CN108085336A - EGFP expression vector establishments based on col2a1 promoters and the application in transgenic zebrafish - Google Patents
EGFP expression vector establishments based on col2a1 promoters and the application in transgenic zebrafish Download PDFInfo
- Publication number
- CN108085336A CN108085336A CN201611021808.3A CN201611021808A CN108085336A CN 108085336 A CN108085336 A CN 108085336A CN 201611021808 A CN201611021808 A CN 201611021808A CN 108085336 A CN108085336 A CN 108085336A
- Authority
- CN
- China
- Prior art keywords
- sequence
- transgenic zebrafish
- col2a1
- expression vector
- nucleic acid
- 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.)
- Pending
Links
- 241000252212 Danio rerio Species 0.000 title claims abstract description 72
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 51
- 101150082216 COL2A1 gene Proteins 0.000 title claims abstract description 45
- 239000013604 expression vector Substances 0.000 title claims abstract description 29
- 108010048367 enhanced green fluorescent protein Proteins 0.000 title claims description 12
- MURGITYSBWUQTI-UHFFFAOYSA-N fluorescin Chemical compound OC(=O)C1=CC=CC=C1C1C2=CC=C(O)C=C2OC2=CC(O)=CC=C21 MURGITYSBWUQTI-UHFFFAOYSA-N 0.000 claims abstract description 56
- 238000000034 method Methods 0.000 claims abstract description 38
- 150000001875 compounds Chemical class 0.000 claims abstract description 31
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 23
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 23
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 23
- 210000000845 cartilage Anatomy 0.000 claims description 26
- 108090000790 Enzymes Proteins 0.000 claims description 22
- 102000004190 Enzymes Human genes 0.000 claims description 22
- 239000003814 drug Substances 0.000 claims description 22
- 229940079593 drug Drugs 0.000 claims description 21
- 201000008482 osteoarthritis Diseases 0.000 claims description 21
- 235000013601 eggs Nutrition 0.000 claims description 20
- 108010043121 Green Fluorescent Proteins Proteins 0.000 claims description 16
- 102000004144 Green Fluorescent Proteins Human genes 0.000 claims description 16
- 239000005090 green fluorescent protein Substances 0.000 claims description 16
- 238000012216 screening Methods 0.000 claims description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 14
- 108091008146 restriction endonucleases Proteins 0.000 claims description 14
- 108091026890 Coding region Proteins 0.000 claims description 13
- 238000012360 testing method Methods 0.000 claims description 13
- 210000003321 cartilage cell Anatomy 0.000 claims description 11
- 102000034287 fluorescent proteins Human genes 0.000 claims description 11
- 108091006047 fluorescent proteins Proteins 0.000 claims description 11
- 210000001519 tissue Anatomy 0.000 claims description 10
- 238000010276 construction Methods 0.000 claims description 8
- 238000004458 analytical method Methods 0.000 claims description 5
- 108091005948 blue fluorescent proteins Proteins 0.000 claims description 5
- 238000010367 cloning Methods 0.000 claims description 5
- 238000000520 microinjection Methods 0.000 claims description 5
- 108091033319 polynucleotide Proteins 0.000 claims description 5
- 102000040430 polynucleotide Human genes 0.000 claims description 5
- 239000002157 polynucleotide Substances 0.000 claims description 5
- 239000012141 concentrate Substances 0.000 claims description 4
- 238000011161 development Methods 0.000 claims description 4
- 230000012010 growth Effects 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 229940000406 drug candidate Drugs 0.000 claims description 3
- 238000011534 incubation Methods 0.000 claims description 3
- 238000003780 insertion Methods 0.000 claims description 3
- 230000037431 insertion Effects 0.000 claims description 3
- 210000001612 chondrocyte Anatomy 0.000 claims description 2
- 230000000295 complement effect Effects 0.000 claims description 2
- 238000003306 harvesting Methods 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 description 12
- 108020004414 DNA Proteins 0.000 description 9
- 210000000988 bone and bone Anatomy 0.000 description 8
- 241001465754 Metazoa Species 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 108010076504 Protein Sorting Signals Proteins 0.000 description 6
- 230000029087 digestion Effects 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 102000008186 Collagen Human genes 0.000 description 5
- 108010035532 Collagen Proteins 0.000 description 5
- 229920001436 collagen Polymers 0.000 description 5
- 230000001276 controlling effect Effects 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 238000013518 transcription Methods 0.000 description 5
- 230000035897 transcription Effects 0.000 description 5
- 108700007698 Genetic Terminator Regions Proteins 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 210000001188 articular cartilage Anatomy 0.000 description 4
- 210000003128 head Anatomy 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 229920001184 polypeptide Polymers 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 206010007710 Cartilage injury Diseases 0.000 description 3
- 241000448255 Congiopodus peruvianus Species 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 206010061218 Inflammation Diseases 0.000 description 3
- 206010003246 arthritis Diseases 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 230000004054 inflammatory process Effects 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- 241000251468 Actinopterygii Species 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Natural products OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 241000283070 Equus zebra Species 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 230000002917 arthritic effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 230000022159 cartilage development Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- GVJHHUAWPYXKBD-UHFFFAOYSA-N d-alpha-tocopherol Natural products OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000012447 hatching Effects 0.000 description 2
- 210000002758 humerus Anatomy 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 230000033001 locomotion Effects 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000002642 osteogeneic effect Effects 0.000 description 2
- 230000036407 pain Effects 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 108010008359 protein kinase C lambda Proteins 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 210000002303 tibia Anatomy 0.000 description 2
- 210000000623 ulna Anatomy 0.000 description 2
- 210000000689 upper leg Anatomy 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- FPIPGXGPPPQFEQ-UHFFFAOYSA-N 13-cis retinol Natural products OCC=C(C)C=CC=C(C)C=CC1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-UHFFFAOYSA-N 0.000 description 1
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- VUVUVNZRUGEAHB-CYBMUJFWSA-N 7-(3,5-dimethyl-4-isoxazolyl)-8-methoxy-1-[(1R)-1-(2-pyridinyl)ethyl]-3H-imidazo[4,5-c]quinolin-2-one Chemical compound C1([C@@H](C)N2C3=C4C=C(C(=CC4=NC=C3NC2=O)C2=C(ON=C2C)C)OC)=CC=CC=N1 VUVUVNZRUGEAHB-CYBMUJFWSA-N 0.000 description 1
- 101150008656 COL1A1 gene Proteins 0.000 description 1
- ZZZCUOFIHGPKAK-UHFFFAOYSA-N D-erythro-ascorbic acid Natural products OCC1OC(=O)C(O)=C1O ZZZCUOFIHGPKAK-UHFFFAOYSA-N 0.000 description 1
- 208000000088 Enchondromatosis Diseases 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241001062009 Indigofera Species 0.000 description 1
- 208000012659 Joint disease Diseases 0.000 description 1
- 102000006830 Luminescent Proteins Human genes 0.000 description 1
- 108010047357 Luminescent Proteins Proteins 0.000 description 1
- 208000026616 Ollier disease Diseases 0.000 description 1
- 208000004286 Osteochondrodysplasias Diseases 0.000 description 1
- 241001098657 Pterois Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-L Sulfate Chemical compound [O-]S([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 108091023045 Untranslated Region Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- FPIPGXGPPPQFEQ-BOOMUCAASA-N Vitamin A Natural products OC/C=C(/C)\C=C\C=C(\C)/C=C/C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-BOOMUCAASA-N 0.000 description 1
- 229930003268 Vitamin C Natural products 0.000 description 1
- 229930003316 Vitamin D Natural products 0.000 description 1
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 1
- 229930003427 Vitamin E Natural products 0.000 description 1
- 108091005971 Wild-type GFP Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- FPIPGXGPPPQFEQ-OVSJKPMPSA-N all-trans-retinol Chemical compound OC\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C FPIPGXGPPPQFEQ-OVSJKPMPSA-N 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 150000001413 amino acids Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 210000004507 artificial chromosome Anatomy 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 230000014461 bone development Effects 0.000 description 1
- 210000003557 bones of lower extremity Anatomy 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 230000008355 cartilage degradation Effects 0.000 description 1
- -1 cell factor Chemical compound 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- 239000003042 chondroprotective agent Substances 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001684 chronic effect Effects 0.000 description 1
- 230000001054 cortical effect Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- XQRLCLUYWUNEEH-UHFFFAOYSA-L diphosphonate(2-) Chemical compound [O-]P(=O)OP([O-])=O XQRLCLUYWUNEEH-UHFFFAOYSA-L 0.000 description 1
- 238000007877 drug screening Methods 0.000 description 1
- 210000004728 ear cartilage Anatomy 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 230000013020 embryo development Effects 0.000 description 1
- 230000035194 endochondral ossification Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000003623 enhancer Substances 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000000695 excitation spectrum Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- WIGCFUFOHFEKBI-UHFFFAOYSA-N gamma-tocopherol Natural products CC(C)CCCC(C)CCCC(C)CCCC1CCC2C(C)C(O)C(C)C(C)C2O1 WIGCFUFOHFEKBI-UHFFFAOYSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000005184 irreversible process Methods 0.000 description 1
- 208000018937 joint inflammation Diseases 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- 210000002184 nasal cartilage Anatomy 0.000 description 1
- 210000002569 neuron Anatomy 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 230000005868 ontogenesis Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 210000004681 ovum Anatomy 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000008488 polyadenylation Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000002787 reinforcement Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 201000003068 rheumatic fever Diseases 0.000 description 1
- 206010039073 rheumatoid arthritis Diseases 0.000 description 1
- 102220132578 rs761410037 Human genes 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000008054 signal transmission Effects 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 210000003625 skull Anatomy 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 235000019155 vitamin A Nutrition 0.000 description 1
- 239000011719 vitamin A Substances 0.000 description 1
- 235000019154 vitamin C Nutrition 0.000 description 1
- 239000011718 vitamin C Substances 0.000 description 1
- 235000019166 vitamin D Nutrition 0.000 description 1
- 239000011710 vitamin D Substances 0.000 description 1
- 150000003710 vitamin D derivatives Chemical class 0.000 description 1
- 235000019165 vitamin E Nutrition 0.000 description 1
- 239000011709 vitamin E Substances 0.000 description 1
- 229940045997 vitamin a Drugs 0.000 description 1
- 108700024526 zebrafish sox32 Proteins 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
-
- 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
- C12N15/65—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2207/00—Modified animals
- A01K2207/15—Humanized animals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- 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
- A01K2227/00—Animals characterised by species
- A01K2227/40—Fish
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Veterinary Medicine (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to the eGFP expression vector establishments based on col2a1 promoters and the applications in transgenic zebrafish.Specifically, the present invention provides a kind of nucleic acid constructs, and the nucleic acid constructs contains the coded sequence of col2a1 promoters and the fluorescin being operatively connected with the promoter.Preferably, the nucleic acid constructs is expression vector.Therefore the present invention provides the transgenic zebrafish using the expression vector establishment.The invention further relates to obtain application of the transgenic zebrafish in related compound is screened using the method for the present invention.
Description
Technical field
The present invention relates to the eGFP expression vector establishments based on col2a1 promoters and the applications in transgenic zebrafish.
Background technology
Collagen is all widely present in animal body Various Tissues and organ, including brain and neuron, blood vessel, skin
Skin, muscle, cartilage and bone etc..Collagen is generally synthesized by closing on cell and is secreted into extracellular, is the master of extracellular matrix
One of component is wanted, it is vital to play the role of iuntercellular tissue, bonding and signal transmission etc..Cartilage is as collagen
One of main carrying histoorgan, while express and include Col1a1, a variety of collagens such as Col2a1 and Col10a1,
Middle Col2a1 remains high expression in whole periods that cartilage cell forms and develops, and becomes cartilage cell and cartilage group
One of specific marker knitted.
Cartilage is the important and special support organ and tissue of animal.In embryonic development period, cartilage is initially formed, and
Osteogenetic process is dominated in growth course and ultimately forms ripe bone (entochondrostosis, Endochondral
ossification).Entochondrostosis is that the bones such as long bone primarily form mode, including femur (Femur), shin bone
(Tibia), the most of main Limb bone of whole body including ulna (Ulna), radius (Radius), humerus (Humerus) etc., with
And the main trunk support bones such as vertebra (Vertebra), rib cage (Rib), it generates and development all derives from entochondrostosis
Process.If dyschondroplasia, subsequent osteogenetic process is just influenced whether, the support and movement for causing body are acted on by broken
It is bad, seriously affect ontogeny and function.In addition, still some tissue maintains cartilage state always in adult,
Including cartilage at nasal cartilages, Ear cartilage and mammal joint etc..Among these, articular cartilage (Articular
Cartilage it is) particularly important, necessary buffering and support can be provided for joint motion.So far, arthritis
(Arthritis) it is one of highest orthopaedic disease of incidence, seriously affects existence and the quality of life of patient.It is arthritic
Pathogenesis is there are many kind, rheumatic arthritis (Rheumatoid arthritis) and pass including inflammatory reaction induction
Osteoarthritis (Osteoarthritis) caused by saving cartilage damage.The damage of adult articular cartilage can cause osteoarthritis, be
It is a kind of using articular cartilage retrogression pathological changes and secondary osteoproliferation as the chronic joint diseases mainly changed.No matter which kind of joint
Inflammation, finally can all cause articular cavity liquid inflammation, and the cartilage of articular surface is damaged.
Cartilage is always without one of organ of blood vessel intrusion energy supply, this results in cartilage damage particularly adult articular soft
The damage of bone, often medically irreversible process.So far, it is arthritic for cartilage damage and replacing bone
Drug mainly includes：
1st, fastoperation relief of symptoms drug:Rapid analgesia and improvement symptom effect, but osteoarthritis pathology and disease are not influenced
Structure changes can not fundamentally treat osteoarthritis.Such drug mainly includes antalgesic, non-steroidal anti-inflammatory drugs and sugared cortical hormone
Element etc..
2nd, relief of symptoms medicine and Chondroprotective agents are acted on slowly:Slow down or reverse the cartilage degradation at osteoarthritis morbidity, delay
It solves pain and improves function of joint, disturb osteoarthritis pathology process.But the cartilage cell being damaged can not be repaiied
Multiple, effect is not strong enough and takes effect slowly.Such drug mainly includes hyaluronic acid, sulfate glucose etc..
3rd, other drugs, including Diphosphonate, cell factor, vitamin A, C, D and E etc..
In summary, it has been found that existing osteoarthritis treatment drug be confined to mostly antagonism inflammation and pain reaction,
Enhance knuckle synovia activity etc.；New drug research and exploitation also tend to be confined in terms of promoting cartilage cell activity in vitro, lack row
Effective animal model.
Compared with mammal, the transparent zebra fish of body is an excellent model of Investigating Cartilage development.Since 1995
Since year first gfp transgene zebra fish is born, polytype transgenosis zebra has had been established both at home and abroad
Fish, for the researchs such as gene function analysis, cell derived and pedigree, drug screening.
The content of the invention
It is contemplated that a kind of visualization of expression vector offer for starting fluorescent protein expression using col2a1 promoters is soft
Bone cell activity and the animal model of position, the method for establishing stable internal screening cartilage and osteoarthritis drugs screening.
Therefore, first aspect present invention provides a kind of nucleic acid constructs, and the nucleic acid constructs contains col2a1 promoters
With the coded sequence of fluorescin being operatively connected with the promoter.
In one or more embodiments, the sequence such as SEQ ID NO of the col2a1 promoters:Shown in 1.
In one or more embodiments, the fluorescin is selected from green fluorescent protein (GFP), enhanced GFP, red
Move fluorescin and blue fluorescent protein.
In one or more embodiments, the coded sequence such as SEQ ID NO of the fluorescin：Shown in 2.
In one or more embodiments, the nucleic acid constructs is also containing Poly A sequences and playback enzyme I-SceI enzymes
Enzyme site.
In one or more embodiments, the sequence such as SEQ ID NO of the Poly A：Shown in 3.
In one or more embodiments, the sequence such as SEQ ID NO of the playback enzyme I-SceI restriction enzyme sites:8 institutes
Show.
In one or more embodiments, the nucleic acid constructs is expression vector.
In one or more embodiments, the expression vector is using carrier pBluescriptII as framework construction.
In one or more embodiments, the expression vector using pBluescriptII as framework construction,
The both ends insertion playback enzyme I-SceI restriction enzyme sites of pBluescriptI I multiple cloning sites, enzyme I-SceI digestions of playbacking at two
Col2a1 promoter sequences, fluorescent protein coding sequence and Poly A sequences are sequentially inserted between site.
Second aspect of the present invention provides a kind of method for providing transgenic zebrafish, the described method comprises the following steps：
(1) by expression vector of the present invention be transferred to zebra fish fertilized egg and
(2) fertilized eggs that incubation step (1) obtains, transgenic zebrafish of the harvest with fluorescence signal.
In one or more embodiments, the method further includes：
(3) transgenic zebrafish with fluorescence signal that step (2) obtains is mated, generates progeny transgenic spot
Horse fish.
In one or more embodiments, in the transgenic zebrafish, fluorescence signal concentrates on vertebra and head, soft
Bone and the position of skeletal system distribution.
In one or more embodiments, at 40~50 minutes after fertilized eggs are given birth to, this is sent out by microinjection
The bright expression vector is transferred in fertilized eggs.
In one or more embodiments, after being transferred to expression vector of the present invention, fertilized eggs are put into 26~28 DEG C of water
Middle culture, so as to obtain transgenic zebrafish.
Third aspect present invention provides a kind of transgenic zebrafish, and the transgenic zebrafish expresses fluorescin, described
Fluorescin is started by external source col2a1 promoters expresses.
In one or more embodiments, the sequence such as SEQ ID NO of the sequence external source col2a1 promoters:1
It is shown.
In one or more embodiments, the transgenic zebrafish has been transferred to the expression vector of the present invention, and expresses
Fluorescin.
In one or more embodiments, the fluorescence signal that the transgenic zebrafish generates concentrates on vertebra and head
Position.
In one or more embodiments, the transgenic zebrafish is prepared using the method for the invention.
Fourth aspect present invention provides a kind of side screened, identify to cartilage generation and develop the compound having an impact
Method the described method includes giving compound described in transgenic zebrafish of the present invention, and analyzes the compound to the transgenosis
The step of influence of zebra fish cartilage cell and tissue.
Fifth aspect present invention provides transgenic zebrafish of the present invention and cartilage is occurred in screening and develops what is had an impact
Application in compound or gene.
Sixth aspect present invention provides the method that screening treats or prevents the drug of osteoarthritis, and the described method includes give
The influence of transgenic zebrafish testing compound of the present invention and the analysis testing compound to cartilage cell and tissue；
Wherein, the testing compound of chondrocyte growth will be promoted as the drug candidate for treating or preventing osteoarthritis.
Seventh aspect present invention provides the drug that transgenic zebrafish of the present invention treats or prevents osteoarthritis in screening
In purposes.
Eighth aspect present invention also provides a kind of polynucleotide sequence, is selected from：
(1) such as SEQ ID NO：Polynucleotide sequence shown in 1；With
(2) complementary series of (1) described sequence.
Description of the drawings
Fig. 1：Build structure diagram.Col2a1 promoters (1.9kb)：The 1.9kb length used in the present invention
Col2a1 promoters；GFP：Start the green fluorescent protein of expression as reporter gene, by col2a1 promoters；Poly A：It turns over
Translate termination signal；I-SceI：Playback enzyme restriction enzyme site.
Fig. 2：Green fluorescence distribution map.After when fertilized eggs formation 24 and 48 is small, the in vivo green of transgenic zebrafish is glimmering
Optical signal.
Fig. 3：Suppressive drug processing influences green fluorescence intensity.24 are formed as a child to zebra fish culture solution in fertilized eggs
In be separately added into different drug (A, B) and respectively solvent control DMSO, be further cultured for 24 it is small when after, pass through fluorescence microscope
Observe the in vivo green florescent signal variation of zebra fish.
Specific embodiment
The present invention, which constructs, a kind of new can be used for cartilage development and the animal of cartilage protection drug or genescreen
Model realizes in situ, the clear and efficient screening in whole animal level.
The animal model of the present invention is transgenic zebrafish model, animal model expression fluorescin, and the fluorescence egg
White expression is controlled be subject to external source col2a1 promoters.
It is a discovery of the invention that the promoter for the II collagen type col2a1 genes with certain length that the present invention obtains
Sequence can accurately control the expression of col2a1 genes in animal body.In certain embodiments, the present invention uses sequence such as
SEQ ID NO:Col2a1 promoters shown in 1.
It is to be understood that in the present invention, " external source " gene refers to for the gene entrained by animal individual itself.Example
Such as, the col2a1 promoters of external source refer to the col2a1 promoters that animal individual is transferred to from outside.
Fluorescin can be various fluorescins well known in the art, include but not limited to green fluorescent protein (GFP),
Enhanced GFP, red shift fluorescin and blue fluorescent protein.For example, green fluorescent protein is made of 238 amino acid residues
Single chain polypeptide.Heim etc. (1995) is by the way of rite-directed mutagenesis, by the 65th mutant serine into threonine or half Guang ammonia
Acid, so as to obtain the GFP mutant of fluorescence intensity raising.Ser65 is then substituted for Thr by Guohon S etc., and Phe64 is substituted for
Leu further improves the fluorescence intensity (i.e. enhanced GFP) of GFP.The Ser65 of wild type GFP is substituted for Thr, is obtained
Only there are one peak in mutant excitation spectrum, and red shift is to 490nm, because of referred to herein as red shift fluorescin.Double-mutant Y66H/
Y154F can generate the blue light of 445nm under the excitation of 381nm, therefore this kind of fluorescin is referred to as blue fluorescent protein.It is all
These fluorescins can be used in the present invention.
In certain embodiments, the coded sequence such as SEQ ID NO for the fluorescin that the present invention uses:Shown in 2.
The col2a1 promoters of the present invention and fluorescent protein coding sequence can be cloned into expression vector, via the expression
Col2a1 promoters and fluorescent protein coding sequence are transferred in zebra fish by carrier.
The method construction of expression vector of this field routine can be used, wherein the col2a1 promoters and fluorescin of the present invention
Coded sequence operability connects.
" operability connection " or the arrangement of similar description finger element, wherein the ingredient is ordered in certain shape, with
Just the function needed for them is performed.Thus, operability is connected to the given promoter of coded sequence, correct transcription because
In the presence of son etc., the coded sequence effective expression can be made.The promoter need not be abutted with the coded sequence, as long as it plays finger
Lead the function of sequence expression.Thus, for example the sequence that is not involved in translating but transcribe may be present in promoter sequence and volume
Between code sequence, as transcribed introne；And still it is believed that the promoter sequence " operability connection " is in the volume
Code sequence.
Other suitable regulating and controlling sequences can also be contained in expression vector, operably connected with the coded sequence of fluorescin
It connects.Suitably " regulating and controlling sequence " refers to the polynucleotide sequence for helping coded sequence expression connected to it, including promoter, transcription
Terminator sequence, upstream regulatory domains, polyadenylation signal, untranslated region (including 5'-UTR and 3'-UTR), when appropriate
Also targeting sequencing and enhancer, these sequences jointly create item for the transcription and translation of the coded sequence in host cell
Part.
For example, regulating and controlling sequence can be suitable transcription terminator, identify to terminate the sequence of transcription for host cell.
3 ' ends of the nucleotide sequence of terminator sequence and encoding fluorescent protein are operatively connected.It is active in the host cell of selection
Any terminator of energy can be used in the present invention.In certain embodiments, the present invention uses Poly A as terminator sequence.
In certain embodiments, the present invention uses SEQ ID NO:Poly A sequences shown in 2 are as terminator sequence.
Regulating and controlling sequence can also be suitable targeting sequencing, the non-translational region of the mRNA important to host cell translation.Before
5 ' the ends for leading the nucleotide sequence of sequence and encoding fluorescent protein are operatively connected.It is functional in the host cell of selection
Any targeting sequencing can be used in the present invention.
Regulating and controlling sequence can also be the amino acid sequence that coding is connected with the amino-terminal end of fluorescin and instruct to be somebody's turn to do
The polypeptide of coding enters the signal peptide coding region of cell secretory pathway.5 ' ends of fluorescent protein coding sequence can inherently include day
So signal peptide coding region of the translation reading frame of code area segment of the connection with coding secrete polypeptide.Alternatively, coded sequence
5 ' ends can include signal peptide coding region with the code area external source.Signal peptide coding region is included when coded sequence non-natural
When, it may be necessary to external signal peptide coding region.Alternatively, external signal peptide coding region can simply replace natural signal
Peptide-coding region is to enhance the secretion of polypeptide.
In the present invention, expression vector can be easily subjected to recombinant DNA method and can cause interested core
Any carrier (such as plasmid or virus) of nucleotide sequence expression.The selection of carrier is generally dependent on carrier and is wherein imported into the load
The compatibility of the host cell of body.The carrier can be circular plasmids that are linear or being closed.
Carrier can be the carrier of autonomous replication, i.e., exist as extrachromosomal entity, replicate independent of chromosome
The carrier of duplication, such as plasmid, extra-chromosomal element, minichromosome or artificial chromosome.Carrier can include to ensure certainly
Any mode that I replicates.Preferably, after carrier of the invention is imported into host cell, can be integrated into genome.It is more excellent
Choosing, can be by occurring homologous recombination, so as to be integrated into gene after carrier of the invention is imported into host cell with genome
In group
In certain embodiments, the present invention uses restructuring of the carrier pBluescriptII as the framework construction present invention
Expression vector.In these embodiments, playback enzyme I-SceI enzymes are first inserted at the both ends of pBluescriptII multiple cloning sites
Then enzyme site is sequentially inserted into col2a1 promoter sequences between two enzyme I-SceI restriction enzyme sites that playback, fluorescin is compiled
Thus code sequence and Poly A sequences build and obtain the recombinant expression carrier of the present invention.
Therefore, as an example, expression vector of the invention is using carrier pBluescriptII as skeleton, in the carrier
Playback enzyme I-SceI restriction enzyme sites, col2a1 containing operability connection between the multiple cloning sites of pBluescriptII start
Subsequence, fluorescent protein coding sequence, Poly A sequences and playback enzyme I-SceI restriction enzyme sites.
The present invention also include a kind of nucleic acid constructs, the construction contain col2a1 promoters and with the promoter operability
The coded sequence of the fluorescin of connection.As it was noted above, in certain embodiments, the sequence of the col2a1 promoters is such as
SEQ ID NO:Shown in 1.In certain embodiments, the fluorescin be selected from green fluorescent protein (GFP), enhanced GFP,
Red shift fluorescin and blue fluorescent protein.In certain embodiments, the coded sequence of the fluorescin such as SEQ ID
NO：Shown in 2.In certain embodiments, the nucleic acid constructs is also containing Poly A sequences and playback enzyme I-SceI digestions position
Point.In certain embodiments, the sequence of the Poly A such as SEQ ID NO：Shown in 3.In certain embodiments, it is described to return
The sequence such as SEQ ID NO of position enzyme I-SceI restriction enzyme sites:Shown in 8.
In certain embodiments, nucleic acid constructs of the invention contains the playback enzyme I-SceI digestions position being operatively connected
Point, col2a1 promoter sequences, fluorescent protein coding sequence, Poly A sequences and playback enzyme I-SceI restriction enzyme sites.Some
In embodiment, the nucleic acid constructs is previously described expression vector.
It can be by the way that by the nucleic acid constructs of the present invention, especially described expression vector be transferred in the fertilized eggs of zebra fish, incubates
Change the fertilized eggs, so as to provide the transgenic zebrafish of the present invention.
The nucleic acid constructs of the present invention is transferred in the fertilized eggs of zebra fish by the mode that microinjection can be used.It in general, will
The expression vector of the present invention is transferred to the zebra fish fertilized egg in unicellular period.Fertilized eggs after injection can be put into 26-28 DEG C
It is cultivated in water, the juvenile fish raising with fluorescent protein expression is then filtered out, so as to obtain the transgenic zebrafish of the present invention.
In certain embodiments, present invention additionally comprises zebra fish individual of the selection with fluorescence, continue culture and arrive adult,
Post-coitum generates the filial generation for still carrying fluorescence.
For the present invention there is thus also provided a kind of transgenic zebrafish, the transgenic zebrafish expresses fluorescin, described glimmering
Photoprotein is started by external source col2a1 promoters expresses.In certain embodiments, the sequence external source col2a1 promoters
Sequence such as SEQ ID NO:Shown in 1.In certain embodiments, the transgenic zebrafish has been transferred to the expression load of the present invention
Body, and express fluorescin.In certain embodiments, the fluorescence signal that the transgenic zebrafish generates concentrate on vertebra and
Head position.In certain embodiments, the transgenic zebrafish is prepared using the method for the invention.
The transgenic zebrafish of the present invention can be used for screening to occur and develop the compound having an impact to cartilage.Therefore,
The present invention provides a kind of method screened and occur and develop the compound having an impact to cartilage, and the described method includes give this hair
Compound described in the bright transgenic zebrafish, and analyze shadow of the compound to transgenic zebrafish cartilage cell and tissue
Loud step.This influence can be the influence proved, such as cartilage is promoted to occur and develop；Can also be negative impact,
Such as known cartilage occurs and development.
In certain embodiments, the present invention provides transgenic zebrafish of the present invention and cartilage is occurred and is developed to produce in screening
Application in the raw compound influenced or gene.
It is described to be used to screen to identify that testing compound that cartilage occurs in it and development has an impact be nucleic acid point
Son, protein such as enzyme or antibody, the molecule or natural extract of chemical synthesis.This kind of testing compound can also be known use
In drug for the treatment of osteoarthritis or derivatives thereof.Gene to be measured can occur and develop relevant gene with cartilage.
The transgenic zebrafish of the present invention can also be used for screening for treating the drug of osteoarthritis.Therefore, the present invention is gone back
A kind of method for the drug for screening and treating or preventing osteoarthritis is provided, this method includes giving transgenosis zebra of the present invention
The influence of fish testing compound and the analysis testing compound to cartilage cell and tissue；Wherein, cartilage cell will be promoted
The testing compound of growth is as the drug candidate for treating or preventing osteoarthritis.
In certain embodiments, the present invention relates to transgenic zebrafish of the present invention osteoarthritis is treated or prevented in screening
Purposes in drug.
Similarly, the testing compound can be nucleic acid molecules, protein such as enzyme or antibody, chemical synthesis molecule or
Natural extract.This kind of testing compound can also be known for treating drug of osteoarthritis or derivatives thereof
In the present invention, the molecule of chemical synthesis refers to the molecule for the pure chemistry synthesis for being different from nucleic acid, peptide and natural extract；
Natural extract be different from native nucleic acid molecule, native protein non-chemical synthesis molecule or mixture.
With reference to specific embodiment, the present invention is further explained.It is to be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.The experimental method of actual conditions is not specified in the following example, usually according to conventional strip
Part such as Sambrook et al., molecular cloning:Lab guide (New York:Cold Spring Harbor Laboratory
Press, 1989) condition described in or according to the condition proposed by manufacturer.
Unless otherwise defined, all professional and scientific terms used in text and meaning known to one skilled in the art
Justice is identical.In addition, any method similar or impartial to described content and material all can be applied in the present invention.It is described in text
Preferred implement methods and materials be for illustrative purposes only.
Embodiment 1：Plasmid construction
First with the zebra fish complete genome DNA extracted as template, zebra fish is obtained by the method for PCR
(1889bp, sequence are shown in SEQ ID NO to the promoter of col2a1：1)；It is utilized respectively that (sequence is shown in SEQ ID NO containing EGFP：2)
(sequence is shown in SEQ ID NO with Poly A sequences：3) pEGFP-N1 (being purchased from Clontech) plasmid is template, passes through the side of PCR
Method obtains EGFP-Poly A sequences (984bp).
Col2a1 promoters pass through I digestion of Xho I and Sma, and EGFP-Poly A pass through Sma I and Spe I digestions；It is carrying
The both ends insertion playback enzyme I-SceI restriction enzyme sites of body pBluescriptII multiple cloning sites, form transformation carrier pEsce1, should
Carrier is transformed by Xho I and Spe I digestions, is connected jointly after recycling, obtains the matter of col2a1 promoter-EGFP-Poly A
Grain carrier.
For building primer used in plasmid：
Col2a1-Xho I-F:AATTAACTCGAGCGGCCCTCTGACACCTGATGCCAATTGC(SEQ ID NO：4)；
Col2a1-Sma I-R:AATTAACCCGGGGTCTAAAGATTAGACATGCAGGT(SEQ ID NO：5)；
GFP-Sma I-F:AATTCCCGGGATGGTGAGCAAGGGCGAGGAGCTG(SEQ ID NO：6)；
PolyA-Spe I-R:AATTAAACTAGTAACGCTTACAATTTACGCC(SEQ ID NO：7).
The schematic diagram for building the carrier part structure of gained is as shown in Figure 1.
Embodiment 2：The raising and microinjection of zebra fish
Experiment is cultivated with zebra fish in Chinese Academy of Sciences's Shanghai school of life and health sciences biochemistry and the spot of Institute of Cell Biology
Horse fish platform, controlled at 26-28 DEG C.In 40-50min after fertilized eggs are given birth to, microinjection is carried out.After injection by
Smart ovum puts into 26-28 DEG C of water and cultivates, 24 and 48 it is small when after, fluorophor formula sem observation EGFP fluorescins can be passed through
Brightness and survival rate.
Embodiment 3：Detection of the EGFP green fluorescent proteins in zebrafish embryo
After when injection 24 and 48 is small, zebrafish embryo is transferred to tablet, is placed under body formula mirror, passes through the indigo plant of 488nm
Light excites, it is observed that the green fluorescence of zebra fish central axes.At 24 hours, green fluorescence is weaker；From 48 it is small when after, can be with
See the green fluorescence of whole vertebra incandescent, it was demonstrated that the activity of col2a1 promoters is properly positioned.In this injection, we amount to
40 fertilized eggs are injected, wherein 25 normal incubations.
It is verified by the PCR for examining fluorescence and GFP, the zebra fish for obtaining 8 successful conversions altogether and having green fluorescence turns
Gene success rates are about 32%.
Embodiment 4：The expansion of transgenic zebrafish is numerous
Zebra fish individual of the selection with green fluorescence, continues culture to adult, post-coitum generates a large amount of filial generations.Filial generation spot
It is still able to see the expression of green fluorescent protein after the hatching of horse fish, can be used for that Large-scale Screening acts on cartilage and cartilage is thin
Drug and gene of born of the same parents etc..
Embodiment 5：The fluorescence intensity of col2a1 1.9Kb promoters connection eGFP transgenic zebrafishes is examined
By fluorescence microscope, it is found that green florescent signal can be observed in pf for 24 hours, focus primarily upon zebra fish
Vertebra, head position, this distribution (skull and vertebra position) with cartilage at this moment is than more consistent；When continuing to observe 48 hours
Fluorescence signal has further reinforcement when finding small compared with 24, illustrates that cartilage multiplication, size are all promoted (Fig. 2).
Embodiment 6：Micromolecular compound processing inhibits the EGFP expression that col2a1 promoters start
It is studied by early stage, inventor has found there is the expression that some micromolecular compounds can directly affect col2a1.This
Wherein, there are two types of the micromolecular compound (A of inhibition:I-BET151, B:(+)-JQ1).Using suitable concentration A and B not between
Disconnected processing zebra fish fertilized egg, it is found that A and B does not interfere with the hatching rate and survival rate of zebra fish, but can be apparent
Inhibit the green fluorescence intensity (Fig. 3) that col2a1 promoters start.It is soft that this shows that this transgenic zebrafish can be used for influencing
The screening of the micromolecular compound and drug of bone development.
Sequence table
<110>Shanghai Inst. of Life Science, CAS
<120>EGFP expression vector establishments based on col2a1 promoters and the application in transgenic zebrafish
<130> 168260
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 1889
<212> DNA
<213>Zebra fish（Barchydanio rerio var）
<220>
<223>Col2a1 promoters
<400> 1
cctctgacac ctgatgccaa ttgcggtcag tgttttgctg gcgacacaga ttcttgtgcc 60
aatggccagg cccctcatca tctgatccgc agcaacccag ccaccctaca cacccctgga 120
gcctctccgt gttctcctca tccctctacc tttccgcact ctccctccat ccacacccgc 180
ggctctcttc tcccccactg cccggtgctc tctcacattc ctcaggtctg cacacagagc 240
cgcattgtgt gtgtgtctta cagagcacac agtcagggct catttcggac acacacacac 300
atccaacagg gtgtgtgcac agtcgcagcg atgcgtacac acacatacac atatcccttc 360
ataaaaccaa tagacatgca ttcatgtgcg cacgcgaccg caaacacaca cactctctga 420
ttttcatatg tgcacactgg atacacactc atctacatac acaggatgtc tcacaccgag 480
gccgagattc cagtatcctt attccgtgtt tcttttttct tttttactct ttcatgattc 540
aattttttca ctcggttctt ggagtttgat cttataatct cttgagcatt gtgatttttc 600
actcctcctc tgttcccaca gtgggacaga tgatatggta gatgctttat tgtgattgga 660
ttgaggagca gttacataaa ggaaataaag acacaacaaa aatacatctg tgatgaagat 720
ttttgggtag ttttagcaat aatatagatc aacaatttta gatactttta gagtatataa 780
ttgaaaggta aatcattttt taacatgttt tgagaaaaat ctgatacttc aacactaata 840
tattatatgt attcatattt taaataactt atactttcca ttttaactga gttttagagt 900
tttatatagt catttgcaaa agtgcttgtt aatagtgttt agttttacgt ttactaaaca 960
ataatcacac tgcatgtgac acaaagagct gctgttaggt tgctttggga gtacaggaca 1020
tttatttgga ttgcaataac aacttctctg aagcagctgg cgctctacca gagaaataat 1080
gtaagcttag tttacattta gtcatgtggt gccaagaatt taaggttcac tgattatcag 1140
ttgataaggt taattcaagc ttggcaagct aaaatggtga cattgttaat taagtggaat 1200
atttataatc agcatgttca acaactttaa caaaataagt taggatgtaa aactatacta 1260
cttacttttt agtttgcagt ttttaaaatg gaatcttcat aagtgtatca tgatgtatca 1320
tgtgattgac tgtacactgt atgcataagg acccatgata tatgctaaaa cttccctgag 1380
gttatctcac gtttttaatg gtcatgtgcg accacagtgg gtcaaaatct ttgaaacaat 1440
gaatgtattt tggttctcca agtctctttt ctgagcaaag gccattatga tgcactttca 1500
tacaaaaggc acagagagaa agcccaccac ttgccaggaa agggcaaaaa ggggaggggg 1560
tggatgggtt tggatggaat tgttcttgag acagaggggt ggtggacttc tttcccaatg 1620
gggtgggctg gggggctcgt atttcagcgc tcatgggggt cggggttgga ctcacttttt 1680
gggatatact aagattcata aatgcgccag tggacataaa accgcggagc tctgactgac 1740
gcggtgggtt ctgtctgtgt gtgcctggcg tcggatcctc ccaaacacat ccacatccac 1800
ttccacactc cccgcgagca ctgggctgcc gctccgccgg gtcttcggcg actttcaccc 1860
cttaggacct gcatgtctaa tctttagac 1889
<210> 2
<211> 720
<212> DNA
<213>Artificial sequence
<220>
<223>The coded sequence of green fluorescent protein
<400> 2
atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60
ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120
ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180
ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240
cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300
ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360
gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420
aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480
ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540
gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600
tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660
ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa 720
<210> 3
<211> 264
<212> DNA
<213>Artificial sequence
<220>
<223>Poly A sequences
<400> 3
agcggccgcg actctagatc ataatcagcc ataccacatt tgtagaggtt ttacttgctt 60
taaaaaacct cccacacctc cccctgaacc tgaaacataa aatgaatgca attgttgttg 120
ttaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 180
caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 240
cttaaggcgt aaattgtaag cgtt 264
<210> 4
<211> 40
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 4
aattaactcg agcggccctc tgacacctga tgccaattgc 40
<210> 5
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 5
aattaacccg gggtctaaag attagacatg caggt 35
<210> 6
<211> 34
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 6
aattcccggg atggtgagca agggcgagga gctg 34
<210> 7
<211> 31
<212> DNA
<213>Artificial sequence
<220>
<223>Primer
<400> 7
aattaaacta gtaacgctta caatttacgc c 31
<210> 8
<211> 18
<212> DNA
<213>Artificial sequence
<220>
<223>Playback enzyme I-SceI restriction enzyme sites
<400> 8
tagggataac agggtaat 18
Claims (10)
1. a kind of nucleic acid constructs, the nucleic acid constructs contain zebra fish col2a1 promoters and with promoter operability
The coded sequence of the fluorescin of connection.
2. nucleic acid constructs as described in claim 1, which is characterized in that
The sequence of the col2a1 promoters such as SEQ ID NO:Shown in 1；And/or
The fluorescin is selected from green fluorescent protein (GFP), enhanced GFP, red shift fluorescin and blue fluorescent protein；It is excellent
Selection of land, the coded sequence such as SEQ ID NO of the fluorescin：Shown in 2；And/or
The nucleic acid constructs is also containing Poly A sequences and playback enzyme I-SceI restriction enzyme sites；
Preferably, the sequence of the Poly A such as SEQ ID NO：Shown in 3；
Preferably, the sequence of the I-SceI restriction enzyme sites such as SEQ ID NO:Shown in 8.
3. such as the nucleic acid constructs any one of claim 1-2, which is characterized in that the nucleic acid constructs is expression
Carrier；
Preferably, the expression vector is using carrier pBluescriptII as framework construction；
It is highly preferred that the expression vector is using pBluescriptII as framework construction, in pBluescriptII multiple cloning sites
Both ends insertion playback enzyme I-SceI restriction enzyme sites, be sequentially inserted into col2a1 between two enzyme I-SceI restriction enzyme sites that playback and open
Promoter sequences, fluorescent protein coding sequence and Poly A sequences.
4. a kind of method for providing transgenic zebrafish, the described method comprises the following steps：
(1) expression vector described in claim 3 is transferred to zebra fish fertilized egg；With
(2) fertilized eggs that incubation step (1) obtains, transgenic zebrafish of the harvest with fluorescence signal；
Optionally, the method further includes：
(3) transgenic zebrafish with fluorescence signal that step (2) obtains is mated, generates progeny transgenic zebra
Fish.
5. method as claimed in claim 4, which is characterized in that the described method includes：
At 40~50 minutes after fertilized eggs are given birth to, the expression vector is transferred in fertilized eggs by microinjection；With
After being transferred to the expression vector, fertilized eggs are put into 26~28 DEG C of water and are cultivated, so as to obtain transgenic zebrafish；
Preferably, in the transgenic zebrafish, fluorescence signal concentrates on vertebra and head position.
6. a kind of screen the method for occurring and developing the compound having an impact to cartilage, the described method includes：
Give compound described in the transgenic zebrafish built using method any one of claim 4-5；With point
Analyse influence of the compound to transgenic zebrafish cartilage cell and tissue.
7. cartilage is occurred in screening using the transgenic zebrafish that method any one of claim 4-5 is built
Application in the compound or gene that are had an impact with development.
8. a kind of method for screening the drug for treating or preventing osteoarthritis, the described method includes：
It gives the transgenic zebrafish testing compound built using method any one of claim 4-5 and divides
Analyse influence of the testing compound to cartilage cell and tissue；
Wherein, the testing compound of chondrocyte growth will be promoted as the drug candidate for treating or preventing osteoarthritis.
9. it is treated or prevented using the transgenic zebrafish that method any one of claim 4-5 is built in screening
Purposes in the drug of osteoarthritis.
10. a kind of polynucleotide sequence, is selected from：
(1) such as SEQ ID NO：Polynucleotide sequence shown in 1；With
(2) complementary series of (1) described sequence.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611021808.3A CN108085336A (en) | 2016-11-21 | 2016-11-21 | EGFP expression vector establishments based on col2a1 promoters and the application in transgenic zebrafish |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611021808.3A CN108085336A (en) | 2016-11-21 | 2016-11-21 | EGFP expression vector establishments based on col2a1 promoters and the application in transgenic zebrafish |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN108085336A true CN108085336A (en) | 2018-05-29 |
Family
ID=62169356
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611021808.3A Pending CN108085336A (en) | 2016-11-21 | 2016-11-21 | EGFP expression vector establishments based on col2a1 promoters and the application in transgenic zebrafish |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108085336A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102084227B1 (en) * | 2018-09-05 | 2020-03-03 | (주)루젠에스씨아이 | Human transformed chondrocyte cell line mediated drug screening system for cartilage disease remedy |
| WO2020050602A1 (en) * | 2018-09-05 | 2020-03-12 | (주)루젠에스씨아이 | Screening system for human-transformed cartilage cell line-based cartilage disease treatment agents |
| CN114166719A (en) * | 2021-11-27 | 2022-03-11 | 北京擎科生物科技有限公司 | Method and device for screening nucleic acid synthetic vector |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090097503A (en) * | 2008-03-11 | 2009-09-16 | 재단법인서울대학교산학협력재단 | Quantification egfp fluorescence as an indicator of arsenite developmental toxicity in mosaic heat shock protein 70 transgenic zebrafish |
| WO2010109168A2 (en) * | 2009-03-23 | 2010-09-30 | Academisch Ziekenhuis Leiden Also Acting Under The Name "Leiden University Medical Center" (Lumc) | Angiogenesis methods, medicaments and agents |
| CN104313043A (en) * | 2014-09-29 | 2015-01-28 | 广东医学院附属医院 | Construction method and application of osteoporosis zebra fish model |
| CN106070063A (en) * | 2016-07-07 | 2016-11-09 | 贵州医科大学 | Transgenic zebrafish system with abcb4 and method for building up thereof |
-
2016
- 2016-11-21 CN CN201611021808.3A patent/CN108085336A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20090097503A (en) * | 2008-03-11 | 2009-09-16 | 재단법인서울대학교산학협력재단 | Quantification egfp fluorescence as an indicator of arsenite developmental toxicity in mosaic heat shock protein 70 transgenic zebrafish |
| WO2010109168A2 (en) * | 2009-03-23 | 2010-09-30 | Academisch Ziekenhuis Leiden Also Acting Under The Name "Leiden University Medical Center" (Lumc) | Angiogenesis methods, medicaments and agents |
| CN104313043A (en) * | 2014-09-29 | 2015-01-28 | 广东医学院附属医院 | Construction method and application of osteoporosis zebra fish model |
| CN106070063A (en) * | 2016-07-07 | 2016-11-09 | 贵州医科大学 | Transgenic zebrafish system with abcb4 and method for building up thereof |
Non-Patent Citations (7)
| Title |
|---|
| NINGNING NIU等: "Bromodomain and Extra-terminal(BET) Protein Inhibitors Suppress Chondrocyte Differentiation and Restrain Bone Growth", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| R.E. MITCHELL等: "New tools for studying osteoarthritis genetics in zebrafish", 《OSTEOARTHRITIS AND CARTILAGE》 * |
| WRIGHT,D: "Zebrafish DNA sequence from clone CH211-216K22 in linkage group 8, complete sequence,Accession ID: BX927114.24", 《GENBANK》 * |
| 张文贤: "《骨性关节炎：膝关节骨性关节炎的病理研究与临床诊治》", 30 September 2008, 甘肃科学技术出版社 * |
| 白俊杰: "《转红色荧光蛋白基因唐鱼研究》", 31 January 2014, 海洋出版社 * |
| 白刚勋: "《大教育视野下的特色课程构建：海洋教育的开发实施》", 30 April 2014, 西南师范大学出版社 * |
| 董武等: "农药福美双对斑马鱼Col2a1基因表达的影响", 《第十届全国家畜环境科学讨论会论文集》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR102084227B1 (en) * | 2018-09-05 | 2020-03-03 | (주)루젠에스씨아이 | Human transformed chondrocyte cell line mediated drug screening system for cartilage disease remedy |
| WO2020050602A1 (en) * | 2018-09-05 | 2020-03-12 | (주)루젠에스씨아이 | Screening system for human-transformed cartilage cell line-based cartilage disease treatment agents |
| CN114166719A (en) * | 2021-11-27 | 2022-03-11 | 北京擎科生物科技有限公司 | Method and device for screening nucleic acid synthetic vector |
| CN114166719B (en) * | 2021-11-27 | 2022-08-12 | 北京擎科生物科技有限公司 | Method and device for screening nucleic acid synthetic vector |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220256819A1 (en) | Chimeric gene constructs for generation of fluorescent transgenic ornamental fish | |
| ES2245454T3 (en) | PATCHED GENES AND ITS USE. | |
| Serano et al. | Comprehensive analysis of Hox gene expression in the amphipod crustacean Parhyale hawaiensis | |
| Ludin et al. | Application of novel vectors for GFP-tagging of proteins to study microtubule-associated proteins | |
| US20100037331A1 (en) | Recombinant constructs and transgenic fluorescent ornamental fish therefrom | |
| CN108085336A (en) | EGFP expression vector establishments based on col2a1 promoters and the application in transgenic zebrafish | |
| CN102604953B (en) | BmCP274 promoter of bombyx mori cuticular protein, recombinant expression vector and application of the promoter | |
| Posner et al. | The zebrafish as a model system for analyzing mammalian and native α-crystallin promoter function | |
| US7358336B2 (en) | Fluorescent protein from Montastraea cavernosa | |
| Suster et al. | A novel conserved evx1 enhancer links spinal interneuron morphology and cis-regulation from fish to mammals | |
| CN104651409B (en) | The high-efficient transgenic method of one species transcription activator effect protein mediation | |
| JP7128525B2 (en) | Transgenic silkworm and method for producing non-natural amino acid-containing protein using the silkworm | |
| Klompen et al. | Venom system variation and the division of labor in the colonial hydrozoan Hydractinia symbiolongicarpus | |
| CN100355888C (en) | New transgenic zebra fish, production method of gene segment and transgenic zebra fish | |
| Liu et al. | Insights into regulatory characteristics of the promoters of Sericin 1 and Sericin 3 in transgenic silkworms | |
| CN111424035A (en) | Method for expressing human connective tissue growth factor with biological activity based on silkworm silk gland, product and application thereof | |
| Poterski | Synthesis of recombinant human relaxin H2 in bacteria and its effects on differentiation of carcinoembryonic stem cells | |
| CN102604954B (en) | Domestic silkworm cuticle protein BmCP231 promoter as well as recombinant expression vector and application thereof | |
| Conway et al. | Fluorescent tagged analysis of neural gene function using mosaics in zebrafish and Xenopus laevis | |
| Islam et al. | Chapter-5 Transgenic Silkworms and Its Scope in Sericulture | |
| Romeo et al. | Modeling Immunoglobulin light chain amyloidosis in Caenorhabditis elegans | |
| Bayramov et al. | Loss of noggin1, a Classic Embryonic Inducer Gene, in Elasmobranchs | |
| Dey | Examining the role of Zfrp8 with respect to iron/heme homeostasis and ecdysone production | |
| Ingersoll | Elucidating the Function of an Evolutionarily Conserved Col2α1 Isoform during Vertebrate Development | |
| TWI402343B (en) | A novel muscle enhancer sequence and applications thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20200708 Address after: 200031 building 35, No. 320, Yueyang Road, Xuhui District, Shanghai Applicant after: Center for excellence and innovation of molecular cell science, Chinese Academy of Sciences Address before: 200031, 319 Yueyang Road, Shanghai, Shanghai, Xuhui District Applicant before: SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCES |
|
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20180529 |