights similar to wild type female mice.
- MC-4R deficient transgenic animals of the present invention can be used in methodology to select for and test potential modulators (e.g., agonists or antagonists) of MC-4R which effect both food intake and metabolic rate.
- test potential modulators e.g., agonists or antagonists
- the MC-4R transgenic animals may be used in methodology to select for and test potential modulators (e.g., agonists or antagonists) of MC-4R involved the regulation of body weight and muscle mass as defined by lean body mass, modulators which may effect such disorders as obesity, diabetes, anorexia, cachexia, cancer, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy, growth disorders relating to reduced GH, IGF1 function, treatment of reduced lean body mass as it occurs in the frail elderly, and other states that are characterized as resulting from GH deficiency and cancer cachexia.
- modulators e.g., agonists or antagonists
- modulators which may effect such disorders as obesity, diabetes, anorexia, cachexia, cancer, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy, growth disorders relating to reduced GH, IGF1 function, treatment of reduced lean body mass as it occurs in the frail elderly, and other states that are characterized as resulting from GH deficiency and
- the transgenic animals of the present invention may be utilized to determine the effect of certain modulators on the activity and/or expression of the MC-4R gene or receptor protein, and aspects of disorders involving regulation of body weight.
- the present invention further relates to animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates heterozygous for a functional MC-4R gene native to that animal which show the male/female obesity phenotypic difference described herein.
- the preferable transgenic animal is a mouse and will be useful as a component or complement to the various assays described herein.
- MC-4R null animals can be used as a strain for the insertion of human MC-4R genes, and provides an animal model useful in the design and assessment of various approaches to modulating MC-4R activity and expression.
- modified transgenic non-human animals can also be used as a source of cells for cell culture. These cells can be used for corresponding in vitro studies of MC-4R expression, activity and the modulation thereof.
- An aspect of this invention is a method to obtain an animal in which the cells lack a functional gene MC-4R native to the animal.
- the method includes providing a gene for an altered form of the MC-4R gene native to the animal in the form of a transgene and targeting the transgene into a chromosome of the animal at the place of the native MC-4R gene or at another chromosomal location.
- the transgene can be introduced into the embryonic stem cells by a variety of methods known in the art, including electroporation, microinjection, and lipofection. Cells carrying the transgene can then be injected into blastocysts which are then implanted into pseudopregnant animals.
- the transgene-targeted embryonic stem cells can be co-incubated with fertilized eggs or morulae followed by implantation into females. After gestation, the animals obtained are chimeric founder transgenic animals.
- the founder animals can be used in further embodiments to cross with wild-type animals to produce FI animals heterozygous for the altered MC-4R gene.
- these heterozygous animals can be interbred to obtain the viable transgenic embryos whose somatic and germ cells are homozygous for the altered MC-4R gene and thereby lack a functional MC-4R gene.
- the heterozygous animals can be used to produce cells lines. In preferred embodiments, the animals are mice.
- a further aspect of the present invention is a transgenic non-human animal which expresses a non-native MC-4R on a native MC-4R null background.
- the null background is generated by producing an animal with an altered native MC-4R gene that is non-functional, i.e. a knockout.
- the animal can be heterozygous (i.e., having a different allelic representation of a gene on each of a pair of chromosomes of a diploid genome), homozygous (i.e., having the same representation of a gene on each of a pair of chromosomes of a diploid genome) for the altered MC-4R gene, hemizygous (i.e., having a gene represented on only one of a pair of chromosomes of a diploid genome), or homozygous for the non-native MC-4R gene.
- the animal is a mouse.
- the non-native MC-4R gene can be a wild-type or mutant allele, preferably a wild-type or mutant human allele.
- the non-native MC-4R gene is operably linked to a promoter.
- operably linked is used to denote a functional connection between two elements whose orientation relevant to one another can vary.
- a promoter can be operably linked to the coding sequence of a gene to direct the expression of the coding sequence while placed at various distances from the coding sequence in a genetic construct.
- Further embodiments are cell lines and cells derived from animals of this aspect of the invention.
- An aspect of this invention are transgenic animals having a transgene including a non-native MC-4R gene on a native MC-4R null background.
- the method includes providing transgenic animals of this invention whose cells are heterozygous for a native gene encoding a functional MC-4R protein and an altered native MC-4R gene. These animals are crossed with transgenic animals of this invention that are hemizygous for a transgene including a non-native MC-4R gene to obtain animals that are both heterozygous for an altered native MC-4R gene and hemizygous for a non-native MC-4R gene.
- the latter animals are interbred to obtain animals that are homozygous or hemizygous for the non-native MC-4R and are homozygous for the altered native MC-4R gene.
- cell lines are produced and cells isolated from any of the animals produced in the steps of the method.
- the transgenic animals and cells of this invention are useful in the determination of the in vivo function of a non-native MC-4R in regulation of body weight.
- the animals are also useful in determining the ability for various forms of wild-type and mutant alleles of a non-native MC-4R to rescue the native MC-4R null deficiency.
- the animals are also useful for identifying and studying the ability of a variety of compounds to act as modulators of the expression or activity of a non-native MC-4R in vivo, or by providing cells for culture, for in vitro studies.
- the genetic information received by the animal can cause the native gene to become non-functional to produce a "knockout" animal.
- the genetic information received by the animal can be foreign to the species of animal to which the recipient belongs, or foreign only to the particular individual recipient. In the last case, the information can be altered or it can be expressed differently than the native gene.
- the non-human transgenic animals of the present invention include non-human mammalian species, including but not limited to transgenic mice, transgenic rats, transgenic guinea pigs, transgenic rabbits, transgenic goats, transgenic non-human primates, such as chimpanzees, rhesus monkeys and green african monkeys, and transgenic cattle. Transgenic mice are preferred and exemplified herein.
- the present invention especially relates to analysis of the complex function(s) of MC-4R as related to obesity and diabetes by generating knock-out transgenic mice and studying how various potential modulators interact within these manipulated animals.
- the native wild type gene is selectively inactivated in totipotent ES cells (such as those described herein) and used to generate the transgenic mice of the present invention. Techniques are available to inactivate or alter any genetic region to any mutation desired by using targeted homologous recombination to insert specific changes into chromosomal alleles.
- the present invention relates to diploid animal cells, non-human transgenic embryos, non-human transgenic animals and non- human transgenic littermates which are heterozygous or homozygous for a disrupted MC- 4R gene resulting in deficient production of the MC-4R protein.
- the cells, embryos and non-human transgenic animals contain two chromosome alleles for MC-4R wherein at least one of the MC-4R alleles is mutated such that less than wild-type levels of MC-4R activity is produced.
- the diploid mouse cell, embryo or non-human transgenic mice homozygous for a disrupted MC-4R gene may show at least from about 50% to about 100% reduction in MC-4R activity compared to a wild type diploid cell.
- the diploid mouse cell, embryo or non-human transgenic mice heterozygous for a disrupted MC-4R gene may show at least from about 10% to about 100% reduction in MC-4R activity compared to a wild type diploid cell. It is within the purview of the artisan of ordinary skill to use known molecular biology techniques to measure the level of transcription, expression and/or functional MR-4R activity in mouse cell homozygous, heterozygous or hemizygous for a mutated MC-4R gene. Therefore, the present invention especially relates to analysis of the complex function(s) of MC-4R as related to obesity by generating homozygous, heterozygous or hemizygous transgenic mice and studying how various potential modulators interact within these manipulated animals.
- the assay is performed by providing an animal of the present invention, exposing the animal to the compound, and measuring the effect of said compound on body weight and other related biochemical and physiological responses.
- the measurement can be compared to these measurements in a genetically similar or identical animal that is not exposed to the compound.
- One way to facilitate such measurements would be to feed both MC-4R knock-out and wild-type mice a high fat diet to promote obesity (diet induced obesity [DIO]). After becoming obese, effects of potential MC-4R agonists may be measured for reduction of body fat in wild-type mice as opposed to no effect in MC-4R knock-out mice. Similar protocols may be useful in studying the effects of MC-4R modulators in other related disorders, such as diabetes.
- MC-4R complex events associated with modulation of the MC-4R.
- the potential role of MC-4R in sexual dysfunction may be studied, in light of the fact that MC-4R is heavily expressed in lamina X of the lumbar and sacral spinal cord a key center for signaling to the penis.
- a MC-4R gene that naturally occurs in the animal is referred to as the native gene, and if it is not mutant, it can also be referred to as wild-type.
- An altered MC-4R gene should not fully encode the same MC-4R as native to the host animal, and its expression product can be altered to a minor or great degree, or absent altogether.
- the altered MC-4R gene induce a null knockout phenotype in the animal.
- a more modestly modified MC-4R gene can also be useful and is within the scope of the present invention.
- the MC-4R mutation may be a targeted deletion mutation, a targeted substitution mutation and/or a targeted insertion mutation.
- the preferred mutation is a deletion mutation, and especially preferred is a deletion mutation which results in a deletion of most if not all of the MC-4R gene.
- Transgenic animals are generated which have an altered, or preferably, completely deleted MC-4R gene.
- MC-4R gene deletions, gene modifications and or gene insertions can render the native gene nonfunctional, producing a "knockout" transgenic animal, or can lead to a MC-4R with altered expression or activity.
- a non-human transgenic animal without an activated MC-4R gene can be used to evaluate the role of MC-4R in obesity and other associated disorders.
- the MC-4R protein is a G-protein coupled receptor comprising a ligand-binding extracellular domain, 7 transmembrane domains and an intracellular domain which couples to activation of adenyl cyclase.
- Melanocortin receptors belong to the rhodopsin sub-family of GPCR's. However, several features in the MC-4R are shared with all other receptors and are absent in most other GPCR's, including the EN motif in TM1, the lack of Cys in the loop between TM2 and TM3 or between TM4 and TM5, the MxxxxxxxY motif in TM5, and the DPxxY motif in TM7.
- interhelical disulfide bond (e.g., between the Cys residues near the top of TM3 and TM5) may play the same function as interloop disulfide bond in most other GPCR's.
- a preferred deletion mutation may contain a deletion of anywhere from 1 nucleotide to deletion of the entire gene, including the open reading frame and associated cis-actmg regulatory sequences associated with wild type MC-4R.
- a smaller deletion within the open reading frame is preferably not divisible by three, so as to result in a frameshift mutation resulting in a protein which most likely is non-functional. It is preferred that any such smaller deletion not divisible by three be targeted toward the 5' region of the open reading frame to increase the possibility of generating a non-functional truncated protein product.
- the deletion mutation encompass most if not all of the MC-4R gene so as to insure prevention of expression of a functional MC-4R protein.
- the transgenic animals which are homozygous, heterozygous or hemizygous for a deficient MC-4R gene are useful for identifying compounds which modulate wild type MC-4R activity or expression in vivo and studying aspects of the regulation of body weight which may be imparted through activation or antagonism of the MC-4R receptor.
- transgenic animals can be used as a strain for the insertion of human MC-4R genes and provides an animal model useful in the design and assessment of various approaches to modulating MC-4R activity and expression.
- An altered MC-4R gene should not fully encode the same MC-4R as native to the host animal, and its expression product can be altered to a minor or great degree, or absent altogether. However a more modestly modified MC-4R gene can also be useful and is within the scope of the present invention.
- the modified cells, embryos and/or non-human transgenic animal of the present invention can also be used as a source of cells for cell culture. These cells can be used for corresponding in vitro studies of MC-4R expression, activity and the modulation thereof.
- the non-human transgenic animals disclosed herein are useful for drug antagonist or agonist studies, for animal models of human diseases, and for testing of treatment of disorders or diseases associated with MC-4R.
- Transgenic animals lacking native MC-4R are useful in characterizing the in vivo function(s) of MC-4R.
- a transgenic animal carrying a non- native MC-4R in the absence of native MC-4R is useful for the establishment of a non- human model for diseases involving MC-4R, such as obesity, for studies of non-native MC-4R, to study modulators of the non-native gene and to distinguish between the activities of the non-native MC-4R in in vivo and in vitro systems.
- antisense RNA transgenes to partially or totally knock out expression of the mouse MC-4R protein.
- the antisense transgene used herein would encode a polynucleotide which is at least partially complementary to all or a part of the host MC-4R gene and which will hybridize to a target sequence encoded by the host
- MC-4R gene most specifically a mRNA transcript expressed from the host MC-4R gene.
- Any such oligonucleotide sequence should be at least about 15 to 30 nucleotides in length and preferably more than about 30 nucleotides, wherein this sequence in substantially complementary to the target host gene.
- the antisense transgene need not be a total complement, but instead should contain adequate sequence identity such that the expressed antisense RNA transgene will effective hybridize with the expressed mRNA from the host target gene so as to efficiently inhibit concomitant protein expression.
- antisense polynucleotides may be produced by subcloning the sequence of interest into an appropriate gene expression vector and transferring this vector to pluripotent embryonic stem cells which may be used as described herein to generate another form of an MC-4R deficient non-human transgenic animal.
- ES cells can be obtained from pre-implantation embryos cultured in vitro and fused with embryos (M. J. Evans et al, 1981, Nature 292: 154-156; Bradley et al., 1984, Nature 309: 255-258; Gossler et al., 1986, Proc. Natl. Acad. Sci. USA 83: 9065-9069; and Robertson et al., 1986, Nature 322: 445-448).
- Transgenes can be efficiently introduced into the ES cells by a variety of standard techniques such as DNA transfection, microinjection, or by retrovirus-mediated transduction.
- the resultant transformed ES cells can thereafter be combined with blastocysts from a non-human animal.
- the introduced ES cells thereafter colonize the embryo and contribute to the germ line of the resulting chimeric animal (Jaenisch, 1988, Science 240: 1468-1474).
- the MC-4R deficient animal cells, non-human transgenic embryos, non-human transgenic animals and non-human transgenic littermates of the present invention may be generated by any techniques known in the art, as sampled in the previous paragraph.
- the generation of a MC-4R knockout mouse with the phenotypic characteristics described herein has not been reported.
- MC-4R knock out mice were generated and evaluated for the effect of the loss of the MC-4R receptor on metabolic rate and the potential role of other melanocortin receptors on the control of food intake. It is disclosed herein that the MC-4R knock out mice have a reduced metabolic rate when compared to their wild type littermates. In addition, following administration of MT-II to MC-4R knock out or wild type mice, conclusive evidence is disclosed to show that the MC-4R mediates the effects of this peptide on metabolic rate and food intake.
- a mouse 129 SJ v lambda genomic library was screened with a 1-Kb rat MC-4R cDNA clone as a probe.
- the final targeting vector targeting vector was constructed in pSP72 (Promega) consisting of a 5' 3.4 Kb Hindlll to Ncol fragment, the 1.6 Kb PGK-neo and a 3' 3.5 kb Hindlll to Sail fragment.
- the linearize vector was electroporated into the AB2.2 embryonic stem cells and G418-resistant clones were selected.
- Targeted ES clones were selected and microinjected into C57B1/6J blastocysts to generate chimeric mice. Two male chimeras showed germline transmission of the targeted allele to their offspring.
- FI heterozygotes were interbred to produce homozygous knockout, heterozygous, and wild type F2 progeny. These F2 littermate mice were used to measure the body weight starting at 5 weeks of age. F2 homozygous knockout mice and wild type littermate mice we also used to produce F3 hybrids.
- In situ hybridization studies of MC-4R transgenic mice showed a lack of MC4-R expression in brain tissue. Expression of MC-4R was seen in wild type brains.
- MC-4R knock out mice exhibit a 16% reduced metabolic rate, when compared to wild type controls.
- the non-selective MC-R agonist MT-II is shown to increase metabolic rate in wild type mice while these effects are not observed in MC-4R knock out mice.
- MT-II is it also shown that the MC-4R knock out mice are resistant to the effects of MT-II on food intake. It is disclosed herein that the MC-4R is involved in both the control of metabolic rate and food intake, making it an ideally suitable target for consideration in the treatment of obesity.
- the MC-4R knock out mice described herein fully recapitulate the effects on body weight first observed and published by Huszar et al. (1997, Cell 88: 141-141, see also U.S. Patent No. 5,942, 779, issued August 4, 1999 to Lee et al.).
- a similar gene dosage sensitivity in male heterozygous MC-4R knock out mice to that of the Huzar et a ⁇ .(id.) knock out mice is disclosed herein.
- a difference between the knock mice described here and those generated by Huszar et al., (id.) exists since the MC-4R knock out mice generated herein do not contain the same gene dosage sensitivity in female heterozygous MC-4R deficient mice.
- mice In this colony female heterozygous MC-4R knockout and wild type littermate mice have similar body weights.
- the targeting vector described in Huzar et al. contains approximately 2.4Kb of additional 3' genomic sequences, a difference in the vectors which might result in altered expression of a closely linked gene differentially affecting body weight gain in heterozygous MC-4R deficient female and male mice.
- the MC-4R deficient mice have an overall reduced metabolic rate (-16 %) when corrected for body mass and increased respiratory coefficient indicative of reduced fat metabolism, when compared to age matched litter-mate controls.
- Intraperitoneal administration of the non-selective MC-R agonist melanotan II increases metabolic rate in wild type mice, while MC-4R deficient mice are insensitive to the effects of MT-II on metabolic rate. MC-4R deficient mice are also insensitive to the effects of MT-II on reducing food intake. Therefore, MC-4R mediates control of both metabolic rate and food intake in mice.
- MT-II non-selective MC-R agonist melanotan II
- the present invention is shown to provide a model system consisting of transgenic animals, cells and assays that are useful in the study of aspects of the etiology of obesity as related to modulation of the MC-4R.
- the various assays are also useful for screening and selecting for compounds that have an effect on body weight regulation, the further study of these compounds and the possible administration of selected compounds to humans in order to regulate disorders which include but are not limited to obesity, diabetes, anorexia, cachexia, cancer, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy, growth disorders relating to reduced GH, IGF1 function, treatment of reduced lean body mass as it occurs in the frail elderly, cancer cachexia, and other states that are characterized as resulting from GH deficiency.
- MC-4R knockout mice can be used to test melanocortin receptor subtype-specific compounds.
- the present invention also relates to cell- and membrane-based methods of identifying selective agonists and/or antagonists of mammalian MC-4R which affect the regulation of body weight through disorders including but not limited to obesity, diabetes, anorexia and cachexia. Therefore, an object of the present invention provides for MC-4R-based assays to select for modulators of this receptor protein which affect regulation of body weight through the various known disorders associated with regulation of body weight.
- the MC-4R modulators may be used to treat these body weight disorders, such as utilizing a MC-4R agonist to treat obesity or a MC-4R antagonist to treat anorexia.
- These assays are preferably cell-based assays whereby a DNA molecule encoding MC-4R is transfected or transformed into a host cell and this recombinant host cell is allowed to grow for a time sufficient to express MC-4R prior to use in various assays described herein.
- any "non-recombinant" cell line which has been genetically modified to overexpress MC-4R may also be utilized to screen and/or select for modulators of MC-4R useful in the treatment of body weight disorders.
- substantially purified membrane fractions from (1) a host cell transfected with a DNA expression vector coding for MC-4R or (2) a cell line genetically manipulated to overexpress MC-4R may be utilized to screen and/or select for modulators useful in the treatment of body weight disorders.
- Any polynucleotide sequence which encodes a functional MC-4R may be utilized in the recombinant cell and membrane-based assays of the present invention.
- a preferred polynucleotide for use in constructing an appropriate DNA expression vector is a DNA molecule which comprises the open reading frame for human MC-4R as set forth in SEQ ID NO:l and disclosed in U.S. Patent No. 5,622,860, issued to Yamada and Gantz on April 22, 1997 and U.S. Patent No. 5,703,220, issued to Yamada and Gantz on December 30, 1997), as disclosed in Figure 1, and as follows:
- the DNA molecule set forth as SEQ ID NO:l or a biologically equivalent polynucleotide may be inserted into an appropriate vector and linked with other DNA molecules, i.e, DNA molecules to which the MC-4R are not naturally linked, to form
- vectors may be comprised of DNA or RNA; for most cloning purposes DNA vectors are preferred. Typical vectors include plasmids, modified viruses, bacteriophage and cosmids, yeast artificial chromosomes and other forms of episomal or integrated DNA that can encode a MC-4R. It is well within the purview of the skilled artisan to determine an appropriate vector for a particular use.
- a variety of mammalian expression vectors may be used to express recombinant MC-4R in mammalian cells.
- expression vectors are defined herein as DNA sequences that are required for the transcription of cloned DNA and the translation of their mRNAs in an appropriate host.
- Such vectors can be used to express eukaryotic DNA in a variety of hosts such as bacteria, blue green algae, plant cells, insect cells and animal cells. Specifically designed vectors allow the shuttling of DNA between hosts such as bacteria- yeast or bacteria-animal cells.
- An appropriately constructed expression vector should contain: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters.
- a promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis.
- a strong promoter is one which causes mRNAs to be initiated at high frequency.
- Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
- mammalian expression vectors which may be suitable for recombinant MC-4R expression, include but are not limited to, pcDNA3.neo (Invitrogen), pcDNA3.1 (Invitrogen), pCI-neo (Promega), pLITMUS28, pLITMUS29, pLITMUS38 and pLITMUS39 (New England Bioloabs), pcDNAI, pcDNAIamp (Invitrogen), pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV- 1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (
- bacterial expression vectors may be used to express recombinant MC-4R in bacterial cells.
- Commercially available bacterial expression vectors which may be suitable for recombinant MC-4R expression include, but are not limited to pCR2.1 (Invitrogen), pETl la (Novagen), lambda gtl l (Invitrogen), and pKK223-3 (Pharmacia).
- fungal cell expression vectors may be used to express recombinant MC-4R in fungal cells.
- Commercially available fungal cell expression vectors which may be suitable for recombinant MC-4R expression include but are not limited to pYES2 (Invitrogen) and Pichia expression vector (Invitrogen).
- a variety of insect cell expression vectors may be used to express recombinant receptor in insect cells.
- Commercially available insect cell expression vectors which may be suitable for recombinant expression of MC-4R include but are not limited to pBlueBacIII and pBlueBacHis2 (Invitrogen), and pAcG2T (Pharmingen).
- Expression of MC-4R DNA may also be performed using in vitro produced synthetic mRNA.
- Synthetic mRNA can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes, with microinjection into frog oocytes being preferred.
- cDNA molecules including but not limited to the following can be constructed: a cDNA fragment containing the full-length open reading frame for MC-4R as well as various constructs containing portions of the cDNA encoding only specific domains of the protein or rearranged domains of the protein.
- All constructs can be designed to contain none, all or portions of the 5' and/or 3' untranslated region of a MC-4R cDNA.
- the expression levels and activity of MC-4R can be determined following the introduction, both singly and in combination, of these constructs into appropriate host cells.
- this MC-4R cDNA construct is transferred to a variety of expression vectors (including recombinant viruses), including but not limited to those for mammalian cells, plant cells, insect cells, oocytes, bacteria, and yeast cells.
- the host cells engineered to contain and/or express DNA sequences encoding the MC-4R can be cultured under suitable conditions to produce MC-4R or a biologically equivalent form.
- Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to, bacteria such as E. coli, fungal cells such as yeast, mammalian cells including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila and silkworm derived cell lines. Therefore, an expression vector containing DNA encoding a MC-4R-like protein may be used for expression of MC-4R in a recombinant host cell.
- Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila- and silkworm-derived cell lines.
- bacteria such as E. coli
- fungal cells such as yeast
- mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin
- insect cells including but not limited to Drosophila- and silkworm-derived cell lines.
- one insect expression system utilizes Spodoptera frugiperda (Sf21) insect cells (Invitrogen) in tandem with a baculovirus expression vector (pAcG2T, Pharmingen).
- pAcG2T baculovirus expression vector
- mammalian species which may be suitable and which are commercially available, include but are not limited to, L cells L-M(TK') (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), Saos-2 (ATCC HTB-85), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26), MRC-5 (ATCC CCL 171) and CPAE (ATCC CCL 209).
- L cells L-M(TK') ATCC CCL 1.3
- L cells L-M ATCC CCL 1.2
- Saos-2 ATCC HTB-85
- 293
- the expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation.
- the expression vector-containing cells are individually analyzed to determine whether they produce MC-4R protein. Identification of MC-4R expressing cells may be done by several means, including but not limited to immunological reactivity with anti-MC-4R antibodies, labeled ligand binding and the presence of host cell-associated MC-4R activity.
- assays described herein can be carried out with cells that have been genetically modified to overexpress host MC-4R, preferably resulting in at least a 5-fold increase over expression in a chosen "wild-type" host cell.
- improvements of overexpression can be brought about by any means presently known in the art, including but not limited to introducing a promoter by homologous recombination while leaving the coding region intact, or by simply selecting for cells that for whatever biological reason express a higher level of the MC-4R.
- assays described herein can be carried out with cells that have been transiently or stably transfected or transformed with an expression vector which directs expression of MC-4R.
- the expression vector may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, protoplast fusion, and electroporation. Transformation is meant to encompass a genetic change to the target cell resulting from an incorporation of DNA. Transfection is meant to include any method known in the art for introducing MC-4R into the test cells. For example, transfection includes calcium phosphate or calcium chloride mediated transfection, lipofection, infection with a retroviral construct containing MC-4R, and electroporation. The expression vector-containing cells are individually analyzed to determine whether they produce MC-4R protein.
- Identification of MC-4R expressing cells may be done by several means, including but not limited to immunological reactivity with anti- MC-4R antibodies, labeled ligand binding and the presence of host cell-associated MC-4R activity.
- the specificity of binding of compounds showing affinity for MC-4R is shown by measuring the affinity of the compounds for recombinant cells expressing the cloned receptor or for membranes from these cells.
- Expression of the cloned receptor and screening for compounds that bind to MC-4R or that inhibit the binding of a known, radiolabeled ligand of MC-4R to these cells, or membranes prepared from these cells provides an effective method for the rapid selection of compounds with high affinity for MC-4R which may be useful in the treatment of body weight disorders.
- Such ligands need not necessarily be radiolabeled but can also be nonisotopic compounds that can be used to displace bound radiolabeled compounds or that can be used as activators in functional assays.
- Compounds identified by the above method are likely to be agonists or antagonists of MC-4R and may be peptides, proteins, or non-proteinaceous organic molecules, all of which may be useful in the treatment of body weight disorders.
- the present invention is directed to methods for screening for compounds which modulate the expression of DNA or RNA encoding a MC-4R protein as well as compounds which effect the function of the MC-4R protein and hence, body weight disorders.
- Methods for identifying agonists and antagonists of other receptors are well known in the art and can be adapted to identify agonists and antagonists of MC-4R.
- Cascieri et al. (1992, Molec. Pharmacol. 41:1096-1099) describe a method for identifying substances that inhibit agonist binding to rat neurokinin receptors and thus are potential agonists or antagonists of neurokinin receptors.
- the method involves transfecting COS cells with expression vectors containing rat neurokinin receptors, allowing the transfected cells to grow for a time sufficient to allow the neurokinin receptors to be expressed, harvesting the transfected cells and resuspending the cells in assay buffer containing a known radioactively labeled agonist of the neurokinin receptors either in the presence or the absence of the substance, and then measuring the binding of the radioactively labeled known agonist of the neurokinin receptor to the neurokinin receptor. If the amount of binding of the known agonist is less in the presence of the substance than in the absence of the substance, then the substance is a potential agonist or antagonist of the neurokinin receptor.
- binding of the substance such as an agonist or antagonist to MC-4R can be measured by employing a labeled substance or agonist.
- the substance or agonist can be labeled in any convenient manner known to the art, e.g., radioactively, fluorescently, enzymatically.
- the specificity of binding of compounds having affinity for MC-4R is shown by measuring the affinity of the compounds for recombinant cells expressing the cloned receptor or for membranes from these cells.
- Expression of the cloned receptor and screening for compounds that bind to MC-4R or that inhibit the binding of a known, radiolabeled ligand of MC-4R to these cells, or membranes prepared from these cells provides an effective method for the rapid selection of compounds with high affinity for MC-4R.
- ligands need not necessarily be radiolabeled but can also be nonisotopic compounds that can be used to displace bound radiolabeled compounds or that can be used as activators in functional assays.
- Compounds identified by the above method are likely to be agonists or antagonists of MC-4R and may be peptides, proteins, or non- proteinaceous organic molecules which may be useful for human administration to treat various maladies , including but in no way limited to obesity, diabetes, anorexia, cachexia, cancer, male and female sexual dysfunction, pain, memory, neuronal regeneration and neuropathy, growth disorders relating to reduced GH, IGF1 function, treatment of reduced lean body mass as it occurs in the frail elderly, cancer cachexia, and other states that are characterized as resulting from GH deficiency.
- Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding MC-4R, or by acting as an agonist or antagonist of the MC-4R receptor protein.
- the assay may be a simple "yes/no" assay to determine whether there is a change in expression or function.
- the assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample. Kits containing MC-4R, antibodies to MC-4R, or modified MC-4R may be prepared by known methods for such uses.
- the present invention relates in part to methods of identifying a substance which modulates MC-4R receptor activity, which involves:
- the present invention includes a method for determining whether a substance is a potential agonist or antagonist of MC-4R useful in the treatment of body weight disorders, comprising: (a) transfecting or transforming cells with an expression vector that directs expression of MC-4R in the cells, resulting in test cells;
- step (c) of the method is practiced are conditions that are typically used in the art for the study of protein-ligand interactions: e.g., physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
- the test cells may be harvested and resuspended in the presence of the substance and the labeled ligand.
- step (c) is modified in that the cells are not harvested and resuspended but rather the radioactively labeled known agonist and the substance are contacted with the cells while the cells are attached to a substratum, e.g., tissue culture plates.
- the present invention also includes a method for determining whether a substance is capable of binding to MC-4R or a mutant MC-4R that is no longer functional but nonetheless may be involved in ligand binding, i.e., whether the substance is a potential agonist, inverse agonist or an antagonist of MC-4R and hence useful in the treatment of body weight disorders, where the method comprises: (a) transfecting or transforming cells with an expression vector that directs the expression of MC-4R in the cells, resulting in test cells;
- step (b) of the method is practiced are conditions that are typically used in the art for the study of protem-ligand interactions: e.g , physiological pH; salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media; a temperature of about 4°C to about 55°C.
- physiological pH e.g , physiological pH
- salt conditions such as those represented by such commonly used buffers as PBS or in tissue culture media
- the test cells are harvested and resuspended in the presence of the substance.
- a MC-4R- ⁇ nduced functional response may be measured in the Xenopus oocyte system, or within any useful eukaryotic cell line.
- the subunit itself or a nucleic acid encoding the subunit may be a DNA expression vector or possible a cRNA transc ⁇ pt generated from an equivalent DNA expression vector, preferably in vitro
- both protein and nucleic ac ⁇ d(s) may be added, and the addition events need not occur together.
- a nucleic acid or pool of nucleic acids wherein at least one nucleic acid encoding a MC-4R is introduced into the cell
- another embodiment of the present invention is the use of promiscuous G-protems (such as G ⁇ l5 , G ⁇ ⁇ 6 , and G q ⁇ 5 ) in assays which rely on phospholipase C activation to measure receptor-mediated changes in mtracellular calcium and which are amenable to high throughput screening.
- G-protems such as G ⁇ l5 , G ⁇ ⁇ 6 , and G q ⁇ 5
- G-protems such as G ⁇ l5 , G ⁇ ⁇ 6 , and G q ⁇ 5
- Measurement in the reduction m cAMP has drawbacks because of a small dynamic range in the assay and low throughput.
- Assays which rely on phospholipase C activation instead measure changes in calcium via assays utilizing detector molecules (e.g., such as aequo ⁇ n bioluminescence [see Example Section 4] or FLIPR), which makes this type of assay amenable to high throughput screening, such as but not limited to detection via a lummometer or a charge-coupled device (CCD) camera system (see Milligan et al, 1996, TiPS 17:235-237 for a review).
- detector molecules e.g., such as aequo ⁇ n bioluminescence [see Example Section 4] or FLIPR
- CCD charge-coupled device
- eukaryotic cell may be used in the assays developed herein These would include oocytes (preferred ones are from Xenopus sp.) but cell lines may be used as well as Examples of preferred cell lines are mammalian cell lines, including COS, HEK-293, CHO, HeLa, NS/0, CV-1, GC, GH3 and VERO.
- the detector molecule is responsive to an mtracellular event which is part of the biochemical cascade initiated by GHS-GHSR binding.
- One class of preferred detector molecules can respond to changes in calcium concentrations.
- a preferred detector molecule which responds to calcium concentrations is aequo ⁇ n (a jellyfish photoprotem; see Inouye et al., 1885, Proc. Natl. Acad. Sci. U.S.A. 82: 3154-3158, hereby incorporated by reference), which emits a characteristic fluoresence in the presence of Ca 2+ , which acts on the substrate coelenterazine.
- detector molecules include calcium chelators with fluorescence capabilities, such as FURA-2 and indo-1.
- FURA-2 calcium chelators with fluorescence capabilities
- indo-1 indo-1.
- modifications to aequorin have disclosed and may also be utilized to practice this portion of the invention (for example, see U.S. Patent Nos. 6,027,881; 5,625,048; and 5,804,387, which are also incorporated by reference).
- the detector molecule itself may be introduced into the cell, or nucleotides which encode the detector molecule may be introduced into the cell, under conditions which will allow the expression of the detector molecule. Generally, it is preferred to introduce nucleotides, such as DNA or cRNA which encode the detector molecule into the cell, under conditions wherein the cell will express the detector molecule.
- Heterotrimeric G proteins consisting of ⁇ , ⁇ , ⁇ subunits, serve to relay information from cell surface receptors to intracellular effectors, such as phospholipase C and adenylate cyclase.
- the G-protein alpha subunit ( ⁇ -subunit) is an essential component of the intracellular signal transduction pathway activated by receptor-ligand interaction.
- the G ⁇ subunit of a trimeric G ⁇ complex will exchange its bound GDP for GTP and dissociate from the ⁇ heterodimer.
- the dissociated G ⁇ -protein serves as the active signal transducer, often in concert with the ⁇ complex, thus starting the activation of the intracellular signal transduction pathway.
- G-alpha subunits are classified into sub-families based on sequence identity and the main type of effectors are coupled: G s activate adenylate cyclase, Gi /0/t , inhibit adenylate cyclase, G q / ⁇ 5 activate ⁇ -class phospholipase C polypeptides (PI-PLC), and G 12/ ⁇ 3 , effector unknown (see Milligan and Rees, TiPS 20: 118-124, for a review of G ⁇ subunit classification and activity. The artisan will realize upon review of this specification that any known G ⁇ subunit may be tested for use in the MC-4R-based assays of this portion of the invention.
- the present invention relates in part to methods of identifying MC-4R agonists or antagonists for use in treating body weight disorders (i.e., body weight regulation) which includes but is by no means limited to the use of promiscuous G-proteins (such as G ⁇ ⁇ 5 , G ⁇ ⁇ 6 , and G q ⁇ 5 ) in assays which rely on phospholipase C activation to receptor-activated changes in intracellular calcium levels.
- body weight disorders i.e., body weight regulation
- a detector molecule which includes but is in no way limited to the exemplified detector molecule, aequorin, such that detection of intracellular calcium as reported by aequorin bioluminescence will be amenable to high throughput screening, as shown in Example Section 4, comprising such steps as follows:
- transfecting or transforming cells such as a Xenopus oocyte or any convenient eukaryotic cell known in the art
- a first expression vector or equivalent cRNA molecule
- a second expression vector or equivalent cRNA molecule
- transfecting or transforming cells such as a Xenopus oocyte or any convenient eukaryotic cell known in the art
- a first expression vector or equivalent cRNA molecule
- a second expression vector or equivalent cRNA molecule
- step (d) subsequently, concurrently or prior to step (c), exposing the test cells to a substance that is a suspected antagonist of MC-4R;
- step (e) determining whether the compound modulates MC-4R activity by monitoring the detector molecule, where a decrease in the level of detection in the test cells in the presence of the suspected antagonist as compared to the level of inositol phosphates in the cells in the absence of the suspected antagonist indicates that the substance is an antagonist of MC-4R.
- compounds selected through such assays may then be administered to non-human animals, such as the transgenic mice described herein, to further characterize potential modulators of MC-4R activity, and hence, the biological events characterized by modulation of this receptor, as described herein.
- Another aspect of this portion of the invention includes a non-radioactive method for determining whether a substance is a potential agonist or antagonist of MC-4R that comprises:
- step (b) transfecting or transforming the test cells of step (a) with an expression vector which comprises a cAMP-inducible promoter fused to a colorimetric gene such a LacZ;
- the expression vector may be a DNA expression vector or a cRNA transcript generated from an equivalent DNA expression vector, preferably in vitro.
- Additional methods of identifying MC-4R agonists or antagonists for use in treating body weight disorders include but are by no means limited to the following: I. (a) transfecting or transforming cells with a first expression vector which directs expression of MC-4R and a second expression vector which directs the expression of a promiscuous G-protein, resulting in test cells;
- step (c) subsequently or concurrently to step (b), exposing the test cells to a substance that is a suspected antagonist of MC-4R;
- step (a) The method of II wherein the first and second expression vectors of step (a) are replaced with a single expression vector which expresses a chimeric MC-4R protein fused at its C-terminus to a promiscuous G-protein.
- membranes can be prepared from the test cells and those membranes can be exposed to the substance.
- a modification utilizing membranes rather than cells is well known in the art and is described in, e.g., Hess et al., 1992, Biochem. Biophys. Res. Comm. 184:260-268.
- another embodiment of the present invention includes a method for determining whether a substance binds and/or is a potential agonist or antagonist of MC-4R wherein membrane preparations from the test cells are utilized in place of the test cells.
- Such methods comprise the following and may utilized the physiological conditions as noted above:
- step (c) subsequently or concurrently to step (b), exposing the membranes from the test cells to a substance;
- the present invention also relates to a method for determining whether a substance is capable of binding to MC-4R comprising:
- a preferred embodiment of the present invention is determining various ligand binding affinities using 125 I-labeled NDP- ⁇ -MSH as the labeled ligand in the presence of varying concentration of unlabeled ligands.
- the activation of the second messenger pathway may be determined by measuring the intracellular cAMP elicited by agonist at various concentration.
- the MC-4R KO and wild-type mice can be used for in vivo testing of candidate compounds for their effects on several different parameters such as food intake, body weight, body composition, glucose, insulin, leptin and cholesterol levels, sexual function, memory, learning, nerve regeneration, and pain.
- DIO diet-induced obesity
- the comparison of the effects on wild type, knock and heterozygote mice is an essential component of the evaluation of the selectivity of said compounds. It is also an essential part of the present invention to measure sensitivity to other melanocortin or other pathways that may have been up or down regulated and the measure changes in sensitivity of compounds that modulate these pathways.
- testing of compounds that affect MC-4R, or other melanocortin receptors, NPY receptors, galanin receptors, MCH receptors, Orexin receptors, Insulin receptors, receptors belonging to the bombesin family of receptors (BRS-3, neuromedin receptors, gastrin releasing peptide receptors), motilin receptors, neuromedin U receptors, adrenergic receptors, leptin receptors, modulators of STATs and SOCs transcription factors, phoshpodiesterase enzymes and others are within the scope of uses for the non-human transgenic animals of the present invention.
- compositions comprising modulators of MC-4R may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences.
- a pharmaceutically acceptable composition suitable for effective administration such compositions will contain an effective amount of the protein, DNA, RNA, modified MC-4R, or either MC-4R agonists or antagonists- Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat or diagnose disorders.
- the effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration.
- compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral and intramuscular.
- chemical derivative describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
- the present invention also has the objective of providing suitable topical, oral, systemic and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention.
- the compositions containing compounds identified according to this invention as the active ingredient can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration.
- the compounds can be administered in such oral dosage forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups and emulsions, or by injection.
- they may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
- compounds of the present invention may be administered in a single daily dose, or the total daily dosage may be administered in divided doses of two, three or four times daily.
- compounds for the present invention can be administered in intranasal form via topical use of suitable intranasal vehicles, or via transdermal routes, using those forms of transdermal skin patches well known to those of ordinary skill in that art.
- the dosage administration will, of course, be continuous rather than intermittent throughout the dosage regimen.
- the active agents can be administered concurrently, or they each can be administered at separately staggered times.
- the dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular compound thereof employed.
- a physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition.
- Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.
- MC-4R targeting vector and gene targeting in ES cells To isolate genomic DNA containing the murine MC-4R gene, a mousel29 SjV lambda genomic library (Lambda FIX II Library, Stratagene, La Jolla,CA) was screened using a 1-Kilobasepair (Kb) rat MC-4R cDNA clone as a probe. One of four clones was mapped in detail by restriction enzyme digestion.
- the clone consisted of the 29-kilobase pair (Kb) lambda vector and a 15 Kb genomic insert encoding the 1 Kb MC-4R coding sequence, between 10 Kb of 5' and 4 Kb of 3' flanking sequences.
- Kb 29-kilobase pair
- An approximately 1.5 Kb fragment extending from an Ncol site located approximately 20 basepair (bp) downstream of the MC-4R translation initiation codon to the Hindlll site situated approximately 0.5 Kb downstream of the ATG stop codon of the MC-4R coding sequence was replaced with a PGK-neo cassette
- pPGKneobpA phosphoglycerokinase promoter
- the targeting vector was constructed in pSP72 (Promega) consisting of a 5' 3.4 Kb Hindlll to Ncol fragment, the 1.6 Kb PGK-neo fragment and a 3' 3.5 kb Hindlll to Sail fragment.
- pSP72 Promega
- the gene targeting vector pAJ7 was linearized at a unique Seal site and electroporated into the AB2.2 embryonic stem cells (Lexicon Genetics) under standard condition using a Gene Pulser (Bio-Rad). Selection of the G418-resistant clones was performed as previously described (Von Koch et al., 1997).
- PCR Analysis of Knockout Mice To facilitate the identification of a large number of knockout and wild type mice, 3 oligonucleotides were designed to distinguish the knockout allele from the wild-type allele by PCR.
- the synthetic oligonucleotides 5'-CTAACCATAAGAAATCAGCAGCCCG-3'(SEQ ID NO:3) and 5'-AGGGAAGTATACATG CCATGGTGGT-3' (SEQ ID NO:4) result in 500 bp PCR product by wild type allele.
- Oligonucleotides 5'-CTAACCATAAGAAATCAG CAGCCCG-3' (SEQ ID NO:5) and 5'-TACCGGTGGATGTGGAATGTGTGC-3' (SEQ ID NO:6) result in 650 bp product derived from the mutant allele.
- mice Animal care and maintenance - All animal protocols used in these studies were approved by Merck Institutional Animal care and Use Committee. The mice were group housed in microisolator cages in a specific pathogen-free facility with an air shower entrance. The mice were maintained on a Teklad 7012 chow diet and ad libitum water.
- mice - Targeted ES clones were identified by Southern blotting analysis, using a 700-bp Ncol -Hindlll fragment located 5' outside of the targeting vector as a probe. Of the 600 clones selected, 3 showed a 7 Kb targeted Apal restriction enzyme fragment in addition to the expected 5 Kb wild-type fragment (the Apal site in the MC-4R coding region was eliminated during the homologous recombination event). These positive clones were microinjected into C57B1/6J blastocysts to generate chimeric mice. Two male chimeras showed germline transmission of the targeted allele to their offspring.
- FI heterozygotes were interbred to produce homozygous knockout, heterozygous, and wild type F2 progeny. These F2 littermate mice were used to measure the body weight starting at 5-week of age. F2 homozygous knockout mice and wild type littermate mice were also used to produce F3 hybrids. For metabolic rate studies, F3 hybrids were used. Since the knock out mice become obese with increasing age, we hypothesized that the homozygous mice might have reduced fertility and therefore would not be suitable for efficient mass-production of knockout mice. However, the litter size of homozygous breeding pairs appeared normal when compared to that of heterozygous breeding pairs.
- the hybridization probes consist of an equal molar mixture of three non-overlapping, antisense oligonucleotides against the coding region of MC-4R. Their nucleotide sequences are 5'-ATCCTCTTAATGTGAAGCCTCGCCATCAGGAACATGT GGACATAGAGAGA -3' (SEQ ID NO:7), 5'-GAAAGCAGGCTGCAAATGGA TGCGAGCAAGGAGCT ACAGATCAC -3' (SEQ ID NO: 8), and
- Indirect calorimetry was measured in male MC-4R knock out and their wild type syngeneic control mice. Metabolic rate was measured by indirect calorimetry using a 16-chamber open-circuit Oxymax system (Columbus Instruments, Columbus, Ohio). Mice (approximately 10 weeks of age) were maintained at 21 to 24°C in a 12 hour light dark cycle, (light period 7:00 am to 7:00 pm). Food (autoclaved pelleted chow: Harlan Teklad 7012, Madison, WI), and water were available ad libitum except where noted below.
- Baseline metabolic rate was measured over a 4 hour period commencing 30 minutes following food removal.
- RER was measured over the 2 hour period prior to food removal.
- food was removed at 12:00 pm and returned at 5 pm that evening.
- the effects of the nonselective MC-R agonist MT-II (Peninsula Laboratories, San Carlos, CA; 20 mg/kg, intraperitoneally administered) or an equivalent volume of vehicle (saline) on metabolic rate were similarly determined over a 4 hour period commencing 30 minutes following food removal and dosing. Each animal received both treatments with at least a 5 day recovery period between treatments.
- mice described above were evaluated for body weight gain in the MC-4R colony. Twenty-four male littermates (4 knockout [diamond symbols], 12 heterozygous [squares] and 8 wild type [triangles] mice) and thirty female littermates (10 knockout, 16 heterozygous and 4 wild type mice) were monitored every week from 5-weeks of age ( Figure 5A-B). The mean body weight of knockout males was statistically significantly different (p ⁇ 0.05) from that of wild type male mice by 6-weeks of age. After 8 weeks, the mean body weight of heterozygous males was also statistically significantly different (p ⁇ 0.05) from that of wild type males.
- Ovarian lobes were broken open, rinsed several times, and oocytes were released from their sacs by collagenase A digestion (Boehringer- Mannheim; 0.2% for 2-3 hours at 18°C) in calcium-free OR-2.
- frogs were pre-tested for their ability to express a control G-protein linked receptor (human gonadotropin- releasing hormone receptor) and show a robust phospholipase C intracellular signaling pathway (incubation with 1% chicken serum which promotes calcium mobilization by activation of phospholipase C). Based on these results, 1-2 frogs were chosen for library pool injection (50 nl of cRNA at a concentration of 25 ng (complex pools) to 0.5 ng (pure clone) per oocyte usually 24 to 48 hours following oocyte isolation.
- G-protein linked receptor human gonadotropin- releasing hormone receptor
- Plasmid DNA Preparation and cRNA Transcription - Plasmid DNA (plasmids included rat MC4R, rat GALRl, G ⁇ 15 and G ⁇ t s) was purified from pellets of bacteria (using the Wizard Miniprep kit according to the manufacturer's instructions (Promega Biotech, Madison, WI).
- the nucleotide sequence encoding respective proteins utilized herein are known in art, for example, see Alvaro, et al., 1996, Mol. Pharmacol. 50 (3): 583-591 (rat MC-4R); Inouye et al., 1985, Proc. Natl. Acad. Sci. U.S.A.
- ABA Aequorin Bioluminescence Assay
- Clone Identification The ABA requires injection of G protein-coupled receptor cRNA (1 ng/egg) with aequorin cRNA (2 ng/egg) supplemented with the G-protein alpha subunits G ⁇ ⁇ 5 (2 ng/egg) or G ⁇ q i 5 (2 ng/egg).
- the expression vector pCDNA-3 was modified (termed pcDNA-3v2) by insertion (in the Apa I restriction enzyme site of the polylinker) of a cassette to append a poly (A) tract on all cRNA's which initiate from the T7 RNA polymerase promoter.
- This cassette includes (5' to 3'): a Bgl II site, pA (20) and a Sfi I site which can be used for plasmid linearization.
- PCR Polymerase chain reaction
- cRNA was injected into oocytes using the motorized "Nanoject" injector (Drummond Sci. Co., Broomall, PA.) in a volume of 50 nl. Injection needles were pulled in a single step using a Flaming/Brown micropipette puller, Model P-87 (Sutter Instrument Co) and the tips were broken using 53X magnification such that an acute angle was generated with the outside diameter of the needle being ⁇ 3 ⁇ m. Following injection, oocytes were incubated in ND-96 medium, with gentle orbital shaking at 18°C in the dark.
- Oocytes were incubated for 24 to 48 hours (depending on the experiment and the time required for expression of the heterologous RNA) before “charging” the expressed aequorin with the essential chromophore coelenterazine.
- Oocytes were "charged” with coelenterazine by transferring them into 35 mm dishes containing 3 ml charging medium and incubating for 2-3 hours with gentle orbital shaking in the dark at 18°C.
- the charging medium contained 10 ⁇ M coelenterazine (Molecular Probes, Inc., Eugene, OR.) and 30 ⁇ M reduced glutathione in OR-2 media (no calcium).
- Oocytes were then returned to ND-86 with calcium medium described above and incubation continued in the dark with orbital shaking until bioluminescence measurements were initiated. Measurement of GHSR expression in oocytes was performed using a Berthold Luminometer LB953 (Wallac Inc.,
- GPC-R G-protein-coupled receptors
- This example shows an alternative assay to measure the activation of melanocortin receptors that is more robust, of a higher throughput, and reduced cost than assays which directly measure cAMP.
- this assay would have the added feature of finding ligands to receptors in which the signaling properties are unknown (orphan receptors) that may represent novel melanocortin receptors.
- the assay described herein relies on the use of G protein alpha subunits which have the unique property of bypassing the normal route of receptor activation and subsequent coupling to intracellular effectors.
- Figure 9 shows an aequorin bioluminescence assay in which the rat melanocortin 4 receptor (MC-4R), upon stimulation by the melanocortin agonist peptide ⁇ NDP-MSH (1 ⁇ M), can functionally couple to activation of phospholipase C (mobilization of intracellular calcium reported by aequorin bioluminescence) when expressed in Xenopus oocytes in the presence of the G ⁇ l5 subunit. Each tracing represents the bioluminescence response (in counts/sec) over time (in seconds) for 1-2 individual oocytes.
- Figure 10 shows a similar experiment using the G ⁇ s subunit.
- the human galanin receptor 1 (hGALRl), which is coupled in its native state to inhibition of adenylate cyclase, can now when co- expressed with the G ⁇ q i 5 subunit, show functional activation in response to agonist treatment (1 ⁇ M human galanin).
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)
- Endocrinology (AREA)
- Cell Biology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Investigating Or Analysing Biological Materials (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 (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2001535977A JP2003525596A (en) | 1999-11-12 | 2000-11-13 | Melanocortin-4 receptor deficient cells, non-human transgenic animals, and method for selecting weight-regulating compounds |
CA002390740A CA2390740A1 (en) | 1999-11-12 | 2000-11-13 | Melanocortin-4 receptor deficient cells, non-human transgenic animals and methods of selecting compounds which regulate body weight |
EP00980352A EP1241934A4 (en) | 1999-11-12 | 2000-11-13 | Melanocortin-4 receptor deficient cells, non-human transgenic animals and methods of selecting compounds which regulate body weight |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US16507499P | 1999-11-12 | 1999-11-12 | |
US60/165,074 | 1999-11-12 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2001033956A1 true WO2001033956A1 (en) | 2001-05-17 |
Family
ID=22597306
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2000/031061 WO2001033956A1 (en) | 1999-11-12 | 2000-11-13 | Melanocortin-4 receptor deficient cells, non-human transgenic animals and methods of selecting compounds which regulate body weight |
Country Status (4)
Country | Link |
---|---|
EP (1) | EP1241934A4 (en) |
JP (1) | JP2003525596A (en) |
CA (1) | CA2390740A1 (en) |
WO (1) | WO2001033956A1 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009144433A1 (en) * | 2008-05-30 | 2009-12-03 | Palatin Technologies, Inc. | Methods for selection of melanocortin 4 receptor-specific agents for treatment of obesity |
WO2009144432A1 (en) * | 2008-05-30 | 2009-12-03 | Palatin Technologies, Inc. | Mc4-r agonists for the treatment of obesity |
WO2013185239A1 (en) * | 2012-06-14 | 2013-12-19 | Université de Montréal | Transgenic mouse models for mc4r |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5622860A (en) * | 1994-02-17 | 1997-04-22 | The Regents Of The University Of Michigan | Genes encoding melanocortin receptors |
US5932779A (en) * | 1996-06-10 | 1999-08-03 | Millennium Pharmaceuticals, Inc. | Screening methods for compounds useful in the regulation of body weight |
-
2000
- 2000-11-13 WO PCT/US2000/031061 patent/WO2001033956A1/en not_active Application Discontinuation
- 2000-11-13 EP EP00980352A patent/EP1241934A4/en not_active Withdrawn
- 2000-11-13 CA CA002390740A patent/CA2390740A1/en not_active Abandoned
- 2000-11-13 JP JP2001535977A patent/JP2003525596A/en not_active Withdrawn
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5622860A (en) * | 1994-02-17 | 1997-04-22 | The Regents Of The University Of Michigan | Genes encoding melanocortin receptors |
US5703220A (en) * | 1994-02-17 | 1997-12-30 | The Regents Of The University Of Michigan | Genes encoding melanocertin-4 receptor and methods of use |
US5932779A (en) * | 1996-06-10 | 1999-08-03 | Millennium Pharmaceuticals, Inc. | Screening methods for compounds useful in the regulation of body weight |
Non-Patent Citations (3)
Title |
---|
FAN ET AL.: "Role of melanocortinergic neurons in feeding and the agouti obesity syndrome", NATURE, vol. 385, 9 January 1997 (1997-01-09), pages 165 - 168, XP002937709 * |
HUSZAR ET AL.: "Targeted disruption of the melanocortin-4 receptor results in obesity in mice", CELL, vol. 88, 10 January 1997 (1997-01-10), pages 131 - 141, XP002937710 * |
See also references of EP1241934A4 * |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2009144433A1 (en) * | 2008-05-30 | 2009-12-03 | Palatin Technologies, Inc. | Methods for selection of melanocortin 4 receptor-specific agents for treatment of obesity |
WO2009144432A1 (en) * | 2008-05-30 | 2009-12-03 | Palatin Technologies, Inc. | Mc4-r agonists for the treatment of obesity |
WO2013185239A1 (en) * | 2012-06-14 | 2013-12-19 | Université de Montréal | Transgenic mouse models for mc4r |
Also Published As
Publication number | Publication date |
---|---|
EP1241934A4 (en) | 2005-04-27 |
EP1241934A1 (en) | 2002-09-25 |
JP2003525596A (en) | 2003-09-02 |
CA2390740A1 (en) | 2001-05-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2461917C (en) | Isolated dna molecules encoding humanized calcitonin gene-related peptide receptor, related non-human transgenic animals and assay methods | |
US6790656B1 (en) | DNA encoding galanin GALR2 receptors | |
US20090013421A1 (en) | Genomic DNA fragments containing regulatory and coding sequences for the B2-subunit of the neuronal nicotinic acetylcholine receptor and transgenic animals made using these fragments or mutated fragments | |
US6639123B1 (en) | Melanocortin-3 receptor deficient cells, non-human trangenic animals and methods of selecting compounds which regulate body weight | |
WO1997026853A9 (en) | Dna encoding galanin galr2 receptors and uses thereof | |
US5750826A (en) | Bradykinin B2 receptor modified transgenic non-human animals | |
CA2488725A1 (en) | Agouti-related protein deficient cells, non-human transgenic animals and methods of selecting compounds which regulate energy metabolism | |
WO2001033956A1 (en) | Melanocortin-4 receptor deficient cells, non-human transgenic animals and methods of selecting compounds which regulate body weight | |
US5817912A (en) | Transgenic mice with disrupted NPY Y1 receptor genes | |
EP1337558B1 (en) | A "bach" g protein coupled receptor polypeptide and polynucleotide encoding this receptor | |
US7321074B1 (en) | Melanocortin-3 receptor deficient cells, non-human transgenic animals and methods of selecting compounds which regulate body weight | |
US20040053812A1 (en) | Methods | |
US20050003407A1 (en) | Genomic DNA fragments containing regulatory and coding sequence for the beta2-subunit of the neuronal nicotinic acetylcholine receptor and transgenic animals made using these fragments or mutated fragments | |
WO2003016495A2 (en) | Transgenic rodents as animal models for modulation of b1 bradykinin receptor protein | |
WO2001083558A1 (en) | Agonists/antagonists of the gpr56 receptor as appetite control agents | |
EP1240199A2 (en) | Method for screening of appetite control agents | |
EP1290449A1 (en) | Use of the g protein coupled receptor gpr3 to identify apetite control agents | |
EP1265922A2 (en) | Agonists and antagonists of the receptor gpr19 and their use in appetite control |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): CA JP US |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
ENP | Entry into the national phase |
Ref country code: JP Ref document number: 2001 535977 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2000980352 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2390740 Country of ref document: CA |
|
WWP | Wipo information: published in national office |
Ref document number: 2000980352 Country of ref document: EP |
|
WWW | Wipo information: withdrawn in national office |
Ref document number: 2000980352 Country of ref document: EP |