CA2305045A1 - Transgenic animals with knocked-in vec receptor genes and uses thereof - Google Patents
Transgenic animals with knocked-in vec receptor genes and uses thereof Download PDFInfo
- Publication number
- CA2305045A1 CA2305045A1 CA002305045A CA2305045A CA2305045A1 CA 2305045 A1 CA2305045 A1 CA 2305045A1 CA 002305045 A CA002305045 A CA 002305045A CA 2305045 A CA2305045 A CA 2305045A CA 2305045 A1 CA2305045 A1 CA 2305045A1
- Authority
- CA
- Canada
- Prior art keywords
- dna sequence
- kdr
- transgenic
- animal
- human
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 241001465754 Metazoa Species 0.000 title claims abstract description 69
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 50
- 108020003175 receptors Proteins 0.000 title claims description 49
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 60
- 108091028043 Nucleic acid sequence Proteins 0.000 claims abstract description 48
- 210000004027 cell Anatomy 0.000 claims abstract description 43
- 238000000034 method Methods 0.000 claims abstract description 39
- 230000033115 angiogenesis Effects 0.000 claims abstract description 29
- 210000003556 vascular endothelial cell Anatomy 0.000 claims abstract description 14
- 108010053099 Vascular Endothelial Growth Factor Receptor-2 Proteins 0.000 claims description 159
- 102100033177 Vascular endothelial growth factor receptor 2 Human genes 0.000 claims description 142
- 102000005962 receptors Human genes 0.000 claims description 45
- 239000000126 substance Substances 0.000 claims description 38
- 239000012634 fragment Substances 0.000 claims description 31
- 241001529936 Murinae Species 0.000 claims description 27
- 241000894007 species Species 0.000 claims description 21
- 210000002889 endothelial cell Anatomy 0.000 claims description 16
- 238000010171 animal model Methods 0.000 claims description 15
- 230000000694 effects Effects 0.000 claims description 15
- 230000001105 regulatory effect Effects 0.000 claims description 10
- 230000004614 tumor growth Effects 0.000 claims description 9
- 230000002401 inhibitory effect Effects 0.000 claims description 8
- 238000010998 test method Methods 0.000 claims description 7
- 101150088608 Kdr gene Proteins 0.000 claims description 4
- 102000004169 proteins and genes Human genes 0.000 claims description 4
- 238000011830 transgenic mouse model Methods 0.000 claims 8
- 101100372765 Homo sapiens KDR gene Proteins 0.000 claims 1
- 238000012360 testing method Methods 0.000 abstract description 18
- 230000001225 therapeutic effect Effects 0.000 abstract description 12
- 239000003153 chemical reaction reagent Substances 0.000 abstract description 10
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 37
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 34
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 34
- 239000002299 complementary DNA Substances 0.000 description 34
- 239000013598 vector Substances 0.000 description 27
- 241000699670 Mus sp. Species 0.000 description 24
- 108020004414 DNA Proteins 0.000 description 21
- 241000699666 Mus <mouse, genus> Species 0.000 description 18
- 230000008685 targeting Effects 0.000 description 14
- 210000001519 tissue Anatomy 0.000 description 13
- 239000003814 drug Substances 0.000 description 12
- 239000013615 primer Substances 0.000 description 12
- 206010028980 Neoplasm Diseases 0.000 description 10
- 238000013459 approach Methods 0.000 description 10
- 238000003752 polymerase chain reaction Methods 0.000 description 10
- 230000029663 wound healing Effects 0.000 description 9
- 238000011161 development Methods 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 8
- 210000003491 skin Anatomy 0.000 description 8
- 238000011144 upstream manufacturing Methods 0.000 description 8
- 108091008605 VEGF receptors Proteins 0.000 description 7
- 102000009484 Vascular Endothelial Growth Factor Receptors Human genes 0.000 description 7
- 210000004204 blood vessel Anatomy 0.000 description 7
- 230000006870 function Effects 0.000 description 7
- 230000006801 homologous recombination Effects 0.000 description 7
- 238000002744 homologous recombination Methods 0.000 description 7
- 108091008598 receptor tyrosine kinases Proteins 0.000 description 7
- 102000027426 receptor tyrosine kinases Human genes 0.000 description 7
- 210000004881 tumor cell Anatomy 0.000 description 7
- 210000005166 vasculature Anatomy 0.000 description 7
- 102100022014 Angiopoietin-1 receptor Human genes 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 101000753291 Homo sapiens Angiopoietin-1 receptor Proteins 0.000 description 6
- 206010029113 Neovascularisation Diseases 0.000 description 6
- 108010053096 Vascular Endothelial Growth Factor Receptor-1 Proteins 0.000 description 6
- 102100033178 Vascular endothelial growth factor receptor 1 Human genes 0.000 description 6
- 201000010099 disease Diseases 0.000 description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 6
- 230000013020 embryo development Effects 0.000 description 6
- 238000003780 insertion Methods 0.000 description 6
- 230000037431 insertion Effects 0.000 description 6
- 239000003446 ligand Substances 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 238000011579 SCID mouse model Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000003112 inhibitor Substances 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 238000010561 standard procedure Methods 0.000 description 5
- 230000005747 tumor angiogenesis Effects 0.000 description 5
- 208000022211 Arteriovenous Malformations Diseases 0.000 description 4
- 108091034117 Oligonucleotide Proteins 0.000 description 4
- 238000002105 Southern blotting Methods 0.000 description 4
- 102000009524 Vascular Endothelial Growth Factor A Human genes 0.000 description 4
- 108010073923 Vascular Endothelial Growth Factor C Proteins 0.000 description 4
- 102000009520 Vascular Endothelial Growth Factor C Human genes 0.000 description 4
- 108010053100 Vascular Endothelial Growth Factor Receptor-3 Proteins 0.000 description 4
- 102100033179 Vascular endothelial growth factor receptor 3 Human genes 0.000 description 4
- 230000002159 abnormal effect Effects 0.000 description 4
- 230000005744 arteriovenous malformation Effects 0.000 description 4
- 210000003038 endothelium Anatomy 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 238000010369 molecular cloning Methods 0.000 description 4
- 230000001575 pathological effect Effects 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000002560 therapeutic procedure Methods 0.000 description 4
- 238000011820 transgenic animal model Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- 241000283707 Capra Species 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- 108700020796 Oncogene Proteins 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 208000034038 Pathologic Neovascularization Diseases 0.000 description 3
- 239000005557 antagonist Substances 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 230000000295 complement effect Effects 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 230000004968 inflammatory condition Effects 0.000 description 3
- 230000002452 interceptive effect Effects 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 230000008488 polyadenylation Effects 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 238000011282 treatment Methods 0.000 description 3
- 238000001262 western blot Methods 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
- WRDABNWSWOHGMS-UHFFFAOYSA-N AEBSF hydrochloride Chemical compound Cl.NCCC1=CC=C(S(F)(=O)=O)C=C1 WRDABNWSWOHGMS-UHFFFAOYSA-N 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 2
- 102000009088 Angiopoietin-1 Human genes 0.000 description 2
- 108010048154 Angiopoietin-1 Proteins 0.000 description 2
- 108010039627 Aprotinin Proteins 0.000 description 2
- 101100481403 Bos taurus TIE1 gene Proteins 0.000 description 2
- 208000005623 Carcinogenesis Diseases 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 108020004635 Complementary DNA Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- 101000808011 Homo sapiens Vascular endothelial growth factor A Proteins 0.000 description 2
- GDBQQVLCIARPGH-UHFFFAOYSA-N Leupeptin Natural products CC(C)CC(NC(C)=O)C(=O)NC(CC(C)C)C(=O)NC(C=O)CCCN=C(N)N GDBQQVLCIARPGH-UHFFFAOYSA-N 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 108091000080 Phosphotransferase Proteins 0.000 description 2
- 108010029485 Protein Isoforms Proteins 0.000 description 2
- 102000001708 Protein Isoforms Human genes 0.000 description 2
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 2
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 2
- 201000004681 Psoriasis Diseases 0.000 description 2
- 241000700159 Rattus Species 0.000 description 2
- 108020004511 Recombinant DNA Proteins 0.000 description 2
- 241000282898 Sus scrofa Species 0.000 description 2
- 102000006601 Thymidine Kinase Human genes 0.000 description 2
- 108020004440 Thymidine kinase Proteins 0.000 description 2
- 108010073925 Vascular Endothelial Growth Factor B Proteins 0.000 description 2
- 102100038217 Vascular endothelial growth factor B Human genes 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 239000004037 angiogenesis inhibitor Substances 0.000 description 2
- 229940121369 angiogenesis inhibitor Drugs 0.000 description 2
- 230000002491 angiogenic effect Effects 0.000 description 2
- 230000003527 anti-angiogenesis Effects 0.000 description 2
- 239000002246 antineoplastic agent Substances 0.000 description 2
- 229960004405 aprotinin Drugs 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000011324 bead Substances 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 230000036952 cancer formation Effects 0.000 description 2
- 231100000504 carcinogenesis Toxicity 0.000 description 2
- 230000033077 cellular process Effects 0.000 description 2
- 108700010039 chimeric receptor Proteins 0.000 description 2
- 208000037976 chronic inflammation Diseases 0.000 description 2
- 210000001072 colon Anatomy 0.000 description 2
- 238000004520 electroporation Methods 0.000 description 2
- 210000005175 epidermal keratinocyte Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 210000004602 germ cell Anatomy 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 102000058223 human VEGFA Human genes 0.000 description 2
- 238000002513 implantation Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000000411 inducer Substances 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 231100000518 lethal Toxicity 0.000 description 2
- 230000001665 lethal effect Effects 0.000 description 2
- GDBQQVLCIARPGH-ULQDDVLXSA-N leupeptin Chemical compound CC(C)C[C@H](NC(C)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C=O)CCCN=C(N)N GDBQQVLCIARPGH-ULQDDVLXSA-N 0.000 description 2
- 108010052968 leupeptin Proteins 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 230000035800 maturation Effects 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- HEGSGKPQLMEBJL-RKQHYHRCSA-N octyl beta-D-glucopyranoside Chemical compound CCCCCCCCO[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HEGSGKPQLMEBJL-RKQHYHRCSA-N 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 102000020233 phosphotransferase Human genes 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 230000001185 psoriatic effect Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- IHIXIJGXTJIKRB-UHFFFAOYSA-N trisodium vanadate Chemical compound [Na+].[Na+].[Na+].[O-][V]([O-])([O-])=O IHIXIJGXTJIKRB-UHFFFAOYSA-N 0.000 description 2
- 241001430294 unidentified retrovirus Species 0.000 description 2
- 230000003827 upregulation Effects 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 206010010356 Congenital anomaly Diseases 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 239000003155 DNA primer Substances 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 206010015218 Erythema multiforme Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 208000010412 Glaucoma Diseases 0.000 description 1
- 208000032612 Glial tumor Diseases 0.000 description 1
- 201000010915 Glioblastoma multiforme Diseases 0.000 description 1
- 206010018338 Glioma Diseases 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 208000032843 Hemorrhage Diseases 0.000 description 1
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001000104 Homo sapiens Myosin-11 Proteins 0.000 description 1
- 101000851007 Homo sapiens Vascular endothelial growth factor receptor 2 Proteins 0.000 description 1
- 108090000144 Human Proteins Proteins 0.000 description 1
- 102000003839 Human Proteins Human genes 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 208000007766 Kaposi sarcoma Diseases 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100351501 Mus musculus Cbfb gene Proteins 0.000 description 1
- 101000808007 Mus musculus Vascular endothelial growth factor A Proteins 0.000 description 1
- 101000851005 Mus musculus Vascular endothelial growth factor receptor 2 Proteins 0.000 description 1
- 229930193140 Neomycin Natural products 0.000 description 1
- 239000000020 Nitrocellulose Substances 0.000 description 1
- 208000022873 Ocular disease Diseases 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 108010038807 Oligopeptides Proteins 0.000 description 1
- 102000015636 Oligopeptides Human genes 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 206010034277 Pemphigoid Diseases 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- 108010089430 Phosphoproteins Proteins 0.000 description 1
- 102000007982 Phosphoproteins Human genes 0.000 description 1
- 241000288906 Primates Species 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 102000001183 RAG-1 Human genes 0.000 description 1
- 108060006897 RAG1 Proteins 0.000 description 1
- 208000007135 Retinal Neovascularization Diseases 0.000 description 1
- 208000017442 Retinal disease Diseases 0.000 description 1
- 206010038923 Retinopathy Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 108090000054 Syndecan-2 Proteins 0.000 description 1
- 102000005450 TIE receptors Human genes 0.000 description 1
- 108010006830 TIE receptors Proteins 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 208000038016 acute inflammation Diseases 0.000 description 1
- 230000006022 acute inflammation Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 239000000556 agonist Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- -1 antibodies Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 206010003246 arthritis Diseases 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 230000035578 autophosphorylation Effects 0.000 description 1
- 238000000376 autoradiography Methods 0.000 description 1
- 239000012148 binding buffer Substances 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 108010006025 bovine growth hormone Proteins 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 208000000594 bullous pemphigoid Diseases 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 210000001043 capillary endothelial cell Anatomy 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008619 cell matrix interaction Effects 0.000 description 1
- 208000025997 central nervous system neoplasm Diseases 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000009402 cross-breeding Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 239000000824 cytostatic agent Substances 0.000 description 1
- 230000001085 cytostatic effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000002828 effect on organs or tissue Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 231100001129 embryonic lethality Toxicity 0.000 description 1
- 210000001671 embryonic stem cell Anatomy 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 230000009762 endothelial cell differentiation Effects 0.000 description 1
- 108091007231 endothelial receptors Proteins 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 231100000321 erythema Toxicity 0.000 description 1
- 238000013401 experimental design Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 230000001605 fetal effect Effects 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- IRSCQMHQWWYFCW-UHFFFAOYSA-N ganciclovir Chemical compound O=C1NC(N)=NC2=C1N=CN2COC(CO)CO IRSCQMHQWWYFCW-UHFFFAOYSA-N 0.000 description 1
- 229960002963 ganciclovir Drugs 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 208000005017 glioblastoma Diseases 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 201000002222 hemangioblastoma Diseases 0.000 description 1
- 210000000777 hematopoietic system Anatomy 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 125000000623 heterocyclic group Chemical group 0.000 description 1
- 102000055590 human KDR Human genes 0.000 description 1
- 102000045989 human MYH11 Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000002390 hyperplastic effect Effects 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 238000007901 in situ hybridization Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000011813 knockout mouse model Methods 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 208000002780 macular degeneration Diseases 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000006510 metastatic growth Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004660 morphological change Effects 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 229960004927 neomycin Drugs 0.000 description 1
- 230000001613 neoplastic effect Effects 0.000 description 1
- 201000003142 neovascular glaucoma Diseases 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000002831 pharmacologic agent Substances 0.000 description 1
- 238000003566 phosphorylation assay Methods 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000000284 resting effect Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000013605 shuttle vector Substances 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 229940048086 sodium pyrophosphate Drugs 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000036561 sun exposure Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- 239000001577 tetrasodium phosphonato phosphate Substances 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 230000005740 tumor formation Effects 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 210000005167 vascular cell Anatomy 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000007998 vessel formation Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0271—Chimeric vertebrates, e.g. comprising exogenous cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- 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
- C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
-
- 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
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Public Health (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Cell Biology (AREA)
- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention provides a non-human transgenic animal whose cells express a foreign DNA sequence that encodes a functional vascular endothelial cell receptor domain, but do not express a substantially homologous native DNA sequence. This invention also provides methods of using these transgenic animals for identifying therapeutic and other reagents that affect angiogenesis. Additionally provided are methods of using transgenic animals whose cells contain any donor genes as models for testing various reagents.
Description
TRANSGENIC ANIMALS WITH KNOCKED-IN VEC RECEPTOR GENES AND USES
THEREOF
BACKGROUND OF THE INVENTION
This invention relates to the development and use of transgenic animals with a "knocked-in" gene. Such animals can be used for testing substances, such as small molecules, antibodies, and other reagents, that have human or veterinary uses.
The knock-in method is a gene replacement technique developed as a tool for developmental studies (Hanks et al., Science, vol. 269, Aug. 4, 9 995), and is a variation of targeted gene inactivation (the "knock-out" method). In a conventional transgenic approach, a gene is introduced into the germ line of the animal at an early developmental stage. In the "knock-out" method, an irrelevant DNA sequence is used to inactivate a gene (Mansour et al., Proc. Natl. Acad. Sci. USA 87: 7688-7692 (1990); Shalaby et al., Nature, vol. 376, July 6, 1995). In the knock-in method, a gene is not merely inactivated, as in the sometimes lethal knock-out method, but is simultaneously replaced at that site by another gene by known methods, such as homologous recombination. For example, in the knock-in method, a cDNA from gene 1 is placed under the control of regulatory elements of another gene (gene 2) while gene 2 is simultaneously inactivated. One advantage of the knock-in technique is that a gene can be introduced into a genome at a specific site. Further, knocking out certain genes can be lethal to an animal, whereas the animal can survive if the appropriate homologous gene is replaced using the knock-in method.
The knock-in method has been used as a powerful tool to discover the functions of specific genes in mammalian pathology. A variation of the knock-in method was used, for example, to understand better the mechanism by which CBFB-MYH11 contributes to leukemogenesis by introducing into mice the human fusion oncogene CBFB-MYH11 by knocking-in a portion of the human MYH11 cDNA into the murine Cbfb locus using homologous recombination (Castilla et al., Cell, 87:687-696, November 15, 1996).
The invention described herein is the first disclosure of the knock-in technique being used to substitute a complete gene from one species with the corresponding homolog from another species or with a chimeric construct. Further, since the knock-in technique has until now been used for discovering functions of genes in mammals, the invention described herein is the first disclosure of knock-in techniques being used for development of an animal model for testing therapeutics.
More specifically, this invention also relates to the development of transgenic animal models with knocked-in vascular endothelial cell-specific receptor genes.
Until now, there have been no sufficient animal models to test therapeutics that are specific for vascular endothelial cell-specific receptors of a particular species. The invention would allow routine testing with quantitative endpoints for therapeutics that inhibit angiogenesis. In contrast, presently available in vivo model systems, such as the SCID mouse model, are difficult to use in a routine manner. For instance, the SCID
mouse model requires laborious surgical manipulations of the animals.
Additionally, these models are often subject to complications that are not easily controlled, such as the unwanted vascularization of the surgically implanted skin grafts by the mouse vasculature.
The knock-in animal models of the invention can be used to test the role of vascular endothelial cell-specific receptors as well as their function in pathological conditions, such as tumorigenesis. Further, the knock-in animal models of the invention are useful for the identification of therapeutics, such as target molecules, antibodies, and other reagents that react with molecules of a specific species only. An example of a knock-in animal model of the invention is one that would be useful specifically for the identification of inhibitors of pathological angiogenesis. Such inhibitors are likely to be specific to human vascular endothelial cell-specific receptors, but need to be tested in an animal model expressing the human receptor gene, such as a knock-in mouse of the invention.
Since one aspect of the invention is the development of knock-in animal models useful for the identification of inhibitors specific to vascular endothelial cell-specific receptors, the following provides a background of the components of angiogenesis, and therapeutic approaches to inhibiting pathological angiogenesis.
Vascular endothelial cells (VECs) play critical roles in blood vessel formation during human development and in many pathological conditions such as tumorigenesis, ocular diseases, arthritis, and arteriosclerosis. Neovascularization, the de novo formation of new blood vessels, as occurs in embryogenesis, is regulated by multiple cellular processes involving endothelial cells, which includes proliferation, migration, cell-cell interactions, cell-matrix interactions, morphological changes and tissue infiltration. Angiogenesis, the formation of new blood vessels from pre-existing blood vessels, involves the same multiple cellular processes, as well as an enhanced need of tissue infiltration of capillary endothelial cells from pre-existing blood vessels. Both of these events, that is, neovascularization and angiogenesis, will herein be referred to as angiogenesis. Angiogenesis is important in normal physiological processes including embryonic development, follicular growth, and wound healing as well as in pathological conditions involving tumor growth and non-neoplastic diseases involving abnormal neovascularization, including neovascular glaucoma (Folkman, ,i. and Klagsbrun, M. Science 235:442-447 (1987), retinal neovascularization of diabetes and macular degeneration of aging, as well as chronic inflammatory diseases such as rheumatoid arthriti .
Many of these diseases currently have few, or no, satisfactory therapies.
Consequently, intense research is underway to explore the hypothesis that blocking the angiogenic component of these diseases is an effective treatment. Many pharmacological agents with angiogenesis-inhibiting properties have been identified but, clinical trials have not progressed sufficiently. Moreover, most known WO 99/18191 PC'T/US98/20717 angiogenesis inhibitors act through poorly understood mechanisms of action. It would .
therefore be desirable to develop new types of angiogenesis inhibitors which act by interfering with known and specific angiogenesis-related mechanisms.
Angiogenesis can be controlled by interfering with a variety of VEC functions, including mitogenesis, migration, adhesion (Cavada, L., et al. J. Clin. Invest. 98: 886-(1996); Brooks, P.C., et al. Cell 79: 1157-1164 (1994)), invasion, and maturation. One approach is to interfere with the action of principal endothelial cell growth factors, especially vascular endothelial growth factor (VEGF).
Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, acts as an angiogenesis inducer by specifically promoting the proliferation of endothelial cells. VEGF is a homodimeric glycoprotein consisting of two 23 kD subunits with structural similarity to PDGF. Several different monomeric isoforms of VEGF
exist, resulting from alternative splicing of mRNA. These include two membrane bound forms (VEGF2o6 and VEGF~89) and two soluble forms (VEGF~65 and VEGF~2~). In all human tissues except placenta, VEGF~65 is the most abundant isoform.
VEGF is expressed in embryonic tissues (Breier et al., Development (Camb.) 114:521 (1992)), macrophages, proliferating epidermal keratinocytes during wound healing (Brown et al., J. Exp. Med., 176:1375 (1992)), and may be responsible for tissue edema associated with inflammation (Ferrara et al., Endocr. Rev. 13:18 (1992}). In situ hybridization studies have demonstrated high VEGF expression in a number of human tumor lines including glioblastoma multiforme, hemangioblastoma, central nervous system neoplasms and AIDS-associated Kaposi's sarcoma (Plate, K. et al.
(1992} Nature 359: 845-848; Plate, K. et al. (1993) Cancer Res. 53: 5822-5827;
Berkman, R. et al. (1993) J. Clin. Invest. 91: 153-159; Nakamura, S. et al.
(1992}
AIDS Weekly, 13 (1 )). High levels of VEGF were also observed in hypoxia induced angiogenesis (Shweiki, D. et al. (1992) Nature 359: 843-845).
THEREOF
BACKGROUND OF THE INVENTION
This invention relates to the development and use of transgenic animals with a "knocked-in" gene. Such animals can be used for testing substances, such as small molecules, antibodies, and other reagents, that have human or veterinary uses.
The knock-in method is a gene replacement technique developed as a tool for developmental studies (Hanks et al., Science, vol. 269, Aug. 4, 9 995), and is a variation of targeted gene inactivation (the "knock-out" method). In a conventional transgenic approach, a gene is introduced into the germ line of the animal at an early developmental stage. In the "knock-out" method, an irrelevant DNA sequence is used to inactivate a gene (Mansour et al., Proc. Natl. Acad. Sci. USA 87: 7688-7692 (1990); Shalaby et al., Nature, vol. 376, July 6, 1995). In the knock-in method, a gene is not merely inactivated, as in the sometimes lethal knock-out method, but is simultaneously replaced at that site by another gene by known methods, such as homologous recombination. For example, in the knock-in method, a cDNA from gene 1 is placed under the control of regulatory elements of another gene (gene 2) while gene 2 is simultaneously inactivated. One advantage of the knock-in technique is that a gene can be introduced into a genome at a specific site. Further, knocking out certain genes can be lethal to an animal, whereas the animal can survive if the appropriate homologous gene is replaced using the knock-in method.
The knock-in method has been used as a powerful tool to discover the functions of specific genes in mammalian pathology. A variation of the knock-in method was used, for example, to understand better the mechanism by which CBFB-MYH11 contributes to leukemogenesis by introducing into mice the human fusion oncogene CBFB-MYH11 by knocking-in a portion of the human MYH11 cDNA into the murine Cbfb locus using homologous recombination (Castilla et al., Cell, 87:687-696, November 15, 1996).
The invention described herein is the first disclosure of the knock-in technique being used to substitute a complete gene from one species with the corresponding homolog from another species or with a chimeric construct. Further, since the knock-in technique has until now been used for discovering functions of genes in mammals, the invention described herein is the first disclosure of knock-in techniques being used for development of an animal model for testing therapeutics.
More specifically, this invention also relates to the development of transgenic animal models with knocked-in vascular endothelial cell-specific receptor genes.
Until now, there have been no sufficient animal models to test therapeutics that are specific for vascular endothelial cell-specific receptors of a particular species. The invention would allow routine testing with quantitative endpoints for therapeutics that inhibit angiogenesis. In contrast, presently available in vivo model systems, such as the SCID mouse model, are difficult to use in a routine manner. For instance, the SCID
mouse model requires laborious surgical manipulations of the animals.
Additionally, these models are often subject to complications that are not easily controlled, such as the unwanted vascularization of the surgically implanted skin grafts by the mouse vasculature.
The knock-in animal models of the invention can be used to test the role of vascular endothelial cell-specific receptors as well as their function in pathological conditions, such as tumorigenesis. Further, the knock-in animal models of the invention are useful for the identification of therapeutics, such as target molecules, antibodies, and other reagents that react with molecules of a specific species only. An example of a knock-in animal model of the invention is one that would be useful specifically for the identification of inhibitors of pathological angiogenesis. Such inhibitors are likely to be specific to human vascular endothelial cell-specific receptors, but need to be tested in an animal model expressing the human receptor gene, such as a knock-in mouse of the invention.
Since one aspect of the invention is the development of knock-in animal models useful for the identification of inhibitors specific to vascular endothelial cell-specific receptors, the following provides a background of the components of angiogenesis, and therapeutic approaches to inhibiting pathological angiogenesis.
Vascular endothelial cells (VECs) play critical roles in blood vessel formation during human development and in many pathological conditions such as tumorigenesis, ocular diseases, arthritis, and arteriosclerosis. Neovascularization, the de novo formation of new blood vessels, as occurs in embryogenesis, is regulated by multiple cellular processes involving endothelial cells, which includes proliferation, migration, cell-cell interactions, cell-matrix interactions, morphological changes and tissue infiltration. Angiogenesis, the formation of new blood vessels from pre-existing blood vessels, involves the same multiple cellular processes, as well as an enhanced need of tissue infiltration of capillary endothelial cells from pre-existing blood vessels. Both of these events, that is, neovascularization and angiogenesis, will herein be referred to as angiogenesis. Angiogenesis is important in normal physiological processes including embryonic development, follicular growth, and wound healing as well as in pathological conditions involving tumor growth and non-neoplastic diseases involving abnormal neovascularization, including neovascular glaucoma (Folkman, ,i. and Klagsbrun, M. Science 235:442-447 (1987), retinal neovascularization of diabetes and macular degeneration of aging, as well as chronic inflammatory diseases such as rheumatoid arthriti .
Many of these diseases currently have few, or no, satisfactory therapies.
Consequently, intense research is underway to explore the hypothesis that blocking the angiogenic component of these diseases is an effective treatment. Many pharmacological agents with angiogenesis-inhibiting properties have been identified but, clinical trials have not progressed sufficiently. Moreover, most known WO 99/18191 PC'T/US98/20717 angiogenesis inhibitors act through poorly understood mechanisms of action. It would .
therefore be desirable to develop new types of angiogenesis inhibitors which act by interfering with known and specific angiogenesis-related mechanisms.
Angiogenesis can be controlled by interfering with a variety of VEC functions, including mitogenesis, migration, adhesion (Cavada, L., et al. J. Clin. Invest. 98: 886-(1996); Brooks, P.C., et al. Cell 79: 1157-1164 (1994)), invasion, and maturation. One approach is to interfere with the action of principal endothelial cell growth factors, especially vascular endothelial growth factor (VEGF).
Vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen, acts as an angiogenesis inducer by specifically promoting the proliferation of endothelial cells. VEGF is a homodimeric glycoprotein consisting of two 23 kD subunits with structural similarity to PDGF. Several different monomeric isoforms of VEGF
exist, resulting from alternative splicing of mRNA. These include two membrane bound forms (VEGF2o6 and VEGF~89) and two soluble forms (VEGF~65 and VEGF~2~). In all human tissues except placenta, VEGF~65 is the most abundant isoform.
VEGF is expressed in embryonic tissues (Breier et al., Development (Camb.) 114:521 (1992)), macrophages, proliferating epidermal keratinocytes during wound healing (Brown et al., J. Exp. Med., 176:1375 (1992)), and may be responsible for tissue edema associated with inflammation (Ferrara et al., Endocr. Rev. 13:18 (1992}). In situ hybridization studies have demonstrated high VEGF expression in a number of human tumor lines including glioblastoma multiforme, hemangioblastoma, central nervous system neoplasms and AIDS-associated Kaposi's sarcoma (Plate, K. et al.
(1992} Nature 359: 845-848; Plate, K. et al. (1993) Cancer Res. 53: 5822-5827;
Berkman, R. et al. (1993) J. Clin. Invest. 91: 153-159; Nakamura, S. et al.
(1992}
AIDS Weekly, 13 (1 )). High levels of VEGF were also observed in hypoxia induced angiogenesis (Shweiki, D. et al. (1992) Nature 359: 843-845).
The biological response of VEGF is mediated through its high affinity VEGF
receptors=
which are selectively expressed on endothelial cells during embryogenesis {Millauer, B., et al., (1993) Cell 72: 835-846) and during tumor formation. Recently, a number of receptor tyrosine kinases (RTKs) that are specifically expressed in endothelial cells have been cloned and characterized. While some RTKs are broadly expressed on diverse cell types, two families have been shown to be primarily restricted to endothelial cells and the early hematopoietic system.
One family includes the VEGF receptors, such as murine FLK-1, and its human homolog KDR; FLT-1; and FLT-4. FLK-11KDR encode a receptor for VEGF-A, VEGF-B, and VEGF-C. FLT-1 and FLT-4 encode a receptor for VEGF-A and VEGF-C, respectively.
The other family includes TIE-1 and TIE-2. T1E-2 is also known as TEK. The ligand for TIE-2, angiopoietin-1, has only recently been cloned (Davis, S., et al., Cell 87:
1161-1169 (1996)). The ligand for TlE-1 has not yet been characterized.
The VEGF receptors, in particular FLK-1/KDR, have been strongly implicated in angiogenesis associated with diverse human pathologies. This realization has led to a major effort to identify inhibitors of tumor angiogenesis with the principal targets being the VEGF molecule and its receptor FLK-1/KDR (Kim, K.J., et al., Nature 362:
(1993); Strawn, L.M., et al., Cancer Research 56: 3540-3545 (1996)).
VEGF receptors typically are class III receptor-type tyrosine kinases characterized by typically having seven immunoglobulin-like loops in their amino-terminal extracellular receptor iigand-binding domains {Kaipainen et al., J. Exp. Med. 178:2077-2088 {1993)). The other two regions include a transmembrane region and a carboxy-terminal intracellular catalytic domain interrupted by an insertion of hydrophilic interkinase sequences of variable lengths, called the kinase insert domain (Vllestermark et al., Prog. Growth Factor Res. 1 (4): 253-266 (1989); Terman et al., Oncogene fi:1677-1683 (1991 )). VEGFs elicit their function as proliferation inducers WO 99!18191 PCT/US98/20717 of endothelial cells by binding to and activating their corresponding receptor tyrosine =~
kinases expressed on the surface of endothelial cells.
Critical roles for these two families of receptors in embryonic development have been conclusively shown by studying knock-out mice of each gene: FLK-1/KDR is critical for endothelial cell differentiation; FLT-1 is important for the organization of primary capillary plexus during the early embryogenesis; TIE-2 was shown to be critical for remodeling of vascular network during angiogenesis in embryos;
was identified as a critical molecule for maturation of the vascular network (Sato, T.N., et al., Nature, 376:70-74 (1995)). However, their roles during later embryonic development and pathological conditions could not be studied since knock-outs of these genes resulted in embryonic lethality. A critical role of FLK-1/KDR in tumor angiogenesis has been clearly shown by retrovirus mediated gene transduction of a dominant negative form of FLK-1 which resulted in prevention of neovascularization in glioma and the consequent prevention of tumor growth.
There are several reasons why KDR is a therapeutic target with highly desirable properties. Of particular importance is that the KDR receptor is expressed almost exclusively on endothelial cells. Further, KDR is strongly up-regulated in activated {proliferating) endothelium as opposed to resting endothelium. In addition, KDR
presents a readily accessible target because of its expression on the surface of blood vessel cells. Accordingly, drugs directed against the extracellular domain of KDR can be particularly useful because they act in a highly specific manner, do not need to enter the endothelial cell, and do not have to reach beyond the vasculature to exert their effects on tissues and thus can be effective at lower doses.
Additionally, these advantages may contribute to favorable safety profiles of anti-KDR drugs.
These properties of KDR also suggest that it is advantageous to interfere with the VEGF-KDR system at the level of KDR rather than VEGF. KDR is localized on the surface of vascular cells in a restricted manner. The VEGF ligand, on the other hand, is present more widely and at higher concentration deep in the interstitial space of tissues. The VEGF ligand is probably found largely in association with heparan sulfate proteoglycans.
Interfering with the formation of new blood vessels by inhibiting the function of KDR
can produce successful new therapies. Further, this approach is advantageous since it offers the possibility of highly specific interference with growing endothelium, as opposed to the generally far less specific treatments now in use. It may be easier to control malignant tumor growth by means of curbing its blood supply with a cytostatic, specific, and potentially non-toxic drug as opposed to directly attacking tumor cells, which is generally done with less specific and frequently cytotoxic drugs.
For example, it is advantageous to interfere with an angiogenic receptor that is specifically expressed on the surface of endothelial cells as opposed to another target (e.g. on tumor cells) which is be distributed more widely and at higher concentrations deep in the interstitial space of tissues. More importantly, the availability of effective, non-toxic anti-angiogenesis drugs can provide long-term or lifetime therapies needed to control a variety of disease, such as, but not limited to the metastatic growth of tumors or rheumatoid arthritis.
Studies by the inventors show that neutralizing antibodies to FLK-1 and to KDR
are species-specific, and therefore do not cross-react. Some anti-KDR antibodies have been shown to have no effect on the binding of FLK-1 to VEGF, and some anti-FLK-1 antibodies have also been shown to have no effect on the binding of KDR to VEGF. Accordingly, it would be futile to attempt to test such anti-KDR
antibodies, and probably other antagonists, for their anti-angiogenic effect in existing murine tumor models.
Until the present invention, it has been difficult to test human antigen-specific antibodies and other potential inhibitors of human angiogenesis, such as tumor angiogenesis, due to the lack of a sufficient animal model. Typically, with testing of various anti-cancer therapeutics, a human tumor cell line is injected into immunodeficient nude mice and the mice are treated with the anti-cancer therapeutic following a period of tumor growth. Potential anti-angiogenesis approaches such as inhibition of the receptor KDR are unique because the target tissue is the host (murine, for example) vasculature rather than the human tumor cells. Most KDft-specific murine monoclonal antibodies, as described above for example, cannot function in such a model.
Possible approaches to circumvent this problem of testing human-specific antagonists in murine tumor models with murine vasculature include: (1 ) searching for tumor models in non-murine mammals whose FLK-1 receptors exhibit higher homology with KDR; and (2) the chimeric human skin/SCID mouse xenograft model (Brooks, P.C., et al., J. Clin. Invest. 96: 1815-1822 (1995)). However, these approaches are unsatisfactory. There are obvious advantages offered by the use of murine tumor models. A large number of syngeneic and xenogeneic murine models of tumor growth have been developed, and the relevance of FLK-1 in these models has been established (Millauer, B., et al., Cancer Research 56: 1615-1820 (1996)).
The substitution of another model (approach (1 )) suffers from major drawbacks, such as: (a) there are no guarantees that anti-KDR antibodies will cross-react with homologous receptors in other species, including primates; {b) establishing consistency and reproducibility would be time-consuming and probably difficult to achieve in non-murine tumor models; (c) satisfactory non-murine tumor models are not common and not readily available.
With regard to approach (2), the chimeric human skin/SCID mouse xenograft model is technically demanding, time consuming and difficult to quantitate (Brooks, P.C., et al., J. Clin. invest. 96: 1815-1822 {1995)). Additionally, there is the added expense associated with the cost of the SCID mice, their maintenance, and the labor needed to conduct the experiments. Further, only limited information is obtained because the duration of treatment is limited due to the finite time of survival of the human skin-grafts on the mouse. Additionally, there is a problem of increasing skin graft s *rB
vascularization by the mouse vasculature as skin grafts age, especially when the skin graft contains a tumor.
Accordingly, there is a need for in vivo transgenic animal models which express the human receptor homolog (e.g., KDR) instead of the native receptor (e.g., FLK-1 ) for testing potential therapeutic molecules to treat humans. There is also a need for in vivo transgenic animal models which express homologous genes from other species for testing potential veterinary therapeutic molecules. An object of this invention is to provide in vivo animal models to test the roles of species-specific receptor tyrosine kinases. This strategy would provide a unique approach to understanding the role of each endothelial cell-specific receptor tyrosine kinase during pathological angiogenesis, and facilitate the identification of therapeutic target molecule(s).
Another object of the invention is to provide transgenic animal models to test potential therapeutic reagents for their effectiveness and specificity, and especially species-sensitive or species-specific reagents.
SUMMARY OF THE INVENTION
These and other objects have been met by providing a method of testing a substance for use in animals comprising administering the substance to a non-human transgenic animal whose cells express a foreign gene or functional gene fragment from a different species, but do not express a substantially homologous native gene or functional gene fragment, and evaluating any effects of the substance on the animal. In this invention, the foreign and native gene or gene fragments can encode vascular endothelial cell receptor domains.
The present invention also provides a non-human transgenic animal whose cells express a foreign gene from a different species; but do not express a substantially homologous native gene. More specifically, the present invention provides a non-human transgenic animal whose cells express a foreign DNA sequence that encodes a functional extracellular vascular endothelial cell receptor domain, but do not express a substantially homologous native DNA sequence.
DESCRIPTION OF THE FIGURE
Figure 1. The targeting strategy for the knock-in of KDR cDNA into the FLK-1 locus.
The outline is a modification of the FLK-1 knock-out targeting strategy of Shalaby et al., (Nature, vol. 376, July 6, 1995). The black rectangle represents the Not I
fragment containing the upstream FLK-1 genomic region. The KDR cDNA and the polyadenylation signal are assembled in an intermediate vector. Approximate lengths of the original genomic FLK-1 DNA and of the restriction fragments relevant for Southern blotting are indicated. Restriction enzymes: B, Bam HI; H, Hind III; N, Nco l; Nt, Not I; P, Pst I, S, Sma I; Sf, Sal I; X, Xha I.
Figure 2. The targeting strategy for the knock-in of a KDR/FLK1-1 chimeric cDNA
into the FLK-1 locus. The outline is a modification of the FLK-1 knock-out targeting strategy of Shalaby et al., (Nature, vol. 376, July 6, 1995). The KDR/FLK-1 chimeric cDNA, including a transmembrane region {TM), and the polyadenylation signal are assembled in an intermediate vector. Approximate lengths of the original genomic FLK-1 DNA and of the restriction fragments relevant for Southern blotting are indicated. Restriction enzymes: B, Bam HI; H, Hind III; N, Nco I; Nt, Not I;
P, Pst I, S, Sma I; SI, Sal l; X, Xho !.
DETAILED DESCRIPTION OF THE INVENTION
*rB
The present invention provides a transgenic animal whose cells express a foreign -v DNA sequence that encodes a functional vascular endothelial cell receptor (VECr) domain. The VECr domain can be a fragment of a VECr, such as the extracellular portion of the receptor, or can be an entire VECr. The foreign DNA sequence is substantially homologous with a native DNA functional VECr domain sequence of the transgenic animal. The cells of the transgenic animal of the invention do not express this homologous native DNA VECr domain sequence.
The VECr foreign DNA sequence can be a fragment or a complete cDNA coding for a VECr of a given species. Further, the VECr foreign DNA sequence can encode a chimeric receptor, wherein the DNA sequence can encode receptors of different species. In one preferred embodiment, the extracellular portion of the receptor is from a species different from the recipient animal.
The transgenic animal of this invention can be produced using the knock-in method.
In the preferred embodiment, homologous recombination using a targeting vector containing the foreign DNA sequence results in insertion of the foreign DNA
sequence at the site of a homologous native DNA sequence, simultaneously inactivating the native DNA sequence. In this specification, the term "replacement,"
as well as various forms of the term "replacement;" refer to the insertion of a foreign DNA sequence at the site of a homologous native DNA sequence, with simultaneous inactivation of the native DNA sequence. The inactivation of the native DNA
sequence occurs upon the disruption of an exon of the native gene when the foreign DNA sequence is inserted into the gene. The foreign DNA sequence is then under the control of the native promoter of the inactivated DNA sequence.
The transgenic animal is preferably a member of a species different than the donor species of the VECr encoded by the foreign DNA. Preferably, both the transgenic animal and the donor are vertebrates, and more preferably, they are mammals.
In one embodiment, the transgenic animal is a non-human mammal, such as a mouse, rat, pig, goat, sheep or monkey, and the donor is a human. In a preferred m embodiment, the transgenic animal is an animal typically used in biomedical or veterinary research, i.e., a laboratory animal. A laboratory animal can be, but is not limited to being, a mouse, rat, rabbit, dog, pig, cow, horse, goat and sheep.
In a more preferred embodiment of the invention, the donor DNA sequence is human, and the transgenic animal is a mouse. In such a preferred embodiment, the human DNA sequence is preferably under the control of murine tissue-specific regulatory elements, such as a murine endothelial cell specific promoter. In the preferred embodiment, the donor human VECr DNA is constructed without a promoter. This promoterless VECr DNA construct is targeted using a vector of the invention into the mouse genome at a site downstream of the promoter for the mouse VECr.
Receptors of the invention can be any VEC receptor. Examples of VEC receptors include, but are not limited to, the protein tyrosine kinase vascular endothelial growth factor (VEGF) receptors KDR, FLK-1, FLT-1, and FLT-4. KDR is the human form of a VEGF receptor having MW 180 kD. FLK-1 is the murine homoiog of KDR. FLT-1 is a form of VEGF receptor different from, but related to, the KDR/FLK-1 receptor.
Both FLK-1 and KDR encode a receptor for VEGF-A , VEGF-B and VEGF-C. FLT-1 and FLT-4 encode a receptor for VEGF-A and VEGF-C, respectively. VEC receptors of the invention also include the TIE family receptor tyrosine kinases, comprising TIE-1 and TIE-2. TIE-2 encodes a receptor for the angiopoietin-1 ligand.
In the preferred embodiment of this invention, the FLK-1 gene of a mouse is replaced with cDNA of KDR from a human donor, under the control of the murine FLK-1 promoter. Murine recipients produced in this manner express both native FLK-1 and KDR receptors. Cross-breeding these murine recipients produces homozygous KDR/KDR mice.
In another embodiment of this invention, the FLK-1 gene of a mouse is replaced with chimeric KDR/FLK-1 cDNA under the control of the murine FLK-1 promoter.
Preferably, in the chimera, sequences coding for the extracellular and transmembrane domains of KDR are fused with those for the intracellular domain of FLK-1, although the transmembrane domains can be from either the KDR or the FLK-1. These clones can be used for the generation of homozygous KDR/FLK-1 mice, so that the intracellular murine FLK-1 domain would be compatible with the murine cell. _ This invention also provides a transgenic animal whose cells contain a donor gene from an animal of a different species that has replaced a substantially homologous native gene of the transgenic animal or of an ancestor of the transgenic animal, wherein the cells no longer express the native gene. The transgenic animal is preferably a mouse and the donor gene is preferably human. The donor gene can be any gene of the animal or any synthetic versions or derivatives thereof that are substantially similar to such donor gene.
UTILITY
The invention provides a method of testing a substance that interacts specifically with a protein expressed by the donor VECr DNA sequence comprising administering the substance to the transgenic recipient of the invention and evaluating any effects of the substance on the recipient. For example, since studies indicate that murine VEGF binds to and activates the human KDR receptor, the homozygous KDR/KDR mice of the invention are useful as animal models for testing various small molecules, antibodies, and other reagents that affect angiogenesis.
Such reagents can either inhibit or increase angiogenesis. Examples of small molecules that can affect angiogenesis include heterocyclic molecules, aromatic molecules, and oligopeptides, among others. Examples of antibodies that can affect angiogenesis are well known in the art.
Further, the invention provides a method of identifying a substance capable of inhibiting abnormal angiogenesis comprising administering the substance to the transgenic KDR animal of the invention and determining whether the substance inhibits abnormal angiogenesis. The invention also provides a method of identifying a substance capable of inhibiting angiogenesis, including tumor angiogenesis, comprising administering the substance to the transgenic KDR animal of the invention and determining whether the substance inhibits angiogenesis. The invention also provides a method of identifying a substance capable of inhibiting tumor growth comprising administering the substance to the transgenic animal of the invention and determining whether the substance inhibits tumor growth. The invention also provides a method of identifying a substance capable of promoting wound healing comprising administering the substance to the transgenic KDR
animal of the invention and determining whether the substance promotes wound healing.
The invention also provides transgenic animals of the invention for use in a method of testing any substance for human or veterinary use. The method of testing a substance for human use comprises administering the substance to a transgenic non-human animal whose cells contain a DNA sequence of a human donor. The donor (foreign) DNA encodes a particular gene of interest, and has replaced a substantially homologous native DNA sequence of the animal or of an ancestor of the animal, wherein the cells no longer express the native DNA sequence. The transgenic animals are then evaluated for any effects of the substance on the animal. In a preferred embodiment, the transgenic non-human animal is a mouse.
Further, in another preferred embodiment, the donor DNA sequence is under the control of the transgenic animal's tissue-specific regulatory elements. The method of testing a substance for veterinary use comprises administering the substance to a transgenic animal whose cells contain a DNA sequence of a donor that has replaced a substantially homologous native DNA sequence of the animal or of an ancestor of the animal, whereby the cells no longer express the native DNA sequence, and evaluating any effects of the substance on the animal. In a preferred embodiment, the transgenic animal and the donor are members of different species. In another preferred embodiment, the donor DNA sequence is under the control of the transgenic animal's tissue-specific regulatory elements.
DNA ENCODING VEC RECEPTORS
Total RNA is prepared by standard procedures from endothelial receptor-containing tissue. The total RNA is used to direct cDNA synthesis. Standard methods for isolating RNA and synthesizing cDNA are provided in standard manuals of molecular biology such as, for example, in Sambrook et al., "Molecular Cloning," Second Edition, Cold Spring Harbor Laboratory Press (1987) and in Ausubel et al., (Eds), "Current Protocols in Molecular Biology," Greene AssociatesNViley Interscience, New York (1990).
The complete gene or the cDNA of the receptors can be amplified by known methods.
For example, the cDNA can be used as a template for amplification by polymerase chain reaction (PCR); see Saiki et al., Science, 239, 487 (1988) or Mullis et al., U.S.
patent 4,fi83,195. The sequences of the oligonucleotide primers for the PCR
amplification are derived from the sequences of mouse and human VEGF receptor respectively. The oligonucleotides are synthesized by methods known in the art.
Suitable methods include those described by Caruthers in Science 230, 281-285 (1985).
Additionally, the complete gene can be obtained by standard methods of isolating genomic clones from genomic phage libraries using standard hybridization techniques.
in order to isolate the entire protein-coding regions for the VEC receptors, the upstream PCR oligonucleotide primer is complementary to the sequence at the 5' end, preferably encompassing the ATG start colon and at least 5-10 nucleotides upstream of the start colon. The downstream PCR oligonucleotide primer is complementary to the sequence at the 3' end of the desired DNA sequence. The desired DNA
sequence preferably encodes the entire extracellular portion of the VEGF receptor, and optionally encodes all or part of the transmembrane region, and/or all or part of the intracellular region, including the stop codon. A mixture of upstream and downstream oligonucleotides are used in the PCR amplification. The conditions are optimized for each particular primer pair according to standard procedures. The PCR product is analyzed by electrophoresis for cDNA having the correct size, corresponding to the sequence between the primers.
Alternatively, the coding region can be amplified in two or more overlapping fragments.
The overlapping fragments are designed to include a restriction site permitting the assembly of the intact cDNA from the fragments.
DNA encoding the VEC receptors of the invention are inserted into a suitable targeting vector and inserted by homologous recombination into a suitable recipient. The DNA
inserted into a recipient can encode the entire VEC receptor, or a fragment of the VEC
receptor.
The nucleic acid molecules that encode the VEC receptors of the invention, or portions thereof, can be inserted into targeting vectors using standard recombinant DNA
techniques. Standard recombinant DNA techniques are described in Sambrook et al., "Molecular Cloning," Second Edition, Cold Spring Harbor Laboratory Press (1987) and by Ausubel et al., (Eds) "Current Protocols in Molecular Biology," Green Publishing Associates/ Wiley-Interscience, New York (1990). A suitable source of cells containing nucleic acid molecules that express the VEC receptor includes VECs.
Suitable vectors for use in mammalian cells are known. Such vectors include well-known derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences and shuttle vectors derived from combination of functional mammalian vectors, such as those described above, and functional plasmids and phage DNA.
EXAMPLES:
The Examples which follow are set forth to aid in understanding the invention but are not intended to, and should not be construed to, limit its scope in any way.
The Examples do not include detailed descriptions of conventional methods employed in the construction of vectors and plasmids, the insertion of genes encoding polypeptides into such vectors and plasmids or the introduction of plasmids into hosts.
Such methods are well known to those of ordinary skill in the art and are described in numerous publications including Sambrook, J., Fritsch, E.F. and Maniatis, T.
(1989) "Molecular Cloning: A Laboratory Manual," 2nd edition, Cold Spring Harbor Laboratory Press.
Knocking-in a KDR Gene Into a Murine Recipient The homologous recombination step occurring in murine embryonic stem cells simultaneously disrupts the first exon of the FLK-1 gene ("knock-out") and replaces it with cDNA for KDR ("knock-in"). The resulting heterozygous mice are expected to express both the native FLK-1 and the KDR receptors. Homozygous KDR/KDR mice are obtained in an F1 intercross.
Experimental Design and Methods:
Production of the targeting construct: The following is a description of the methods of obtaining the necessary DNA components for the targeting vectors (FLK-1 genomic fragments and KDR cDNA). Also described are the preparation of both a full-length KDR construct and a novel chimeric cDNA consisting of the extracellular portion of KDR fused to the intracellular portion of FLK-1.
i~
FLK-1 genomic and cDNA clones: A 15 kb FLK-1 genomic clone is obtained. A 200 v by cDNA fragment that includes the signal peptide sequence was obtained by PCR
from a mouse lung cDNA library using primers complimentary to the published sequence (Matthews, W., et al. Proc. Natl. Acad. Sci. USA 88: 9026-9030 {1997)).
This fragment was random primed with 32P-dCTP and used as a probe to screen a 129/SV mouse genomic library (Stratagene). Purified phage DNA from clone #8 was digested with Sal I and an approximately 15 kb fragment was inserted in both orientations into the Sal I site of pBluescript SK II (+) vector (Stratagene) to give the plasmid pmgFLK1.2. This clone was confirmed by restriction analysis to encode the mouse FLK-1 genomic DNA (Shalaby, F., et al., Nature 376: 62-66 (1995)). Full length FLK-1 cDNA was obtained in accordance with Matthews, W., et al., Proc.
Natl.
Acad. Sci. USA 88: 9026-9030 (1991 ).
KDR cDNA clone: Full length KDR cDNA was obtained by RT-PCR using primers complementary to the published sequence (Terman, B.I., Oncogene 6: 1677-1683 (1991 )). The template for RT-PCR was human fetal kidney mRNA obtained from spontaneously aborted human fetuses (Clontech). Two overlapping fragments encoding 5' and 3' regions of the cDNA were obtained and assembled in the expression vector pcDNA 3 (Invitrogen) using a unique Bam HI site to give the vector KB 113. The cDNA was completely sequenced on both strands.
Vectors for taraetingi construct: Neomycin (pPGK neo bpA) and thymidine kinase (TKpSL1190) (Sato, T.N., et al., Nature 376: 70-74 (1995)) vectors are used for the construction of the targeting vector.
Preparation of the chimeric KDR/FLK-1 cDNA: FLK-1 and KDR cDNAs share a unique Bam HI site located at the codon for methionine 806 of the KDR
sequence. To amplify the sequence coding for the FLK-1 cytoplasmic domain, PCR primers are designed such that the 5' primer is located just upstream of the Bam HI site and the 3' primer just downstream of the termination codon. In addition, the 3' primer is designed to encode a Not I site. Full length FLK-1 cDNA serves as the template for the amplification. The PCR product is cloned into the vector pCR 2.1 (Invitrogen) and w sequenced on both strands. The FLK-1 cDNA is digested with Bam HI and Not I
and subcloned into the KDR expression vector KB 113 (see above) replacing the sequence coding for KDR cytoplasmic domain. In the resulting chimeric cDNA, the first 20 amino acids of the cytoplasmic domain are derived from the human sequence.
However, this region contains only a single difference between the murine and human proteins with glycine (KDR) and glutamic acid (FLK-1 ) at amino acid 794 of the KDR
sequence.
Verification of the expression constructs: The expression constructs tested for the ability to mediate the expression of a functional hybrid receptor molecule by transient transfections into COS 7 cells. The full length KDR expression construct KB
serves as a positive control. 48 hours after transfection, the expression of KDR or KDR/FLK-1 is tested by 1251-VEGF binding, Fluorescence Activated Cell Sorting (FAGS), Western blotting and in a receptor autophosphorylation assay.
For 1251-VEGF binding, VEGF165 is iodinated with 1251. COS 7 cells are plated at near confluency in 24-well plates. All subsequent steps are carried out at 4°C. The cells are washed 1X with binding buffer (BB = MCDB-131/15 mM HEPES/0.1%
gelatin/1pg/ml heparin), then incubated for 2 hours in 0.5 m! BB containing 2 ng/ml of 1251-VEGF. Following incubation, the cells are washed 3X with BB, 2X with PBS, dissolved in 1 % Triton X-100 and counted for radioactivity.
For FAGS, the cells are removed with 2 mM EDTA in PBS, washed with cold Hanks balanced salt solution supplemented with 1 % BSA (HBSS-BSA) and then resuspended in 100 NI of the same buffer at a concentration of 105 cells per sample.
The cells are incubated for 30 minutes with 10 pg of the appropriate anti-KDR
or control FLK-1 specific monoclonal antibody. After washing, a 1:40 dilution of goat anti-mouse or anti-rat IgG conjugated to FITC (TAGO) is added for a final 30 minute incubation on ice. Cells are then analyzed on a Coulter Epics Elite Cytometer.
Data is expressed as the measurement of the mean fluorescent intensity of anti-KDR
WO 99!18191 PCT/US98/20717 monoclonal antibody binding to cells relative to the control measurement of anti-FLK-1=' monoclonal antibody binding.
For Western blot analyses, transfected and control COS 7 cells are lysed in a buffer containing 20 mM Tris-HCI pH 7.4, 1 % N-octylglucoside, 137 mM NaCI, 10%
glycerol, mM EDTA, 100 Ng/ml Pefabloc (Boehringer Mannheim), 100 Ng/ml aprotinin, and 100 Ng/ml leupeptin. Following low speed centrifugation the lysates are separated by SDS-PAGE and transferred to nitrocellulose. The KDR and chimeric KDR/FLK-1 receptor proteins are detected with affinity-purified polycional rabbit antibodies developed at ImClone against the soluble KDR extracellular domain. The blots are incubated with 1251-labeled Protein A (Amersham) and detected by autoradiography.
For the receptor phosphorylation assay, the control and transfected COS 7 cells are starved for 24 hours in DMEM containing 0.5% CS and then stimulated with 20 ng/mi VEGF for 10 minutes at room temperature. Following ligand stimulation, cells are washed with cold PBS containing 1 mM sodium orthovanadate, lysed in a buffer containing 20 mM Tris-HCI pH 7.4, 1 % N-octylglucoside, 137 mM NaCI, 10%
glycerol, 10 mM EDTA, 0.1 mM sodium orthovanadate, 10 mM NaF, 100 mM sodium pyrophosphate, 100 Irg/ml Pefabloc (Boehringer Mannheim), 100 Ng/ml aprotinin, and 100 Ng/ml leupeptin. Following centrifugation at 14,000 x g for 10 minutes, receptors are immunoprecipitated from cleared lysates with Protein A Sepharose beads coupled to rabbit anti-KDR antibodies. The beads are washed, mixed with SDS loading buffer and subjected to Western blot analysis. The phosphoprotein patterns of the stimulated receptors are detected using an anti-phosphotyrosine monoclonal antibody (UBI) and developed by chemiluminescence (ECL; Amersham).
All routine molecular biology procedures such as restriction digests, ligations, PCR and Southern blotting is performed using standard procedures (Sambrook J, Fritsch EF, and Maniatis T, editors. "Molecular Cloning. A Laboratory Manual." 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (19$9)).
Assemblingi the target vector: The targeting vectors that direct the homologous recombination of the two KDR receptor forms (full-length KDR and chimeric KDR/FLK-1 ) into the FLK-1 locus are assembled as follows.
The strategy for disruption of the FLK-1 gene in mouse embryonic stem (ES) cells and the expression of KDR or chimeric receptors under the control of the FLK-1 regulatory elements is outlined in Figure 1. The cDNA in the targeting construct in Figure 1 and in the discussion below is referred to as KDR but represents either KDR or chimeric forms of the receptor. The strategy is broadly based on that used by Shalaby et al., for the targeted disruption of the FLK-1 gene (Nature 376: 62-66 (1995)).
An upstream FLK-1 genomic DNA fragment and KDR cDNA are assembled in a cloning vector designed for this task. The assembly vector consists of the pCR
II
backbone piasmid (Invitrogen) in which the multiple cloning site (MCS) between the Nsi I and Xba I sites are replaced with a synthetic MCS containing the required restriction sites in the following order: Not I, Bam HI, Nsi I, Sma I, Kpn ), Eco RV and Not I. The correct construction of the assembly vector is verified by digesting with Sma I as this enzyme is not present in the parental pCR II plasmid and by sequencing.
A 1.8 kb genomic FLK-1 fragment extending from an upstream Bam HI site to the Pst I
site in the first coding exon (Figure 1 ) is cloned into the Bam HI and Nsi I
sites of the assembly vector with the simultaneous inactivation of the compatible Pst I and Nsi I
sites. The KDR cDNA and bovine growth hormone polyadenylation signal (pA) are excised from the KB 113 expression plasmid with Kpn I and Pvu II and cloned into the Kpn I and Eco RV sites of the assembly vector (Pvu II and Eco RV are compatible, blunt-cutting enzymes).
A Not I fragment from the assembly vector containing the upstream FLK-1 genomic sequence and the KDR cDNA are inserted into the unique Not I site of pPGK neo bpA
and clones are selected in which the orientation of the inserted DNA matches that indicated in Figure 1. A 5.7 kb fragment of genomic FLK-1 DNA extending from a Sma I site downstream from the first coding exon to a Sal I site further downstream is cloned into the Hind III and Sal I sites of pPGK neo bpA by filling the digested Hind II I
site with the Kienow fragment of DNA Polymerase I. Finally, a blunted 2 kb Hind II1 fragment containing the thymidine kinase expression cassette is cloned into a blunted unique Sac II site of pPGK neo bpA.
The correct assembly of the final targeting vector is verified by PCR with primers located on adjacent DNA fragments and by restriction digests.
Production of the Mice with a Knocked-In KDR Gene: The following describes a method of producing lines of ES cells in which at least one allele of the FLK-1 gene is inactivated and in which the expression of the KDR or KDR/FLK-1 chimeric mRNA
is under the control of the native FLK-1 regulatory elements.
1291sv ES (Nagy, A., et al., Proc. Natl. Acad. Sci. USA 90: 8424-8428 (1993)) cells are electroporated with the targeting vector using the ECM 600 electroporator (Gentronics) in HEPES-buffered saline at 160 V, 50 pF capacitance and 360 ohms resistance.
After electroporation, 2 x104 cells are cultured on a 100-mm dish containing feeder STO fibroblasts (Mansour, S.L., et al., Proc. Natl. Acad. Sci. USA 87:7688-(1990)). At 48 hours post electroporation, the cells are selected with gancyclovir and 6418 and individual colonies are isolated.
Genomic DNA from double-selected ES clones is prepared and tested by Southern blotting for homologous recombination. Genomic DNA is digested with either Nco I or Xho I. A probe is generated from a FLK-1 Pst I-Xho I fragment downstream of the targeted locus (see Figure 1 ) and labeled with 32P. This probe is expected to detect a 6.5 kb Nco I fragment in the wild-type locus (Shalaby, F., et al. Nature 376:
(1995)) and a much larger fragment in the targeted locus resulting from the insertion of the KDR and Neo cDNAs. Similarly, this probe should detect an approximately 3.8 kb Xho I fragment generated by the insertion of a novel Xho I site just downstream of the KDR cDNA.
The targeted ES cells are used for the subsequent development of mice that express w the chimeric KDR/FLK-1 or the full length KDR receptors under the control of the FLK-1 promoter. First, heterozygous germ-line chimeric (KDR/FLK-1 )IFLK-1 or full length KDR/FLK-1 mice can be produced using conventional knock-out procedures.
Expression of KDR in the endothelium of heterozygous mice is confirmed by immunocytochemistry using KDR-specific polyclonal and monoclonal antibodies and by RT-PCR with KDR specific primers. The mice are then cross-bred to produce homozygous full length KDR/KDR or homozygous chimeric (KDR/FLK-1 )/(KDR/FLK-1 ) mice.
These mice are cross-bred with an immunodeficient mouse strain such as RAG -I-and the progeny can serve as recipients for the implantation of various murine and human tumor cell lines. The efficacy of administration of monoclonal antibodies that inhibit KDR-VEGF binding or the administration of other KDR-directed agents can then be determined.
Transaenic Animal Models with Knocked-In VEC Receptors and Their Uses:
The following assays can be used to identify target molecules for therapeutic intervention. In addition, the angiogenesis models described below can be used to test the resulting therapeutic reagents as to their effectiveness and specificity.
a. Tumor Angiogenesis:
The mice with knocked-in VECr genes described above are crossed with RAG-1 (-I-) mice and the resulting progeny used for implantation of tumor cell lines.
Various human tumor cell lines are injected into these immuno-compromised knock-in mice and the effect of therapeutic antibodies, target molecules, and other human species-specific reagents is evaluated.
b. Ocular Neovascularization:
The locally induced expression of each knock-in form of receptor tyrosine kinase is tested in the mice with knocked-in VECr genes during ocular neovascularization induced by various angiogenic factors such as VEGF and FGF. These models would be useful in the study of ocular conditions such as retinopathy.
c. Acute and Chronic Inflammation Models:
The mice with knocked-in VECr genes described above are used to study the effects of therapeutic agents on a variety of induced inflammatory conditions. These knock-in mice, in which an inflammatory condition has been induced, would be of particular value in studying therapies for a variety of acute and/or chronic inflammatory conditions, such as rheumatoid arthritis.
d. Psoriasis Models:
Psoriatic skin is characterized by microvascular hyperpermeability and angioproliferation. The hyperplastic epidermis of psoriatic skin expresses strikingly increased amounts of vascular endothelial growth factor. Accordingly, the mice with knocked-in VECr genes described above are useful to study the effects of therapeutic agents on psoriasis, which is often characterized by an increase in vascular endothelial growth factor.
e. Bullous Disease:
Vascular endothelial growth factor is strongly expressed by epidermal keratinocytes in bullous diseases such as erythema multiforme and bullous pemphigoid. These conditions are characterized by increased microvascular permeability and angiogenesis. The development of erythema as a result of hyperpermeable blood vessels is also a common feature after excess sun exposure. To test various therapeutic compounds that have an effect upon these various conditions the mice with knocked-in VECr genes described above are useful.
Wound-Healing:
The role of angiogenesis in wound healing is well-known. In particular, an increase in VEGF expression has been reported in wound healing. The mice with knocked-in VECr genes described above are useful in testing the effects of agonists and antagonists on the expressed receptors, as they relate to wound healing. Such effects would further an understanding of the wound healing process, and would allow therapeutic intervention of the process.
g. Arteriovenous Malformations:
Arteriovenous malformations (AVMs) are congenital lesions composed of abnormal vasculature, with no capillary component, and are clinically significant due to their tendency to spontaneously hemorrhage. The endothelial cell-specific receptor tyrosine kinase, TIE, has been shown to be elevated in AVM
and surrounding brain vasculature. Additionally, upregulation of VEGF mRNA was observed in the cells of brain parenchyma adjacent to the AVM, and VEGF
protein was detected in this tissue as well as in AVM endothelia. Normal brain, in comparison, expressed little or no TIE or VEGF. The significant upregulation of VEGF and TIE in AVMs indicates ongoing angiogenesis, contributing to the slow growth and maintenance of the AVM. Accordingly, the mice with knocked-in VECr genes described above are used to study the effects of therapeutic agents on these congential lesions.
receptors=
which are selectively expressed on endothelial cells during embryogenesis {Millauer, B., et al., (1993) Cell 72: 835-846) and during tumor formation. Recently, a number of receptor tyrosine kinases (RTKs) that are specifically expressed in endothelial cells have been cloned and characterized. While some RTKs are broadly expressed on diverse cell types, two families have been shown to be primarily restricted to endothelial cells and the early hematopoietic system.
One family includes the VEGF receptors, such as murine FLK-1, and its human homolog KDR; FLT-1; and FLT-4. FLK-11KDR encode a receptor for VEGF-A, VEGF-B, and VEGF-C. FLT-1 and FLT-4 encode a receptor for VEGF-A and VEGF-C, respectively.
The other family includes TIE-1 and TIE-2. T1E-2 is also known as TEK. The ligand for TIE-2, angiopoietin-1, has only recently been cloned (Davis, S., et al., Cell 87:
1161-1169 (1996)). The ligand for TlE-1 has not yet been characterized.
The VEGF receptors, in particular FLK-1/KDR, have been strongly implicated in angiogenesis associated with diverse human pathologies. This realization has led to a major effort to identify inhibitors of tumor angiogenesis with the principal targets being the VEGF molecule and its receptor FLK-1/KDR (Kim, K.J., et al., Nature 362:
(1993); Strawn, L.M., et al., Cancer Research 56: 3540-3545 (1996)).
VEGF receptors typically are class III receptor-type tyrosine kinases characterized by typically having seven immunoglobulin-like loops in their amino-terminal extracellular receptor iigand-binding domains {Kaipainen et al., J. Exp. Med. 178:2077-2088 {1993)). The other two regions include a transmembrane region and a carboxy-terminal intracellular catalytic domain interrupted by an insertion of hydrophilic interkinase sequences of variable lengths, called the kinase insert domain (Vllestermark et al., Prog. Growth Factor Res. 1 (4): 253-266 (1989); Terman et al., Oncogene fi:1677-1683 (1991 )). VEGFs elicit their function as proliferation inducers WO 99!18191 PCT/US98/20717 of endothelial cells by binding to and activating their corresponding receptor tyrosine =~
kinases expressed on the surface of endothelial cells.
Critical roles for these two families of receptors in embryonic development have been conclusively shown by studying knock-out mice of each gene: FLK-1/KDR is critical for endothelial cell differentiation; FLT-1 is important for the organization of primary capillary plexus during the early embryogenesis; TIE-2 was shown to be critical for remodeling of vascular network during angiogenesis in embryos;
was identified as a critical molecule for maturation of the vascular network (Sato, T.N., et al., Nature, 376:70-74 (1995)). However, their roles during later embryonic development and pathological conditions could not be studied since knock-outs of these genes resulted in embryonic lethality. A critical role of FLK-1/KDR in tumor angiogenesis has been clearly shown by retrovirus mediated gene transduction of a dominant negative form of FLK-1 which resulted in prevention of neovascularization in glioma and the consequent prevention of tumor growth.
There are several reasons why KDR is a therapeutic target with highly desirable properties. Of particular importance is that the KDR receptor is expressed almost exclusively on endothelial cells. Further, KDR is strongly up-regulated in activated {proliferating) endothelium as opposed to resting endothelium. In addition, KDR
presents a readily accessible target because of its expression on the surface of blood vessel cells. Accordingly, drugs directed against the extracellular domain of KDR can be particularly useful because they act in a highly specific manner, do not need to enter the endothelial cell, and do not have to reach beyond the vasculature to exert their effects on tissues and thus can be effective at lower doses.
Additionally, these advantages may contribute to favorable safety profiles of anti-KDR drugs.
These properties of KDR also suggest that it is advantageous to interfere with the VEGF-KDR system at the level of KDR rather than VEGF. KDR is localized on the surface of vascular cells in a restricted manner. The VEGF ligand, on the other hand, is present more widely and at higher concentration deep in the interstitial space of tissues. The VEGF ligand is probably found largely in association with heparan sulfate proteoglycans.
Interfering with the formation of new blood vessels by inhibiting the function of KDR
can produce successful new therapies. Further, this approach is advantageous since it offers the possibility of highly specific interference with growing endothelium, as opposed to the generally far less specific treatments now in use. It may be easier to control malignant tumor growth by means of curbing its blood supply with a cytostatic, specific, and potentially non-toxic drug as opposed to directly attacking tumor cells, which is generally done with less specific and frequently cytotoxic drugs.
For example, it is advantageous to interfere with an angiogenic receptor that is specifically expressed on the surface of endothelial cells as opposed to another target (e.g. on tumor cells) which is be distributed more widely and at higher concentrations deep in the interstitial space of tissues. More importantly, the availability of effective, non-toxic anti-angiogenesis drugs can provide long-term or lifetime therapies needed to control a variety of disease, such as, but not limited to the metastatic growth of tumors or rheumatoid arthritis.
Studies by the inventors show that neutralizing antibodies to FLK-1 and to KDR
are species-specific, and therefore do not cross-react. Some anti-KDR antibodies have been shown to have no effect on the binding of FLK-1 to VEGF, and some anti-FLK-1 antibodies have also been shown to have no effect on the binding of KDR to VEGF. Accordingly, it would be futile to attempt to test such anti-KDR
antibodies, and probably other antagonists, for their anti-angiogenic effect in existing murine tumor models.
Until the present invention, it has been difficult to test human antigen-specific antibodies and other potential inhibitors of human angiogenesis, such as tumor angiogenesis, due to the lack of a sufficient animal model. Typically, with testing of various anti-cancer therapeutics, a human tumor cell line is injected into immunodeficient nude mice and the mice are treated with the anti-cancer therapeutic following a period of tumor growth. Potential anti-angiogenesis approaches such as inhibition of the receptor KDR are unique because the target tissue is the host (murine, for example) vasculature rather than the human tumor cells. Most KDft-specific murine monoclonal antibodies, as described above for example, cannot function in such a model.
Possible approaches to circumvent this problem of testing human-specific antagonists in murine tumor models with murine vasculature include: (1 ) searching for tumor models in non-murine mammals whose FLK-1 receptors exhibit higher homology with KDR; and (2) the chimeric human skin/SCID mouse xenograft model (Brooks, P.C., et al., J. Clin. Invest. 96: 1815-1822 (1995)). However, these approaches are unsatisfactory. There are obvious advantages offered by the use of murine tumor models. A large number of syngeneic and xenogeneic murine models of tumor growth have been developed, and the relevance of FLK-1 in these models has been established (Millauer, B., et al., Cancer Research 56: 1615-1820 (1996)).
The substitution of another model (approach (1 )) suffers from major drawbacks, such as: (a) there are no guarantees that anti-KDR antibodies will cross-react with homologous receptors in other species, including primates; {b) establishing consistency and reproducibility would be time-consuming and probably difficult to achieve in non-murine tumor models; (c) satisfactory non-murine tumor models are not common and not readily available.
With regard to approach (2), the chimeric human skin/SCID mouse xenograft model is technically demanding, time consuming and difficult to quantitate (Brooks, P.C., et al., J. Clin. invest. 96: 1815-1822 {1995)). Additionally, there is the added expense associated with the cost of the SCID mice, their maintenance, and the labor needed to conduct the experiments. Further, only limited information is obtained because the duration of treatment is limited due to the finite time of survival of the human skin-grafts on the mouse. Additionally, there is a problem of increasing skin graft s *rB
vascularization by the mouse vasculature as skin grafts age, especially when the skin graft contains a tumor.
Accordingly, there is a need for in vivo transgenic animal models which express the human receptor homolog (e.g., KDR) instead of the native receptor (e.g., FLK-1 ) for testing potential therapeutic molecules to treat humans. There is also a need for in vivo transgenic animal models which express homologous genes from other species for testing potential veterinary therapeutic molecules. An object of this invention is to provide in vivo animal models to test the roles of species-specific receptor tyrosine kinases. This strategy would provide a unique approach to understanding the role of each endothelial cell-specific receptor tyrosine kinase during pathological angiogenesis, and facilitate the identification of therapeutic target molecule(s).
Another object of the invention is to provide transgenic animal models to test potential therapeutic reagents for their effectiveness and specificity, and especially species-sensitive or species-specific reagents.
SUMMARY OF THE INVENTION
These and other objects have been met by providing a method of testing a substance for use in animals comprising administering the substance to a non-human transgenic animal whose cells express a foreign gene or functional gene fragment from a different species, but do not express a substantially homologous native gene or functional gene fragment, and evaluating any effects of the substance on the animal. In this invention, the foreign and native gene or gene fragments can encode vascular endothelial cell receptor domains.
The present invention also provides a non-human transgenic animal whose cells express a foreign gene from a different species; but do not express a substantially homologous native gene. More specifically, the present invention provides a non-human transgenic animal whose cells express a foreign DNA sequence that encodes a functional extracellular vascular endothelial cell receptor domain, but do not express a substantially homologous native DNA sequence.
DESCRIPTION OF THE FIGURE
Figure 1. The targeting strategy for the knock-in of KDR cDNA into the FLK-1 locus.
The outline is a modification of the FLK-1 knock-out targeting strategy of Shalaby et al., (Nature, vol. 376, July 6, 1995). The black rectangle represents the Not I
fragment containing the upstream FLK-1 genomic region. The KDR cDNA and the polyadenylation signal are assembled in an intermediate vector. Approximate lengths of the original genomic FLK-1 DNA and of the restriction fragments relevant for Southern blotting are indicated. Restriction enzymes: B, Bam HI; H, Hind III; N, Nco l; Nt, Not I; P, Pst I, S, Sma I; Sf, Sal I; X, Xha I.
Figure 2. The targeting strategy for the knock-in of a KDR/FLK1-1 chimeric cDNA
into the FLK-1 locus. The outline is a modification of the FLK-1 knock-out targeting strategy of Shalaby et al., (Nature, vol. 376, July 6, 1995). The KDR/FLK-1 chimeric cDNA, including a transmembrane region {TM), and the polyadenylation signal are assembled in an intermediate vector. Approximate lengths of the original genomic FLK-1 DNA and of the restriction fragments relevant for Southern blotting are indicated. Restriction enzymes: B, Bam HI; H, Hind III; N, Nco I; Nt, Not I;
P, Pst I, S, Sma I; SI, Sal l; X, Xho !.
DETAILED DESCRIPTION OF THE INVENTION
*rB
The present invention provides a transgenic animal whose cells express a foreign -v DNA sequence that encodes a functional vascular endothelial cell receptor (VECr) domain. The VECr domain can be a fragment of a VECr, such as the extracellular portion of the receptor, or can be an entire VECr. The foreign DNA sequence is substantially homologous with a native DNA functional VECr domain sequence of the transgenic animal. The cells of the transgenic animal of the invention do not express this homologous native DNA VECr domain sequence.
The VECr foreign DNA sequence can be a fragment or a complete cDNA coding for a VECr of a given species. Further, the VECr foreign DNA sequence can encode a chimeric receptor, wherein the DNA sequence can encode receptors of different species. In one preferred embodiment, the extracellular portion of the receptor is from a species different from the recipient animal.
The transgenic animal of this invention can be produced using the knock-in method.
In the preferred embodiment, homologous recombination using a targeting vector containing the foreign DNA sequence results in insertion of the foreign DNA
sequence at the site of a homologous native DNA sequence, simultaneously inactivating the native DNA sequence. In this specification, the term "replacement,"
as well as various forms of the term "replacement;" refer to the insertion of a foreign DNA sequence at the site of a homologous native DNA sequence, with simultaneous inactivation of the native DNA sequence. The inactivation of the native DNA
sequence occurs upon the disruption of an exon of the native gene when the foreign DNA sequence is inserted into the gene. The foreign DNA sequence is then under the control of the native promoter of the inactivated DNA sequence.
The transgenic animal is preferably a member of a species different than the donor species of the VECr encoded by the foreign DNA. Preferably, both the transgenic animal and the donor are vertebrates, and more preferably, they are mammals.
In one embodiment, the transgenic animal is a non-human mammal, such as a mouse, rat, pig, goat, sheep or monkey, and the donor is a human. In a preferred m embodiment, the transgenic animal is an animal typically used in biomedical or veterinary research, i.e., a laboratory animal. A laboratory animal can be, but is not limited to being, a mouse, rat, rabbit, dog, pig, cow, horse, goat and sheep.
In a more preferred embodiment of the invention, the donor DNA sequence is human, and the transgenic animal is a mouse. In such a preferred embodiment, the human DNA sequence is preferably under the control of murine tissue-specific regulatory elements, such as a murine endothelial cell specific promoter. In the preferred embodiment, the donor human VECr DNA is constructed without a promoter. This promoterless VECr DNA construct is targeted using a vector of the invention into the mouse genome at a site downstream of the promoter for the mouse VECr.
Receptors of the invention can be any VEC receptor. Examples of VEC receptors include, but are not limited to, the protein tyrosine kinase vascular endothelial growth factor (VEGF) receptors KDR, FLK-1, FLT-1, and FLT-4. KDR is the human form of a VEGF receptor having MW 180 kD. FLK-1 is the murine homoiog of KDR. FLT-1 is a form of VEGF receptor different from, but related to, the KDR/FLK-1 receptor.
Both FLK-1 and KDR encode a receptor for VEGF-A , VEGF-B and VEGF-C. FLT-1 and FLT-4 encode a receptor for VEGF-A and VEGF-C, respectively. VEC receptors of the invention also include the TIE family receptor tyrosine kinases, comprising TIE-1 and TIE-2. TIE-2 encodes a receptor for the angiopoietin-1 ligand.
In the preferred embodiment of this invention, the FLK-1 gene of a mouse is replaced with cDNA of KDR from a human donor, under the control of the murine FLK-1 promoter. Murine recipients produced in this manner express both native FLK-1 and KDR receptors. Cross-breeding these murine recipients produces homozygous KDR/KDR mice.
In another embodiment of this invention, the FLK-1 gene of a mouse is replaced with chimeric KDR/FLK-1 cDNA under the control of the murine FLK-1 promoter.
Preferably, in the chimera, sequences coding for the extracellular and transmembrane domains of KDR are fused with those for the intracellular domain of FLK-1, although the transmembrane domains can be from either the KDR or the FLK-1. These clones can be used for the generation of homozygous KDR/FLK-1 mice, so that the intracellular murine FLK-1 domain would be compatible with the murine cell. _ This invention also provides a transgenic animal whose cells contain a donor gene from an animal of a different species that has replaced a substantially homologous native gene of the transgenic animal or of an ancestor of the transgenic animal, wherein the cells no longer express the native gene. The transgenic animal is preferably a mouse and the donor gene is preferably human. The donor gene can be any gene of the animal or any synthetic versions or derivatives thereof that are substantially similar to such donor gene.
UTILITY
The invention provides a method of testing a substance that interacts specifically with a protein expressed by the donor VECr DNA sequence comprising administering the substance to the transgenic recipient of the invention and evaluating any effects of the substance on the recipient. For example, since studies indicate that murine VEGF binds to and activates the human KDR receptor, the homozygous KDR/KDR mice of the invention are useful as animal models for testing various small molecules, antibodies, and other reagents that affect angiogenesis.
Such reagents can either inhibit or increase angiogenesis. Examples of small molecules that can affect angiogenesis include heterocyclic molecules, aromatic molecules, and oligopeptides, among others. Examples of antibodies that can affect angiogenesis are well known in the art.
Further, the invention provides a method of identifying a substance capable of inhibiting abnormal angiogenesis comprising administering the substance to the transgenic KDR animal of the invention and determining whether the substance inhibits abnormal angiogenesis. The invention also provides a method of identifying a substance capable of inhibiting angiogenesis, including tumor angiogenesis, comprising administering the substance to the transgenic KDR animal of the invention and determining whether the substance inhibits angiogenesis. The invention also provides a method of identifying a substance capable of inhibiting tumor growth comprising administering the substance to the transgenic animal of the invention and determining whether the substance inhibits tumor growth. The invention also provides a method of identifying a substance capable of promoting wound healing comprising administering the substance to the transgenic KDR
animal of the invention and determining whether the substance promotes wound healing.
The invention also provides transgenic animals of the invention for use in a method of testing any substance for human or veterinary use. The method of testing a substance for human use comprises administering the substance to a transgenic non-human animal whose cells contain a DNA sequence of a human donor. The donor (foreign) DNA encodes a particular gene of interest, and has replaced a substantially homologous native DNA sequence of the animal or of an ancestor of the animal, wherein the cells no longer express the native DNA sequence. The transgenic animals are then evaluated for any effects of the substance on the animal. In a preferred embodiment, the transgenic non-human animal is a mouse.
Further, in another preferred embodiment, the donor DNA sequence is under the control of the transgenic animal's tissue-specific regulatory elements. The method of testing a substance for veterinary use comprises administering the substance to a transgenic animal whose cells contain a DNA sequence of a donor that has replaced a substantially homologous native DNA sequence of the animal or of an ancestor of the animal, whereby the cells no longer express the native DNA sequence, and evaluating any effects of the substance on the animal. In a preferred embodiment, the transgenic animal and the donor are members of different species. In another preferred embodiment, the donor DNA sequence is under the control of the transgenic animal's tissue-specific regulatory elements.
DNA ENCODING VEC RECEPTORS
Total RNA is prepared by standard procedures from endothelial receptor-containing tissue. The total RNA is used to direct cDNA synthesis. Standard methods for isolating RNA and synthesizing cDNA are provided in standard manuals of molecular biology such as, for example, in Sambrook et al., "Molecular Cloning," Second Edition, Cold Spring Harbor Laboratory Press (1987) and in Ausubel et al., (Eds), "Current Protocols in Molecular Biology," Greene AssociatesNViley Interscience, New York (1990).
The complete gene or the cDNA of the receptors can be amplified by known methods.
For example, the cDNA can be used as a template for amplification by polymerase chain reaction (PCR); see Saiki et al., Science, 239, 487 (1988) or Mullis et al., U.S.
patent 4,fi83,195. The sequences of the oligonucleotide primers for the PCR
amplification are derived from the sequences of mouse and human VEGF receptor respectively. The oligonucleotides are synthesized by methods known in the art.
Suitable methods include those described by Caruthers in Science 230, 281-285 (1985).
Additionally, the complete gene can be obtained by standard methods of isolating genomic clones from genomic phage libraries using standard hybridization techniques.
in order to isolate the entire protein-coding regions for the VEC receptors, the upstream PCR oligonucleotide primer is complementary to the sequence at the 5' end, preferably encompassing the ATG start colon and at least 5-10 nucleotides upstream of the start colon. The downstream PCR oligonucleotide primer is complementary to the sequence at the 3' end of the desired DNA sequence. The desired DNA
sequence preferably encodes the entire extracellular portion of the VEGF receptor, and optionally encodes all or part of the transmembrane region, and/or all or part of the intracellular region, including the stop codon. A mixture of upstream and downstream oligonucleotides are used in the PCR amplification. The conditions are optimized for each particular primer pair according to standard procedures. The PCR product is analyzed by electrophoresis for cDNA having the correct size, corresponding to the sequence between the primers.
Alternatively, the coding region can be amplified in two or more overlapping fragments.
The overlapping fragments are designed to include a restriction site permitting the assembly of the intact cDNA from the fragments.
DNA encoding the VEC receptors of the invention are inserted into a suitable targeting vector and inserted by homologous recombination into a suitable recipient. The DNA
inserted into a recipient can encode the entire VEC receptor, or a fragment of the VEC
receptor.
The nucleic acid molecules that encode the VEC receptors of the invention, or portions thereof, can be inserted into targeting vectors using standard recombinant DNA
techniques. Standard recombinant DNA techniques are described in Sambrook et al., "Molecular Cloning," Second Edition, Cold Spring Harbor Laboratory Press (1987) and by Ausubel et al., (Eds) "Current Protocols in Molecular Biology," Green Publishing Associates/ Wiley-Interscience, New York (1990). A suitable source of cells containing nucleic acid molecules that express the VEC receptor includes VECs.
Suitable vectors for use in mammalian cells are known. Such vectors include well-known derivatives of SV-40, adenovirus, retrovirus-derived DNA sequences and shuttle vectors derived from combination of functional mammalian vectors, such as those described above, and functional plasmids and phage DNA.
EXAMPLES:
The Examples which follow are set forth to aid in understanding the invention but are not intended to, and should not be construed to, limit its scope in any way.
The Examples do not include detailed descriptions of conventional methods employed in the construction of vectors and plasmids, the insertion of genes encoding polypeptides into such vectors and plasmids or the introduction of plasmids into hosts.
Such methods are well known to those of ordinary skill in the art and are described in numerous publications including Sambrook, J., Fritsch, E.F. and Maniatis, T.
(1989) "Molecular Cloning: A Laboratory Manual," 2nd edition, Cold Spring Harbor Laboratory Press.
Knocking-in a KDR Gene Into a Murine Recipient The homologous recombination step occurring in murine embryonic stem cells simultaneously disrupts the first exon of the FLK-1 gene ("knock-out") and replaces it with cDNA for KDR ("knock-in"). The resulting heterozygous mice are expected to express both the native FLK-1 and the KDR receptors. Homozygous KDR/KDR mice are obtained in an F1 intercross.
Experimental Design and Methods:
Production of the targeting construct: The following is a description of the methods of obtaining the necessary DNA components for the targeting vectors (FLK-1 genomic fragments and KDR cDNA). Also described are the preparation of both a full-length KDR construct and a novel chimeric cDNA consisting of the extracellular portion of KDR fused to the intracellular portion of FLK-1.
i~
FLK-1 genomic and cDNA clones: A 15 kb FLK-1 genomic clone is obtained. A 200 v by cDNA fragment that includes the signal peptide sequence was obtained by PCR
from a mouse lung cDNA library using primers complimentary to the published sequence (Matthews, W., et al. Proc. Natl. Acad. Sci. USA 88: 9026-9030 {1997)).
This fragment was random primed with 32P-dCTP and used as a probe to screen a 129/SV mouse genomic library (Stratagene). Purified phage DNA from clone #8 was digested with Sal I and an approximately 15 kb fragment was inserted in both orientations into the Sal I site of pBluescript SK II (+) vector (Stratagene) to give the plasmid pmgFLK1.2. This clone was confirmed by restriction analysis to encode the mouse FLK-1 genomic DNA (Shalaby, F., et al., Nature 376: 62-66 (1995)). Full length FLK-1 cDNA was obtained in accordance with Matthews, W., et al., Proc.
Natl.
Acad. Sci. USA 88: 9026-9030 (1991 ).
KDR cDNA clone: Full length KDR cDNA was obtained by RT-PCR using primers complementary to the published sequence (Terman, B.I., Oncogene 6: 1677-1683 (1991 )). The template for RT-PCR was human fetal kidney mRNA obtained from spontaneously aborted human fetuses (Clontech). Two overlapping fragments encoding 5' and 3' regions of the cDNA were obtained and assembled in the expression vector pcDNA 3 (Invitrogen) using a unique Bam HI site to give the vector KB 113. The cDNA was completely sequenced on both strands.
Vectors for taraetingi construct: Neomycin (pPGK neo bpA) and thymidine kinase (TKpSL1190) (Sato, T.N., et al., Nature 376: 70-74 (1995)) vectors are used for the construction of the targeting vector.
Preparation of the chimeric KDR/FLK-1 cDNA: FLK-1 and KDR cDNAs share a unique Bam HI site located at the codon for methionine 806 of the KDR
sequence. To amplify the sequence coding for the FLK-1 cytoplasmic domain, PCR primers are designed such that the 5' primer is located just upstream of the Bam HI site and the 3' primer just downstream of the termination codon. In addition, the 3' primer is designed to encode a Not I site. Full length FLK-1 cDNA serves as the template for the amplification. The PCR product is cloned into the vector pCR 2.1 (Invitrogen) and w sequenced on both strands. The FLK-1 cDNA is digested with Bam HI and Not I
and subcloned into the KDR expression vector KB 113 (see above) replacing the sequence coding for KDR cytoplasmic domain. In the resulting chimeric cDNA, the first 20 amino acids of the cytoplasmic domain are derived from the human sequence.
However, this region contains only a single difference between the murine and human proteins with glycine (KDR) and glutamic acid (FLK-1 ) at amino acid 794 of the KDR
sequence.
Verification of the expression constructs: The expression constructs tested for the ability to mediate the expression of a functional hybrid receptor molecule by transient transfections into COS 7 cells. The full length KDR expression construct KB
serves as a positive control. 48 hours after transfection, the expression of KDR or KDR/FLK-1 is tested by 1251-VEGF binding, Fluorescence Activated Cell Sorting (FAGS), Western blotting and in a receptor autophosphorylation assay.
For 1251-VEGF binding, VEGF165 is iodinated with 1251. COS 7 cells are plated at near confluency in 24-well plates. All subsequent steps are carried out at 4°C. The cells are washed 1X with binding buffer (BB = MCDB-131/15 mM HEPES/0.1%
gelatin/1pg/ml heparin), then incubated for 2 hours in 0.5 m! BB containing 2 ng/ml of 1251-VEGF. Following incubation, the cells are washed 3X with BB, 2X with PBS, dissolved in 1 % Triton X-100 and counted for radioactivity.
For FAGS, the cells are removed with 2 mM EDTA in PBS, washed with cold Hanks balanced salt solution supplemented with 1 % BSA (HBSS-BSA) and then resuspended in 100 NI of the same buffer at a concentration of 105 cells per sample.
The cells are incubated for 30 minutes with 10 pg of the appropriate anti-KDR
or control FLK-1 specific monoclonal antibody. After washing, a 1:40 dilution of goat anti-mouse or anti-rat IgG conjugated to FITC (TAGO) is added for a final 30 minute incubation on ice. Cells are then analyzed on a Coulter Epics Elite Cytometer.
Data is expressed as the measurement of the mean fluorescent intensity of anti-KDR
WO 99!18191 PCT/US98/20717 monoclonal antibody binding to cells relative to the control measurement of anti-FLK-1=' monoclonal antibody binding.
For Western blot analyses, transfected and control COS 7 cells are lysed in a buffer containing 20 mM Tris-HCI pH 7.4, 1 % N-octylglucoside, 137 mM NaCI, 10%
glycerol, mM EDTA, 100 Ng/ml Pefabloc (Boehringer Mannheim), 100 Ng/ml aprotinin, and 100 Ng/ml leupeptin. Following low speed centrifugation the lysates are separated by SDS-PAGE and transferred to nitrocellulose. The KDR and chimeric KDR/FLK-1 receptor proteins are detected with affinity-purified polycional rabbit antibodies developed at ImClone against the soluble KDR extracellular domain. The blots are incubated with 1251-labeled Protein A (Amersham) and detected by autoradiography.
For the receptor phosphorylation assay, the control and transfected COS 7 cells are starved for 24 hours in DMEM containing 0.5% CS and then stimulated with 20 ng/mi VEGF for 10 minutes at room temperature. Following ligand stimulation, cells are washed with cold PBS containing 1 mM sodium orthovanadate, lysed in a buffer containing 20 mM Tris-HCI pH 7.4, 1 % N-octylglucoside, 137 mM NaCI, 10%
glycerol, 10 mM EDTA, 0.1 mM sodium orthovanadate, 10 mM NaF, 100 mM sodium pyrophosphate, 100 Irg/ml Pefabloc (Boehringer Mannheim), 100 Ng/ml aprotinin, and 100 Ng/ml leupeptin. Following centrifugation at 14,000 x g for 10 minutes, receptors are immunoprecipitated from cleared lysates with Protein A Sepharose beads coupled to rabbit anti-KDR antibodies. The beads are washed, mixed with SDS loading buffer and subjected to Western blot analysis. The phosphoprotein patterns of the stimulated receptors are detected using an anti-phosphotyrosine monoclonal antibody (UBI) and developed by chemiluminescence (ECL; Amersham).
All routine molecular biology procedures such as restriction digests, ligations, PCR and Southern blotting is performed using standard procedures (Sambrook J, Fritsch EF, and Maniatis T, editors. "Molecular Cloning. A Laboratory Manual." 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (19$9)).
Assemblingi the target vector: The targeting vectors that direct the homologous recombination of the two KDR receptor forms (full-length KDR and chimeric KDR/FLK-1 ) into the FLK-1 locus are assembled as follows.
The strategy for disruption of the FLK-1 gene in mouse embryonic stem (ES) cells and the expression of KDR or chimeric receptors under the control of the FLK-1 regulatory elements is outlined in Figure 1. The cDNA in the targeting construct in Figure 1 and in the discussion below is referred to as KDR but represents either KDR or chimeric forms of the receptor. The strategy is broadly based on that used by Shalaby et al., for the targeted disruption of the FLK-1 gene (Nature 376: 62-66 (1995)).
An upstream FLK-1 genomic DNA fragment and KDR cDNA are assembled in a cloning vector designed for this task. The assembly vector consists of the pCR
II
backbone piasmid (Invitrogen) in which the multiple cloning site (MCS) between the Nsi I and Xba I sites are replaced with a synthetic MCS containing the required restriction sites in the following order: Not I, Bam HI, Nsi I, Sma I, Kpn ), Eco RV and Not I. The correct construction of the assembly vector is verified by digesting with Sma I as this enzyme is not present in the parental pCR II plasmid and by sequencing.
A 1.8 kb genomic FLK-1 fragment extending from an upstream Bam HI site to the Pst I
site in the first coding exon (Figure 1 ) is cloned into the Bam HI and Nsi I
sites of the assembly vector with the simultaneous inactivation of the compatible Pst I and Nsi I
sites. The KDR cDNA and bovine growth hormone polyadenylation signal (pA) are excised from the KB 113 expression plasmid with Kpn I and Pvu II and cloned into the Kpn I and Eco RV sites of the assembly vector (Pvu II and Eco RV are compatible, blunt-cutting enzymes).
A Not I fragment from the assembly vector containing the upstream FLK-1 genomic sequence and the KDR cDNA are inserted into the unique Not I site of pPGK neo bpA
and clones are selected in which the orientation of the inserted DNA matches that indicated in Figure 1. A 5.7 kb fragment of genomic FLK-1 DNA extending from a Sma I site downstream from the first coding exon to a Sal I site further downstream is cloned into the Hind III and Sal I sites of pPGK neo bpA by filling the digested Hind II I
site with the Kienow fragment of DNA Polymerase I. Finally, a blunted 2 kb Hind II1 fragment containing the thymidine kinase expression cassette is cloned into a blunted unique Sac II site of pPGK neo bpA.
The correct assembly of the final targeting vector is verified by PCR with primers located on adjacent DNA fragments and by restriction digests.
Production of the Mice with a Knocked-In KDR Gene: The following describes a method of producing lines of ES cells in which at least one allele of the FLK-1 gene is inactivated and in which the expression of the KDR or KDR/FLK-1 chimeric mRNA
is under the control of the native FLK-1 regulatory elements.
1291sv ES (Nagy, A., et al., Proc. Natl. Acad. Sci. USA 90: 8424-8428 (1993)) cells are electroporated with the targeting vector using the ECM 600 electroporator (Gentronics) in HEPES-buffered saline at 160 V, 50 pF capacitance and 360 ohms resistance.
After electroporation, 2 x104 cells are cultured on a 100-mm dish containing feeder STO fibroblasts (Mansour, S.L., et al., Proc. Natl. Acad. Sci. USA 87:7688-(1990)). At 48 hours post electroporation, the cells are selected with gancyclovir and 6418 and individual colonies are isolated.
Genomic DNA from double-selected ES clones is prepared and tested by Southern blotting for homologous recombination. Genomic DNA is digested with either Nco I or Xho I. A probe is generated from a FLK-1 Pst I-Xho I fragment downstream of the targeted locus (see Figure 1 ) and labeled with 32P. This probe is expected to detect a 6.5 kb Nco I fragment in the wild-type locus (Shalaby, F., et al. Nature 376:
(1995)) and a much larger fragment in the targeted locus resulting from the insertion of the KDR and Neo cDNAs. Similarly, this probe should detect an approximately 3.8 kb Xho I fragment generated by the insertion of a novel Xho I site just downstream of the KDR cDNA.
The targeted ES cells are used for the subsequent development of mice that express w the chimeric KDR/FLK-1 or the full length KDR receptors under the control of the FLK-1 promoter. First, heterozygous germ-line chimeric (KDR/FLK-1 )IFLK-1 or full length KDR/FLK-1 mice can be produced using conventional knock-out procedures.
Expression of KDR in the endothelium of heterozygous mice is confirmed by immunocytochemistry using KDR-specific polyclonal and monoclonal antibodies and by RT-PCR with KDR specific primers. The mice are then cross-bred to produce homozygous full length KDR/KDR or homozygous chimeric (KDR/FLK-1 )/(KDR/FLK-1 ) mice.
These mice are cross-bred with an immunodeficient mouse strain such as RAG -I-and the progeny can serve as recipients for the implantation of various murine and human tumor cell lines. The efficacy of administration of monoclonal antibodies that inhibit KDR-VEGF binding or the administration of other KDR-directed agents can then be determined.
Transaenic Animal Models with Knocked-In VEC Receptors and Their Uses:
The following assays can be used to identify target molecules for therapeutic intervention. In addition, the angiogenesis models described below can be used to test the resulting therapeutic reagents as to their effectiveness and specificity.
a. Tumor Angiogenesis:
The mice with knocked-in VECr genes described above are crossed with RAG-1 (-I-) mice and the resulting progeny used for implantation of tumor cell lines.
Various human tumor cell lines are injected into these immuno-compromised knock-in mice and the effect of therapeutic antibodies, target molecules, and other human species-specific reagents is evaluated.
b. Ocular Neovascularization:
The locally induced expression of each knock-in form of receptor tyrosine kinase is tested in the mice with knocked-in VECr genes during ocular neovascularization induced by various angiogenic factors such as VEGF and FGF. These models would be useful in the study of ocular conditions such as retinopathy.
c. Acute and Chronic Inflammation Models:
The mice with knocked-in VECr genes described above are used to study the effects of therapeutic agents on a variety of induced inflammatory conditions. These knock-in mice, in which an inflammatory condition has been induced, would be of particular value in studying therapies for a variety of acute and/or chronic inflammatory conditions, such as rheumatoid arthritis.
d. Psoriasis Models:
Psoriatic skin is characterized by microvascular hyperpermeability and angioproliferation. The hyperplastic epidermis of psoriatic skin expresses strikingly increased amounts of vascular endothelial growth factor. Accordingly, the mice with knocked-in VECr genes described above are useful to study the effects of therapeutic agents on psoriasis, which is often characterized by an increase in vascular endothelial growth factor.
e. Bullous Disease:
Vascular endothelial growth factor is strongly expressed by epidermal keratinocytes in bullous diseases such as erythema multiforme and bullous pemphigoid. These conditions are characterized by increased microvascular permeability and angiogenesis. The development of erythema as a result of hyperpermeable blood vessels is also a common feature after excess sun exposure. To test various therapeutic compounds that have an effect upon these various conditions the mice with knocked-in VECr genes described above are useful.
Wound-Healing:
The role of angiogenesis in wound healing is well-known. In particular, an increase in VEGF expression has been reported in wound healing. The mice with knocked-in VECr genes described above are useful in testing the effects of agonists and antagonists on the expressed receptors, as they relate to wound healing. Such effects would further an understanding of the wound healing process, and would allow therapeutic intervention of the process.
g. Arteriovenous Malformations:
Arteriovenous malformations (AVMs) are congenital lesions composed of abnormal vasculature, with no capillary component, and are clinically significant due to their tendency to spontaneously hemorrhage. The endothelial cell-specific receptor tyrosine kinase, TIE, has been shown to be elevated in AVM
and surrounding brain vasculature. Additionally, upregulation of VEGF mRNA was observed in the cells of brain parenchyma adjacent to the AVM, and VEGF
protein was detected in this tissue as well as in AVM endothelia. Normal brain, in comparison, expressed little or no TIE or VEGF. The significant upregulation of VEGF and TIE in AVMs indicates ongoing angiogenesis, contributing to the slow growth and maintenance of the AVM. Accordingly, the mice with knocked-in VECr genes described above are used to study the effects of therapeutic agents on these congential lesions.
Claims (21)
1. A non-human transgenic animal whose cells express a foreign DNA sequence that encodes a functional vascular endothelial cell receptor domain, but do not express a substantially homologous native DNA sequence.
2. The transgenic animal of claim 1, wherein the transgenic animal is a laboratory animal.
3. The transgenic animal of claim 2, wherein the laboratory animal is a mouse.
4. The transgenic animal of claim 1, wherein the foreign vascular endothelial cell receptor DNA sequence is chimeric.
5. The transgenic animal of claim 4, wherein the chimeric DNA sequence is a FLK-1/KDR gene.
6. The transgenic animal of claim 1, wherein the foreign DNA sequence is mammalian.
7. The transgenic mouse of claim 3, wherein the foreign DNA sequence is human.
8. The transgenic mouse of claim 7, wherein the human foreign DNA sequence is under the control of murine tissue-specific regulatory elements.
9. The transgenic mouse of claim 8, wherein the human foreign DNA sequence is under the control of a murine endothelial cell specific promoter.
10. The transgenic mouse of claim 9, wherein the promoter is a FLK-1 or a TIE-promoter.
11. The transgenic mouse of claim 10, wherein the promoter is a FLK-1 promoter.
12. The transgenic mouse of claim 7, wherein the native DNA sequence is the FLK-1 gene.
13. The transgenic mouse of claim 12, wherein the human foreign DNA sequence is a KDR gene.
14. The transgenic mouse of claim 7, wherein the foreign DNA sequence is an extracellular human KDR gene fragment.
15. A non-human transgenic animal whose cells express a foreign gene from a different species, but do not express a substantially homologous native gene.
16. A method of testing a substance that interacts with a protein expressed by the foreign DNA sequence of claim 1 comprising administering the substance to the transgenic animal of claim 1 and evaluating any effects of the substance on the animal.
17. A method of identifying a substance capable of inhibiting angiogenesis comprising administering the substance to the transgenic animal of claim 1 and determining whether the substance inhibits angiogenesis.
18. A method of identifying a substance capable of inhibiting tumor growth comprising administering the substance to the transgenic animal of claim 1 and determining whether the substance inhibits tumor growth.
19. A method of testing a substance for use in animals comprising administering the substance to a non-human transgenic animal whose cells express a foreign gene or functional gene fragment from a different species, but do not express a substantially homologous native gene or functional gene fragment, and evaluating any effects of the substance on the animal.
20. The method of claim 19, wherein the transgenic non-human animal is a mouse.
21. The method of claim 19, wherein the donor DNA sequence is under the control of the transgenic animal's tissue-specific regulatory elements.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US94338197A | 1997-10-02 | 1997-10-02 | |
US08/943,381 | 1997-10-02 | ||
PCT/US1998/020717 WO1999018191A1 (en) | 1997-10-02 | 1998-10-02 | Transgenic animals with knocked-in vec receptor genes and uses thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2305045A1 true CA2305045A1 (en) | 1999-04-15 |
Family
ID=25479555
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002305045A Abandoned CA2305045A1 (en) | 1997-10-02 | 1998-10-02 | Transgenic animals with knocked-in vec receptor genes and uses thereof |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP1007652A1 (en) |
JP (1) | JP2001519145A (en) |
AU (1) | AU9598598A (en) |
CA (1) | CA2305045A1 (en) |
WO (1) | WO1999018191A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AUPR446701A0 (en) | 2001-04-18 | 2001-05-17 | Gene Stream Pty Ltd | Transgenic mammals for pharmacological and toxicological studies |
DE10130657A1 (en) * | 2001-06-27 | 2003-01-16 | Axaron Bioscience Ag | New endothetially expressed protein and its use |
JP2013102747A (en) * | 2011-11-16 | 2013-05-30 | Ehime Univ | Vascular endothelial cell-specific promotor |
-
1998
- 1998-10-02 AU AU95985/98A patent/AU9598598A/en not_active Abandoned
- 1998-10-02 CA CA002305045A patent/CA2305045A1/en not_active Abandoned
- 1998-10-02 JP JP2000514989A patent/JP2001519145A/en not_active Withdrawn
- 1998-10-02 EP EP98949716A patent/EP1007652A1/en not_active Withdrawn
- 1998-10-02 WO PCT/US1998/020717 patent/WO1999018191A1/en not_active Application Discontinuation
Also Published As
Publication number | Publication date |
---|---|
AU9598598A (en) | 1999-04-27 |
JP2001519145A (en) | 2001-10-23 |
EP1007652A1 (en) | 2000-06-14 |
WO1999018191A1 (en) | 1999-04-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Korhonen et al. | Endothelial-specific gene expression directed by the tie gene promoter in vivo | |
Dziennis et al. | The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1; 85 (7): 1983] | |
Reiss et al. | Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice. | |
Dumont et al. | Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo. | |
Kelly et al. | Myosin light chain 3F regulatory sequences confer regionalized cardiac and skeletal muscle expression in transgenic mice. | |
Giovannini et al. | Conditional biallelic Nf2 mutation in the mouse promotes manifestations of human neurofibromatosis type 2 | |
Greer et al. | The Fps/Fes protein-tyrosine kinase promotes angiogenesis in transgenic mice | |
JP2014111624A (en) | Artery- and vein-specific proteins and use methods therefor | |
US8846386B2 (en) | sVEGFR-2 and its role in lymphangiogenesis modulation | |
US7196190B2 (en) | Methods and compositions for screening for angiogenesis modulating compounds | |
Lieuw et al. | Temporal and spatial control of murine GATA-3 transcription by promoter-proximal regulatory elements | |
US7011973B1 (en) | Regulatory sequences capable of conferring expression of a heterologous DNA sequence in endothelial cells in vivo and uses thereof | |
EP1692935A1 (en) | Transgenic animal as a model for human pulmonary disease | |
CA2305045A1 (en) | Transgenic animals with knocked-in vec receptor genes and uses thereof | |
JP4855265B2 (en) | RAS inactivation kinase suppressor for the treatment of RAS-mediated tumorigenesis | |
EP1670308B1 (en) | Chimeric cancer models | |
JPH08509863A (en) | Human C / EBP gene and vector for its expression | |
JP2008525050A (en) | Zebrafish heterotrimeric G protein γ2 subunit (GNG2) | |
US20040091913A1 (en) | Composition and method for imaging cells | |
US7427677B2 (en) | Expression of zebrafish bone morphogenetic protein 4 | |
JP2009526861A (en) | GPCR as an angiogenic target | |
JP2006524049A (en) | Animals and cells containing mutant α2 / δ1 genes | |
CA2518627A1 (en) | Method of screening antiobesity agents and animal model of obesity | |
JPH0779773A (en) | Cancer cell line | |
JP2005187408A (en) | Angiogenesis regulator |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request | ||
FZDE | Dead |