CN106070063B - Transgenic zebrafish system and its method for building up with abcb4 - Google Patents
Transgenic zebrafish system and its method for building up with abcb4 Download PDFInfo
- Publication number
- CN106070063B CN106070063B CN201610531446.6A CN201610531446A CN106070063B CN 106070063 B CN106070063 B CN 106070063B CN 201610531446 A CN201610531446 A CN 201610531446A CN 106070063 B CN106070063 B CN 106070063B
- Authority
- CN
- China
- Prior art keywords
- tol2
- abcb4
- plasmid
- egfp
- gene
- 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.)
- Active
Links
- 241000252212 Danio rerio Species 0.000 title claims abstract description 80
- 238000000034 method Methods 0.000 title claims abstract description 45
- 230000009261 transgenic effect Effects 0.000 title claims abstract description 33
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 64
- 239000013612 plasmid Substances 0.000 claims description 104
- 241000894006 Bacteria Species 0.000 claims description 33
- 210000001161 mammalian embryo Anatomy 0.000 claims description 28
- 230000029087 digestion Effects 0.000 claims description 25
- 241000588724 Escherichia coli Species 0.000 claims description 24
- 108020004999 messenger RNA Proteins 0.000 claims description 23
- 238000000605 extraction Methods 0.000 claims description 22
- 239000000243 solution Substances 0.000 claims description 17
- 102000008579 Transposases Human genes 0.000 claims description 14
- 108010020764 Transposases Proteins 0.000 claims description 14
- 238000007877 drug screening Methods 0.000 claims description 14
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 14
- 238000005086 pumping Methods 0.000 claims description 14
- 229960003022 amoxicillin Drugs 0.000 claims description 12
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 239000012634 fragment Substances 0.000 claims description 12
- 238000002347 injection Methods 0.000 claims description 12
- 239000007924 injection Substances 0.000 claims description 12
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 claims description 12
- 238000004064 recycling Methods 0.000 claims description 12
- 230000003321 amplification Effects 0.000 claims description 11
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 10
- 238000000338 in vitro Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 10
- 238000000520 microinjection Methods 0.000 claims description 9
- 238000012216 screening Methods 0.000 claims description 8
- 206010059866 Drug resistance Diseases 0.000 claims description 7
- 102000003960 Ligases Human genes 0.000 claims description 7
- 108090000364 Ligases Proteins 0.000 claims description 7
- 229960000723 ampicillin Drugs 0.000 claims description 7
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 claims description 7
- 239000011259 mixed solution Substances 0.000 claims description 6
- 238000012360 testing method Methods 0.000 claims description 5
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 4
- 239000001110 calcium chloride Substances 0.000 claims description 4
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 4
- 238000010276 construction Methods 0.000 claims description 4
- 108010042407 Endonucleases Proteins 0.000 claims description 3
- 102000004533 Endonucleases Human genes 0.000 claims description 3
- 101150063830 abcB4 gene Proteins 0.000 claims description 3
- 239000000499 gel Substances 0.000 claims description 3
- 238000012856 packing Methods 0.000 claims description 3
- 238000004445 quantitative analysis Methods 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- 238000004458 analytical method Methods 0.000 claims description 2
- 238000013518 transcription Methods 0.000 claims description 2
- 230000035897 transcription Effects 0.000 claims description 2
- 230000009466 transformation Effects 0.000 claims description 2
- 108010048367 enhanced green fluorescent protein Proteins 0.000 claims 3
- 230000036457 multidrug resistance Effects 0.000 abstract description 22
- 230000014509 gene expression Effects 0.000 abstract description 16
- 230000007246 mechanism Effects 0.000 abstract description 8
- 102000004169 proteins and genes Human genes 0.000 abstract description 7
- 101150119038 ABCB1 gene Proteins 0.000 abstract description 6
- 108700007489 zebrafish Abcb4 Proteins 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 27
- 239000003814 drug Substances 0.000 description 27
- 230000008569 process Effects 0.000 description 21
- 206010028980 Neoplasm Diseases 0.000 description 19
- 238000010367 cloning Methods 0.000 description 19
- 239000000047 product Substances 0.000 description 18
- 238000011160 research Methods 0.000 description 16
- 102100033350 ATP-dependent translocase ABCB1 Human genes 0.000 description 15
- 229940079593 drug Drugs 0.000 description 15
- 108010047230 Member 1 Subfamily B ATP Binding Cassette Transporter Proteins 0.000 description 11
- 239000000872 buffer Substances 0.000 description 10
- 230000000694 effects Effects 0.000 description 10
- 101100321990 Homo sapiens ABCD4 gene Proteins 0.000 description 9
- 238000011161 development Methods 0.000 description 8
- 230000018109 developmental process Effects 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 241000251468 Actinopterygii Species 0.000 description 7
- 210000000941 bile Anatomy 0.000 description 7
- 239000002609 medium Substances 0.000 description 7
- 238000010171 animal model Methods 0.000 description 6
- 230000008499 blood brain barrier function Effects 0.000 description 6
- 238000005119 centrifugation Methods 0.000 description 6
- 239000002699 waste material Substances 0.000 description 6
- 108010078791 Carrier Proteins Proteins 0.000 description 5
- 241000283070 Equus zebra Species 0.000 description 5
- 210000000013 bile duct Anatomy 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 238000009630 liquid culture Methods 0.000 description 5
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 5
- 210000004881 tumor cell Anatomy 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 239000012124 Opti-MEM Substances 0.000 description 4
- 210000001218 blood-brain barrier Anatomy 0.000 description 4
- 238000004925 denaturation Methods 0.000 description 4
- 230000036425 denaturation Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 210000005229 liver cell Anatomy 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 3
- 229910021529 ammonia Inorganic materials 0.000 description 3
- QGZKDVFQNNGYKY-UHFFFAOYSA-N ammonia Natural products N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 3
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 229940041181 antineoplastic drug Drugs 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 239000003613 bile acid Substances 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 231100000167 toxic agent Toxicity 0.000 description 3
- 101150031010 Abcb1a gene Proteins 0.000 description 2
- 108700028369 Alleles Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000448255 Congiopodus peruvianus Species 0.000 description 2
- 206010064571 Gene mutation Diseases 0.000 description 2
- 108060001084 Luciferase Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- 101100400864 Mus musculus Abcb1a gene Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 2
- 206010061902 Pancreatic neoplasm Diseases 0.000 description 2
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 2
- 208000006735 Periostitis Diseases 0.000 description 2
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000007622 bioinformatic analysis Methods 0.000 description 2
- 210000005013 brain tissue Anatomy 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 230000004069 differentiation Effects 0.000 description 2
- 210000002889 endothelial cell Anatomy 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 239000010977 jade Substances 0.000 description 2
- 210000003460 periosteum Anatomy 0.000 description 2
- YNPNZTXNASCQKK-UHFFFAOYSA-N phenanthrene Chemical compound C1=CC=C2C3=CC=CC=C3C=CC2=C1 YNPNZTXNASCQKK-UHFFFAOYSA-N 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 239000000700 radioactive tracer Substances 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000009897 systematic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- 101710153593 Albumin A Proteins 0.000 description 1
- 208000019838 Blood disease Diseases 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 206010009944 Colon cancer Diseases 0.000 description 1
- 239000010282 Emodin Substances 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 244000153234 Hibiscus abelmoschus Species 0.000 description 1
- 241000254158 Lampyridae Species 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 241000242743 Renilla reniformis Species 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 108700009124 Transcription Initiation Site Proteins 0.000 description 1
- 108700025716 Tumor Suppressor Genes Proteins 0.000 description 1
- 102000044209 Tumor Suppressor Genes Human genes 0.000 description 1
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 description 1
- 210000004100 adrenal gland Anatomy 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000000711 cancerogenic effect Effects 0.000 description 1
- 231100000315 carcinogenic Toxicity 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 231100000481 chemical toxicant Toxicity 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- VNFPBHJOKIVQEB-UHFFFAOYSA-N clotrimazole Chemical compound ClC1=CC=CC=C1C(N1C=NC=C1)(C=1C=CC=CC=1)C1=CC=CC=C1 VNFPBHJOKIVQEB-UHFFFAOYSA-N 0.000 description 1
- 229960004022 clotrimazole Drugs 0.000 description 1
- 210000001072 colon Anatomy 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 239000002619 cytotoxin Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000009509 drug development Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- RHMXXJGYXNZAPX-UHFFFAOYSA-N emodin Chemical compound C1=C(O)C=C2C(=O)C3=CC(C)=CC(O)=C3C(=O)C2=C1O RHMXXJGYXNZAPX-UHFFFAOYSA-N 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000001952 enzyme assay Methods 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 208000010824 fish disease Diseases 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 230000004907 flux Effects 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000003209 gene knockout Methods 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 208000014951 hematologic disease Diseases 0.000 description 1
- 210000005096 hematological system Anatomy 0.000 description 1
- 208000018706 hematopoietic system disease Diseases 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 201000001441 melanoma Diseases 0.000 description 1
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- UPSFMJHZUCSEHU-JYGUBCOQSA-N n-[(2s,3r,4r,5s,6r)-2-[(2r,3s,4r,5r,6s)-5-acetamido-4-hydroxy-2-(hydroxymethyl)-6-(4-methyl-2-oxochromen-7-yl)oxyoxan-3-yl]oxy-4,5-dihydroxy-6-(hydroxymethyl)oxan-3-yl]acetamide Chemical compound CC(=O)N[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](O)[C@@H](NC(C)=O)[C@H](OC=2C=C3OC(=O)C=C(C)C3=CC=2)O[C@@H]1CO UPSFMJHZUCSEHU-JYGUBCOQSA-N 0.000 description 1
- HYIMSNHJOBLJNT-UHFFFAOYSA-N nifedipine Chemical compound COC(=O)C1=C(C)NC(C)=C(C(=O)OC)C1C1=CC=CC=C1[N+]([O-])=O HYIMSNHJOBLJNT-UHFFFAOYSA-N 0.000 description 1
- 229960001597 nifedipine Drugs 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 201000002528 pancreatic cancer Diseases 0.000 description 1
- 230000001991 pathophysiological effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 229960003048 vinblastine Drugs 0.000 description 1
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 description 1
- 229960004528 vincristine Drugs 0.000 description 1
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 1
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- 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
-
- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/89—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
-
- 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
- A01K2267/0393—Animal model comprising a reporter system for screening tests
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Environmental Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a kind of transgenic zebrafish system and its method for building up, the transgenic zebrafish system be premised on not changing zebra fish abcb4 gene expression, and by enhanced green fluorescence protein (EGFP) monitor abcb4 changes in gene expression situation transgenic zebrafish system.That is the transgenic zebrafish system is Tg (abcb4:EGFP) the transgenic zebrafish system with abcb4 gene promoter to drive EGFP to express.The present invention establishes one premised on not changing zebra fish abcb4 gene (with derived from mankind ABCB1 gene) expression, and the transgenic zebrafish system of abcb4 changes in gene expression situation is monitored by enhanced green fluorescence protein (EGFP).Tg (abcb4:EGFP) the transgenic zebrafish system of EGFP expression is driven with abcb4 gene promoter.Previous experiments basis is provided in multidrug resistance Mechanism Study for next abcb4 gene.
Description
Technical field
The present invention relates to a kind of transgenic zebrafish system and its method for building up, belong to gene engineering technology field.
Background technique
Multidrug resistance (multidrug-resistance, MDR) is the common cause of chemotherapy of tumors failure, however for mesh
Before for, chemotherapy remains unchanged the main method for the treatment of of cancer.Therefore on judging cancer prognosis and survival rate, multidrug resistance whether production
It generates for important index.And the definition of multidrug resistance refers to, a variety of drugs different for structure and pharmacology, tumour is simultaneously
Produce drug resistance.There are many mechanism that multidrug resistance generates, and traditional viewpoint is considered because certain cells in tumour occur
Caused by gene mutation, but some pathophysiologic factors, such as anoxic, regulation oncogene, tumor suppressor gene, cell wither
The variation for dying the factor also all may generation of the promotion to multidrug resistance.However, by ATP combination box (ATP-binding
Cassette, ABC) transport protein family mediated cell drug outlet effect, be only multidrug resistance generation main mechanism.
ABCB1 is as one of ATP combination box (ATP-binding cassette, ABC) transporter member, in physiological status
Under, it is primarily involved in absorption, distribution, the excretion etc. of inside and outside source substance and toxin, protection and defense reaction are risen to histoorgan,
It is distributed mainly on the surface epithelial cell top layer of mammal, including colon, small intestine, pancreas, bile duct, bile, renal proximal are small
Pipe, adrenal gland etc..It is also distributed in the endothelial cell of blood-brain barrier (bloodbrain barrier, BBB), can be by endothelial cell
Toxin and excretion of drug into lumen of vessels, to protect brain tissue.
Numerous studies show that the high expression of ABCB1 is generally existing in the tumour cell for generating multidrug resistance, especially exist
In hematological system tumor and entity tumor.The mRNA expression of ABCB1 in patient tumors cell is detected, finds that multiple medicine easily occurs
In drug resistant patient, the mRNA expression of ABCB1 is all higher than other patients.And a large amount of Vitro Tumor drug-resistant cell strains
In studies have shown that ABCB1 gene and its coding albumen be all increased significantly in tumor drug resistance cell, in multidrug resistance
In play key effect.
Zebra fish ABCD4 gene promoter and its prediction and cloning process are similarly ATP combination box (ATP-binding
One of cassette, ABC) transporter member, zebra fish ABCD4 gene promoter and its prediction and cloning process gene are located at people
On No. 7 chromosome of class.Liver cell is mainly acted on the outlet of various compounds by abc transport body protein mediate transport.
And zebra fish ABCD4 gene promoter and its prediction and cloning process are present on the small periosteum of the liver cell of the liver of the mankind,
It is same that energy is obtained by hydrolysising ATP, phosphatidyl choline (phosphatidylcholine, PC) is penetrated into lobuli hepatis bile duct
Film is transported into bile and forms protomere with bile acid, so as to protect bile duct not by damage caused by bile acid, just
Important role in normal bile.
While studying tumor multi-medicine drug-resistant mechanism, the always numerous scholars of tumor multi-medicine drug-resistant how are overcome the problems, such as
The problem of extensive concern.Especially during drug development, the multidrug resistance formation of anti-tumor drug how is predicted
Possibility? therefore the primary dcreening operation research for using ABCB1 to carry out drug resistance as the target spot that multidrug resistance is screened has become hot spot.Such as swollen
ABCB1 is over-expressed in tumor cell strain, so that the cell strain is judged its curative effect as the target spot of screening anti-tumor medicine, and in body
In outer tumor cell line, studies for the inhibitor of ABCB1 and its related abc transport albumen and overcome tumor multi-medicine drug-resistant.Afterwards
The current approved of person carries out clinical research, and clinical application effect is still unsatisfactory.
In Research of Animal Model for Study, early in 1994, the Schinkel etc. of Dutch institute of oncology just established Abcb1a gene
The mouse of (with mankind ABCB1 gene is derived from) deletion allele.By the mouse and normal mouse it was found that, P-gp is big
Amount is distributed in blood-brain barrier (bloodbrain barrier, BBB)；Meanwhile the drug concentration in many tissues of the mouse is bright
It shows and increases, especially brain tissue.And drug itself clearance rate is significantly reduced.And about P-gp Abcb1a(-/-) mouse
In research, so far still be concerned.And the ABCB1(or P-gp being directed to) inhibitor research and development, also on mouse also
It carries out.And zebra fish abcd4 gene promoter and its prediction and cloning process gene are (the same as derived from mankind zebra fish ABCD4 gene
Promoter and its prediction and cloning process gene) deletion allele mouse, also established in 1993 by Smit etc., by this
Mouse and normal mouse it was found that, this mouse cannot secrete lecithin into human bile.And zebra fish abcd4 gene so far
Promoter and its prediction and cloning process (-/-) mouse model still as research liver, pancreatic neoplasm occur, be in progress in it is related
The tool of carcinogenic factor be equally also concerned.
However, there are still many limitations for ongoing research at present.The in vitro study of tumor cell line is relative to internal
Not system, cannot effect in systematic reaction ABCB1 genosome in multidrug resistance so that tumour medicine multidrug resistance is sieved
The one-sidedness of choosing.And use the modeling of small muroid mammal since the growth of animal period is long, it continuous can not be stained with effectively tracer
The expression of gene and the differentiation and development process of cell are contaminated, Abcb1a gene is similarly and is brought in the screening of tumour medicine multidrug resistance
Very big difficulty.So still lacking effective high-throughput, living body animal sieve in the screening process of tumour medicine multidrug resistance
Modeling type, therefore effective, high-throughput, living body a, multidrug resistance screening animal model is established, being one has novelty
Research work, not only can deeply systematically carry out ABCB1 gene and its encode albumin A BCB1(or P-gp) function grind
Study carefully, simultaneously for the targeted approach and drug for being directed to multidrug resistance is found, develops selective height, toxic side effect is small and is not easy to send out
The anti-cancer drugs of raw multidrug resistance provides important living body high flux screening model, with important scientific value and huge answers
Use prospect.
Zebra fish (zebrafish) is " four-dimension " model organism emerging in recent years, is the bony fish for originating in India
Class.The advantages that having embodied small in size, easy absorption and relatively economical in high-throughput, small molecule screening, and reproductive capacity is strong (every
200 pieces of embryo or so can be produced week), growth and development it is fast (can grow up within 2 months), this is not available for other experimental animals.Spot
Horse fish has also embodied specific advantage in transgenosis sieve, gene knockout, gene mutation, is research vertebrate animal development mechanism
And its idealized model biology of gene function.Zebra fish during evolution, protect by development height as with other vertebrates
It keeps, is model organism important in current biological development research.Meanwhile zebra fish is as emerging model organism, it complete
Gene order-checking work has been completed, and is had the development highly conserved with the mankind and disease gene and signal transduction pathway, is
It studies human developmental and disease signal conducting path and in vivo carries out the optimal mode biology of high-throughput lead drug screening
One.
Zebra fish is increasingly becoming the advantage experiment model biology of life science since the last century end.In recent years, spot
The Application and research progress of horse fish is especially rapid.Current many zebra fish disease models are it has been established that be related to heart disease, white blood
Disease and multiple organ tumour include melanoma, cancer of pancreas, liver cancer and colon cancer etc..These disease models can help researcher
Go deep into the mechanism and intervention means of systematically study of various disease development, current this kind of research achievement emerges one after another, public
Think the research tool of life science hot topic.
Bioinformatic analysis confirms that abcb1 gene is not present in zebra fish, but has homology with mankind's ABCB1 gene
Zebra fish abcd4 gene promoter and its prediction and the research of cloning process, abcb5 gene and its related gene but reported
Road.Root according to foreign literature, zebra fish zebra fish abcd4 gene promoter and its prediction and cloning process in fish body in and
A variety of toxic compounds transhipments are closely related, and participate in hindering the absorption of chemical substance.And mankind's zebra fish ABCD4 gene promoter
Son and its prediction and cloning process are similarly one of ATP combination box (ATP-binding cassette, ABC) transporter member,
Zebra fish ABCD4 gene promoter and its prediction and cloning process encoding gene are located on No. 7 chromosome of the mankind.Liver cell pair
In the outlet of various compounds, mainly acted on by abc transport body protein mediate transport.And zebra fish ABCD4 gene promoter
And its prediction and cloning process are present on the small periosteum of the liver cell of the liver of the mankind, it is same that energy is obtained by hydrolysising ATP,
Phosphatidyl choline (phosphatidylcholine, PC) is penetrated into lobuli hepatis bile duct film, is transported into bile and and bile acid
Form protomere, so as to protect bile duct not by damage caused by bile acid, the important role in normal bile.But
Phosphatidyl choline or bile are not detected at present in zebra fish, therefore compared to mankind's zebra fish ABCD4 gene promoter
And its prediction and cloning process, zebra fish zebra fish abcd4 gene promoter and its prediction and the function of cloning process may be more
It is similar to ABCB1 and is different from zebra fish ABCD4 gene promoter and its prediction and cloning process.And abcb5 gene is come
It says, the drugs such as nifedipine, clotrimazole, rheum emodin also can induce the mRNA of abcb5 gene and protein expression level increases.But
It is the zebra fish abcd4 gene promoter in the research of the drugs such as vincristine, vinblastine, Moschus and phenanthrene and compound
And its prediction and cloning process are reported as the cell toxicant transporter for fighting chemical toxicant.It is mentioned that passing through mortifier
With the zebra fish abcd4 gene promoter of Morpholino downward zebra fish and its prediction and cloning process gene, can increase
Cumulant of the fluorogenic substrate in embryonic tissue and the sensibility to toxic compounds, but be but not present compared to abcb5 gene
This influence.So zebra fish abcd4 gene promoter and its prediction and cloning process, abcb5 gene are resistance in zebra fish multiple medicine
Role in medicine mechanism is not fully understood.And zebra fish abcd4 gene promoter based on multidrug resistance research and
It predicts that there has been no foundation with cloning process transgenic zebrafish animal model.
Summary of the invention
The main technical problem to be solved in the present invention is to provide the transgenic zebrafish system and its foundation side of a kind of band abcb4
Method by predicting, cloning zebra fish abcd4 gene promoter, and constructs transgenic zebrafish system, so that establishing one kind can be with
For assessing the animal model of tumor drug resistance.
Technical solution of the present invention includes the following contents:
Firstly, carrying out the prediction of zebra fish abcb4 gene promoter and clone, and carry out zebra fish abcb4 gene promoter
After surveying effect, establishes Tg (abcb4:EGFP) transgenic zebrafish and it is identified, so that obtaining one kind can be used in assessing
The animal model of anti-tumor drug drug resistance.
Specifically, transgenic zebrafish system of the invention is such that the transgenic zebrafish system is not change zebra
Fish abcb4 gene expression premise, and abcb4 changes in gene expression situation is monitored by enhanced green fluorescence protein (EGFP)
Transgenic zebrafish system.The transgenic zebrafish system be with abcb4 gene promoter come drive EGFP express Tg (abcb4:
EGFP) transgenic zebrafish system.
The method for building up of the transgenic zebrafish system, includes the following steps:
Step 1: the building of the Tol2 recombinant plasmid with abcb4 gene promoter；
Step 2:Tol2 transposase mRNA is transcribed in vitro；
Step 3: by transposons Tol2 system, carrying out embryo's microinjection.
Wherein, abcb4 gene promoter Tol2 construction of recombinant plasmid follows the steps below in step 1:
(1) by Calcium Chloride Method, E.coli DH5 α is prepared into competence bacteria；
(2) it by transform mode, is transferred into E.coli DH5 α competence bacteriaTol2Plasmid, and amoxicillin is anti-
Property drug screening, selects positive colony；
(3) monoclonal for choosing the amicillin resistance positive, moves to 3ml added with the LB Liquid Culture of ampicillin
In base, be placed on constant-temperature table, 190rpm, 37 DEG C shaken cultivation 10 ~ 14 hours；
(4) bacterium solution being incubated overnight is taken out to take by the small pumping kit of Axygen plasmid and propose amplificationpCS2 + Plasmid
DNA；
(5) EGFP gene PCR fragment and extraction are obtainedTol2Plasmid carries out respectivelyNheI andEcoR It is double
Digestion；
(6) after digestion, the EGFP gene PCR fragment of digestion and extraction are obtainedTol2Plasmid is cut
Glue recycling；
(7) by the warp of recyclingNheI andEcoRWhat the EGFP gene PCR fragment of double digestion and extraction obtainedTol2
Plasmid passes through T4 Ligase is attached, and 25 DEG C of connections are overnight；
(8) connection product is transferred to by way of conversion in E.coli DH5 α competence bacteria, and amoxicillin
Resistance drug screening, selects positive colony；Plasmid DNA is extracted with the small pumping kit of Axgen plasmid after bacterium colony amplification；
(9) Tol2-EGFPThe identification of recombinant plasmid:
(10) by transform mode, it is correct that identification is transferred into E.coli DH5 α competence bacteriaTol2-EGFPPlasmid,
And amoxicillin resistance drug screening, select positive colony；
(11) monoclonal for choosing the amicillin resistance positive, moves to 3ml added with the LB Liquid Culture of ampicillin
In base, be placed on constant-temperature table, 190rpm, 37 DEG C shaken cultivation 10 ~ 14 hours；
(12) it takes out the bacterium solution being incubated overnight and amplification is extracted by the small pumping kit of Axygen plasmidTol2-EGFP
Plasmid；
(13) willabcb4What gene promoter PCR product and extraction obtainedTol2-EGFPPlasmid carries out respectivelyXhoI
AndNheI double digestion；
(14) after digestion, by digestionabcb4What gene promoter PCR product and extraction obtainedTol2- EGFPPlasmid is tapped and recovered；
(15) by the warp of recyclingXhoI andNheI double digestionabcb4Gene promoter PCR product and extraction obtain
'sTol2-EGFPPlasmid passes through T4 Ligase is attached, and 25 DEG C of connections are overnight；
(16) connection product is transferred to by way of conversion in E.coli DH5 α competence bacteria, and amoxicillin
Resistance drug screening, selects positive colony.Plasmid DNA is extracted with the small pumping kit of Axgen plasmid after bacterium colony amplification；
(17) Tol2-abcb4:EGFPThe identification of recombinant plasmid.
Wherein, Tol2 transposase mRNA in-vitro transcription follows the steps below in step 2:
(1) willpCS-TPRecombinant plasmid linearization plasmid DNA after NotI single endonuclease digestion, the identification of 1% agarose gel electrophoresis
Afterwards, then be tapped and recovered linearisation after Plasmid DNA；
It (2) will linearisationpCS-TPPlasmid was transcribed in vitro as template using Sp6 polymerase, through 2 hours, 37 DEG C
After reaction, Tol2 transposase mRNA is obtained；
(3) through NucAwayTMSpin Columns purify adsorption column recycling mRNA after, through 2% agarose gel electrophoresis into
Row qualitative analysis, and quantitative analysis is carried out through One Drop instrument, it will be saved after packing mRNA in -70 DEG C.
Wherein, the embryo's microinjection of transposons Tol2 system follows the steps below in step 3:
(1) willTol2-abcb4:EGFPRecombinant plasmid dna and Tol2 transposase mRNA are purified and are mixed, on ice to
With；
(2) normotrophic wild-type zebrafish one cell stage is chosen, is placed in Ago-Gel injection plate；
(3) zebrafish embryo finished will be put to be placed under stereomicroscope, injection needle draws about 2 μ of mixed solution
l；
(4) by microinjection instrument, into the cell of zebra fish one cell stage, with about 0.005 μ l of every piece of embryo
InjectionTol2-abcb4:EGFPRecombinant plasmid dna and Tol2 transposase mRNA mixed solution；
(5) zebrafish embryo that finishes of injection is raised, used in testing later.
The present invention establishes one premised on not changing zebra fish abcb4 gene (with derived from mankind ABCB1 gene) expression,
And the transgenic zebrafish system of abcb4 changes in gene expression situation is monitored by enhanced green fluorescence protein (EGFP).I.e. with
Abcb4 gene promoter come drive EGFP express Tg (abcb4:EGFP) transgenic zebrafish system.For next abcb4 base
Because providing previous experiments basis in multidrug resistance Mechanism Study.Also solving ongoing research at present simultaneously, there are still many offices
It is sex-limited.It, cannot be more in systematic reaction ABCB1 genosome if the in vitro study of tumor cell line is relative to not system in vivo
Effect in medicine drug resistance, so that the one-sidedness of tumour medicine multidrug resistance screening.And mouse quasi-mode animal is used to model, exist
The growth of animal period is long, can not it is continuous and effectively the differentiation and development process of the expression of tracer rotaring redyeing gene and cell the defects of.
Detailed description of the invention
Fig. 1 is Tol2(abcb4-EGFP) recombinant plasmid figure；
Fig. 2 is transgenic zebrafish(F 1 ) tg(Abcb4:EGFP) fluorescence shows schematic diagram.
Specific embodiment
The present invention is described in further detail with reference to the accompanying drawings and examples.
Embodiment 1:
1 zebra fishabcb4Gene promoter prediction and clone
1.1 zebra fishabcb4Gene promoter prediction
According to the website NCBI, by bioinformatic analysis, by zebra fishabcb4Gene transcription start site is to upstream
The gene order of about 8000bp or so is as template between adam15 gene, through 2.0 Prediction Server of Promoter
Promoter prediction website carries out promoter horizon prediction.
1.2 zebra fishabcb4Gene promoter clone
(1) about 20 ~ 30 pieces of 5dpf zebrafish embryo are chosen, is put into the 1.5ml Epp pipe of RNAse-Free, 1 × PBS
Solution cleans embryo；
(2) after cleaning, 500 μ l Lysis Buffer(is added and wherein contain Proteinase K, final concentration of 0.17mg/
Ml), 55 DEG C of water-baths digest 1 hour after sealing；
(3) room temperature 14000rpm is centrifuged 10 minutes, takes supernatant into new RNAse-Free 1.5ml Epp pipe；
(4) 500 μ l phenol/chloroform mixed liquor (1:1) is added in Xiang Shangshu Epp pipe, acutely concussion 15 seconds, room temperature
14000rpm is centrifuged 10 minutes；
(5) supernatant is taken, is moved in the 1.5ml Epp pipe of new RNAse-Free, 900 μ l 75% being pre-chilled on ice are added
+ 100 μ l sodium acetate of ethyl alcohol (concentration 3M, PH6.0), it is soft to mix, it places on ice 10 minutes, 14000rpm, 4 DEG C and is centrifuged 30 points
Clock；
(6) supernatant to be abandoned, 70% ethyl alcohol that 1ml is pre-chilled on ice is added, after washing precipitates, 7500rpm, 4 DEG C are centrifuged 5 minutes,
This step repeats twice；
(7) abandon supernatant, drying at room temperature after twenty minutes, adds 50 μ l DEPC water, after sealing in 55 DEG C water-bath 10 minutes, it is heavy to help
It forms sediment and dissolves；
(8) 1 μ l genome DNA sample is taken, by 1% agarose gel electrophoresis, detects extracted DNA fragmentation size feelings
Condition.It is saved after detection in -70 DEG C of refrigerators.
(9) it according to estimation range, and is addedXhoI、NheI restriction enzyme site and protection base sequence pass through Primer
6.0 software design specific primer of Premier, and synthesized by Beijing Ao Weisen Gene Tech. Company Limited.Sequence is as follows: F:
5’- CCGCTCGAGGGCCTGGAAACATTTCTTC-3’
R: 5’-CTAGCTAGCTTGCTGTTATCTCAGATGCT-3’
(10) it using the zebra fish genomic DNA of extraction as template, carries outabcb4Gene promoter PCR amplification.Condition is such as
Under: 94 DEG C of initial denaturation 2min；94 DEG C of denaturation 15s, 60 DEG C of annealing 30s, 68 DEG C of extension 1min are recycled for 35 times totally；Finally extend 68 DEG C
10min.PCR product size is about 1532bp, and 1% agarose gel electrophoresis is identified.
2 zebra fishabcb4Gene promoter surveys effect
2.1 pGL3-abcb4PConstruction of recombinant plasmid
(1) by Calcium Chloride Method, E.coli DH5 α is prepared into competence bacteria；
(2) it by transform mode, is transferred into E.coli DH5 α competence bacteriapGL3-basicPlasmid, and it is green through ammonia benzyl
Chloramphenicol resistance drug screening, selects positive colony.Transform mode is as follows:
1) it in -70 DEG C of refrigerators, takes the 0.6ml Epp equipped with 200 μ l E.coli DH5 α competence bacterias to manage, is placed in ice
It is upper to melt 12 minutes；
2) after wait melt, into E.coli DH5 α competence bacteria, 0.5 μ l is addedpGL3-basicPlasmid, and pressure-vaccum
It mixes, is placed in 30 minutes on ice；
3) on ice after 30 minutes, the Epp seal of tube is placed in 42 DEG C of water baths, after heat shock 90 seconds, is immediately inserted into ice
In 5 minutes；
4) 300 μ l non-resistant LB liquid mediums are added into Epp pipe, are placed on constant-temperature table, 160rpm, 37 DEG C of temperature
It educates 45 minutes；
5) after mixing well, by way of streak inoculation, E.coli DH5 α competence bacteria is applied to green added with ammonia benzyl
In the LB solid medium tablets of mycin；
6) it is just putting after five minutes, 37 DEG C of inversion overnight incubations；
(3) monoclonal for choosing the amicillin resistance positive, moves to 3ml added with the LB Liquid Culture of ampicillin
In base, be placed on constant-temperature table, 190rpm, 37 DEG C shaken cultivation 10 ~ 14 hours；
(4) it takes out the bacterium solution being incubated overnight and amplification is extracted by the small pumping kit of Axygen plasmidpCS2 + Plasmid
DNA.Method is as follows:
1) take 3ml bacterium solution into 1.5ml Epp pipe, 12000rpm is centrifuged 1 minute；
2) supernatant liquor is abandoned, 250 μ l are added and are resuspended through the processed Buffer S1 of RNase A；
3) after being sufficiently resuspended, 250 μ l Buffer S2 are added immediately, up and down mild overturning 4 ~ 6 times；
4) after solution is bright, 350 μ l Buffer S3 are added, after equally up and down mild overturning 6 ~ 8 times, 12000rpm
Centrifugation 15 minutes；
5) supernatant liquor is drawn, moves to and puts on the DNA of recovery tube and prepare in pipe, 12000rpm is centrifuged 1 minute；
6) waste liquid is discarded, and 500 μ l Buffer W1,12000rpm is added centrifugation 1 minute；
7) waste liquid is discarded, and 700 μ l Buffer W2,12000rpm is added centrifugation 1 minute；This step is repeated twice；
8) after discarding waste liquid, 12000rpm sky throws away the heart 1 minute；
9) recovery tube is changed to new 1.5ml Epp to manage, 50 μ l DEPC water is added in the film center that DNA prepares pipe, it is quiet
Only after 2 minutes, 12000rpm is centrifuged 1 minute；
10) preparation pipe is abandoned, 1 μ l is drawn from Epp pipe and extracts product, is quantified through One Drop, and with 1% agarose
Gel carries out electroresis appraisal；
(5) willabcb4What gene promoter PCR product and extraction obtainedpGL3-basicPlasmid carries out respectivelyNheI
AndXhoI double digestion；
(6) after digestion, by digestionabcb4What gene promoter PCR product and extraction obtainedpGL3- basicPlasmid is tapped and recovered, and specific method is the same；
(7) by the warp of recyclingNheI andXhoI double digestionabcb4Gene promoter PCR product and extraction obtain
'spGL3-basicPlasmid passes through T4 Ligase is attached, and 25 DEG C of connections are overnight；
(8) connection product is transferred to by way of conversion in E.coli DH5 α competence bacteria, and amoxicillin
Resistance drug screening, selects positive colony.Plasmid DNA, specific side are extracted with the small pumping kit of Axgen plasmid after bacterium colony amplification
Method is the same；
(9) pGL3-abcb4PThe identification of recombinant plasmid:
1) it passes throughNheI andXhoI double digestionpGL3-abcb4PRecombinant plasmid carries out digestion identification, and passes through 1% fine jade
Whether sepharose electroresis appraisal target DNA fragment size is correct；
2) willpGL3-abcb4PRecombinant plasmid send to Beijing Ao Weisen Gene Tech. Company Limited and carries out sequencing mirror
It is fixed.
2.2 K562 cytolipin plasmidspGL3-abcb4PRecombinant plasmid
Using 1640 culture medium of Gibco RPMI (it contains 10% fetal calf serum) to K562 cell in 5%CO2,37 DEG C,
Cell culture is carried out under conditions of saturated humidity.Cell suspension growth in culture medium chooses logarithmic growth phase cell for real
It tests.
(1) Lipofectamine is taken®2000 Reagent, 10 μ l adds to Opti-MEM®It is mixed in 100 μ l of Medium
It is even.
(2) two groups of plasmids are taken (i.e.pGL3-basicWithpRL-SV40Plasmid group,pGL3-abcb4PWithpRL-SV40Plasmid
Group) every kind of plasmid 500ng of each 1 μ g() it is separately added into 50 μ l Opti-MEM® It is mixed in Medium.
(3) it takes and mixes each 50 μ l of reagent in step (1), mixed respectively with each 50 μ l of two mixing groups in step 2, mixed simultaneously
It is incubated at room temperature 5min.
(4) after being centrifuged K562 cell with 800rpm/min, Opti-MEM is used®Medium is resuspended.Be resuspended sufficiently after with
Every hole 2 × 105A cell inoculation is in 24 orifice plates.Two plasmid groups, every group of 5 holes.
(5) Incubating Solution in step 3 is separately added into step 4 in corresponding aperture in 25min, Incubating Solution 100 is added in every hole
μl.Guarantee that amount is about 500ng, Lipofectamine to every kind of every hole plasmid eventually®The whole amount in the every hole 2000 Reagent about 1.0 ~
2.5μl.After gently shaking up, 37 DEG C are placed in, 4 ~ 6h is cultivated in 5%CO2.Opti-MEM is mended in every hole later® Medium 500μ
L continues to be placed in 37 DEG C, cultivates 3 days in 5%CO2.It is carried out after 3 daysabcb4Gene promoter surveys effect analysis.(note: culture body
It is not plus dual anti-in system, do not influence experimental result.)
K562 cell fluorescence element enzyme assay after 2.3 transfections
(1) by two groups of K562 cells of plasmid transfection in 24 orifice plates, and the K562 cell normally cultivated is from constant temperature incubator
It takes out, it is each to draw into sterile 25ml centrifuge tube, cell is cleaned using 1 × PBS solution after being centrifuged with 800rpm/min, 2 times；
(2) Gibco is respectively added®1640 Medium of RPMI is resuspended, and is added lighttight 96 after being resuspended sufficiently with 75 μ l
In orifice plate, every group of every hole cell measures about 1 × 10 eventually4A cell；
(3) 75 μ l Dual-Glo are added into every hole®Luciferase Reagent, mixes well；
(4) it is incubated for 1.5 hours to 25 DEG C, after allowing cell sufficiently to crack, in BioTek Synergy2 multi-function microplate reader
Middle measurement firefly fluorescent；
(5) 75 μ l Dual-Glo are added into every hole® Stop & Glo® Reagent is mixed well；
After (6) 25 DEG C are incubated for 1.5 hours, sea pansy fluorescent is measured in BioTek Synergy2 multi-function microplate reader；
(7) sample data is handled by Gen5 software, calculation formula are as follows:
3 Tg(abcb4:EGFP)The foundation of transgenic zebrafish
3.1 abcb4Gene promoterTol2Construction of recombinant plasmid
(1) by Calcium Chloride Method, E.coli DH5 α is prepared into competence bacteria；
(2) it by transform mode, is transferred into E.coli DH5 α competence bacteriaTol2Plasmid, and amoxicillin is anti-
Property drug screening, selects positive colony.Transform mode is as follows:
1) it in -70 DEG C of refrigerators, takes the 0.6ml Epp equipped with 200 μ l E.coli DH5 α competence bacterias to manage, is placed in ice
It is upper to melt 12 minutes；
2) after wait melt, into E.coli DH5 α competence bacteria, 0.5 μ l is addedTol2Plasmid, and pressure-vaccum mixes,
It is placed in 30 minutes on ice；
3) on ice after 30 minutes, the Epp seal of tube is placed in 42 DEG C of water baths, after heat shock 90 seconds, is immediately inserted into ice
In 5 minutes；
4) 300 μ l non-resistant LB liquid mediums are added into Epp pipe, are placed on constant-temperature table, 160rpm, 37 DEG C of temperature
It educates 45 minutes；
5) after mixing well, by way of streak inoculation, E.coli DH5 α competence bacteria is applied to green added with ammonia benzyl
In the LB solid medium tablets of mycin；
6) it is just putting after five minutes, 37 DEG C of inversion overnight incubations；
(3) monoclonal for choosing the amicillin resistance positive, moves to 3ml added with the LB Liquid Culture of ampicillin
In base, be placed on constant-temperature table, 190rpm, 37 DEG C shaken cultivation 10 ~ 14 hours；
(4) bacterium solution being incubated overnight is taken out to take by the small pumping kit of Axygen plasmid and propose amplificationpCS2 + Plasmid
DNA.Method is as follows:
1) take 3ml bacterium solution into 1.5ml Epp pipe, 12000rpm is centrifuged 1 minute；
2) supernatant liquor is abandoned, 250 μ l are added and are resuspended through the processed Buffer S1 of RNase A；
3) after being sufficiently resuspended, 250 μ l Buffer S2 are added immediately, up and down mild overturning 4 ~ 6 times；
4) after solution is bright, 350 μ l Buffer S3 are added, after equally up and down mild overturning 6 ~ 8 times, 12000rpm
Centrifugation 15 minutes；
5) supernatant liquor is drawn, moves to and puts on the DNA of recovery tube and prepare in pipe, 12000rpm is centrifuged 1 minute；
6) waste liquid is discarded, and 500 μ l Buffer W1,12000rpm is added centrifugation 1 minute；
7) waste liquid is discarded, and 700 μ l Buffer W2,12000rpm is added centrifugation 1 minute；This step is repeated twice；
8) after discarding waste liquid, 12000rpm sky throws away the heart 1 minute；
9) recovery tube is changed to new 1.5ml Epp to manage, 50 μ l DEPC water is added in the film center that DNA prepares pipe, it is quiet
Only after 2 minutes, 12000rpm is centrifuged 1 minute；
10) preparation pipe is abandoned, 1 μ l is drawn from Epp pipe and extracts product, is quantified through One Drop, and with 1% agarose
Gel carries out electroresis appraisal；
(5) EGFP gene PCR fragment and extraction are obtainedTol2Plasmid carries out respectivelyNheI andEcoRDouble enzymes
It cuts；
(6) after digestion, the EGFP gene PCR fragment of digestion and extraction are obtainedTol2Plasmid is cut
Glue recycling, specific method are the same；
(7) by the warp of recyclingNheI andEcoRWhat the EGFP gene PCR fragment of double digestion and extraction obtainedTol2
Plasmid passes through T4 Ligase is attached, and 25 DEG C of connections are overnight；
(8) connection product is transferred to by way of conversion in E.coli DH5 α competence bacteria, and amoxicillin
Resistance drug screening, selects positive colony.Plasmid DNA, specific side are extracted with the small pumping kit of Axgen plasmid after bacterium colony amplification
Method is the same；
(9) Tol2-EGFPThe identification of recombinant plasmid:
1) it passes throughNheI andEcoRDouble digestionTol2-EGFPRecombinant plasmid carries out digestion identification, and passes through 1% fine jade
Whether sepharose electroresis appraisal target DNA fragment size is correct；
2) willTol2-EGFPRecombinant plasmid send to Beijing Ao Weisen Gene Tech. Company Limited and carries out sequencing identification.
(10) by transform mode, it is correct that identification is transferred into E.coli DH5 α competence bacteriaTol2-EGFPPlasmid,
And amoxicillin resistance drug screening, select positive colony；
(11) monoclonal for choosing the amicillin resistance positive, moves to 3ml added with the LB Liquid Culture of ampicillin
In base, be placed on constant-temperature table, 190rpm, 37 DEG C shaken cultivation 10 ~ 14 hours；
(12) it takes out the bacterium solution being incubated overnight and amplification is extracted by the small pumping kit of Axygen plasmidTol2-EGFP
Plasmid；
(13) willabcb4What gene promoter PCR product and extraction obtainedTol2-EGFPPlasmid carries out respectivelyXhoI
AndNheI double digestion；
(14) after digestion, by digestionabcb4What gene promoter PCR product and extraction obtainedTol2- EGFPPlasmid is tapped and recovered, and specific method is the same；
(15) by the warp of recyclingXhoI andNheI double digestionabcb4Gene promoter PCR product and extraction obtain
'sTol2-EGFPPlasmid passes through T4 Ligase is attached, and 25 DEG C of connections are overnight；
(16) connection product is transferred to by way of conversion in E.coli DH5 α competence bacteria, and amoxicillin
Resistance drug screening, selects positive colony.Plasmid DNA, specific side are extracted with the small pumping kit of Axgen plasmid after bacterium colony amplification
Method is the same；
(17) Tol2-abcb4:EGFPThe identification of recombinant plasmid:
1) it passes throughXhoI andNheI double digestionTol2-abcb4:EGFPRecombinant plasmid carries out digestion identification, and passes through 1%
Agarose gel electrophoresis identification target DNA fragment size it is whether correct；
2) willTol2-abcb4:EGFPRecombinant plasmid send to Beijing Ao Weisen Gene Tech. Company Limited and carries out sequence survey
Fixed identification.
3.2 Tol2 transposase mRNA are transcribed in vitro
(1) willpCS-TPRecombinant plasmid linearization plasmid DNA after NotI single endonuclease digestion, the identification of 1% agarose gel electrophoresis
Afterwards, then be tapped and recovered linearisation after Plasmid DNA；
It (2) will linearisationpCS-TPPlasmid was transcribed in vitro as template using Sp6 polymerase, through 2 hours, 37 DEG C
After reaction, Tol2 transposase mRNA is obtained；
(3) through NucAwayTMSpin Columns purify adsorption column recycling mRNA after, through 2% agarose gel electrophoresis into
Row qualitative analysis, and quantitative analysis is carried out through One Drop instrument, it will be saved after packing mRNA in -70 DEG C.
3.3 transposons Tol2 system embryo's microinjections
(1) willTol2-abcb4:EGFPRecombinant plasmid dna and Tol2 transposase mRNA are purified and are mixed.After mixing
The two final concentration is 250ng/ μ l, on ice for use；
(2) normotrophic wild-type zebrafish one cell stage is chosen, is placed in Ago-Gel injection plate；
(3) zebrafish embryo finished will be put to be placed under stereomicroscope, injection needle draws about 2 μ of mixed solution
l；
(4) by microinjection instrument, into the cell of zebra fish one cell stage, with about 0.005 μ l of every piece of embryo
InjectionTol2-abcb4:EGFPRecombinant plasmid dna and Tol2 transposase mRNA mixed solution；
(5) zebrafish embryo that finishes of injection is raised, used in testing later.
4 Tg(abcb4:EGFP)The identification of transgenic zebrafish
4.1 Tg(abcb4:EGFP)Transgenic zebrafish luciferase expression and gene order-checking identification
(1) embryo after microinjection is collected, there are the embryos of green florescent signal by fluorescence microscope selection
It is raised.It is set toTg(abcb4:EGFP)Transgenic zebrafish F0Generation.
(2) then by F0The adult fish in generation mates with wild-type zebrafish adult fish, output F1For zebrafish embryo, choose
There are the F of correct green florescent signal1Genomic DNA is extracted for embryo, according toabcb4:EGFPTransgenosis particular sequence, warp
6.0 software Design primers of Primer Premier carry out PCR identification:
F:5'-GGCCTGGAAACATTTCTTC-3', R:5'-GTCCATGCCGAGAGTGAT-3'
PCR amplification condition: 94 DEG C of initial denaturation 2min；94 DEG C of denaturation 15s, 60 DEG C of annealing 30s, 68 DEG C extension 1min totally 35 times
Circulation；Finally extend 68 DEG C of 10min.PCR product size is that about 2260bp, 1% agarose gel electrophoresis are identified.
(3) again by the correct F of genome identification1Raised for embryo, after for adult fish with wild-type zebrafish adult fish
It mates, by embryo's green fluorescence situation, filters out output F1For homozygote.
(4) pass through F1For the mode of homozygote selfing, output F2For homozygote embryo, there are correct fluorescence signals for selection
F2In generation, carries out same genome identification work.
(5) again by the correct F of genome identification2For homozygote, embryo is raised, and is mated after for adult fish, is produced
F out3For homozygote embryo, there are the F of correct fluorescence signal for selection3Same genome identification is carried out for homozygote embryo.
4.2 Tg(abcb4:EGFP)Transgenic zebrafish functionality Preliminary Identification
Under Stereo microscope, select normotrophicTg(abcb4:EGFP)It is transgenic zebrafish homozygote 4 ~ 8 thin
The fertilized eggs embryo in born of the same parents' stage phase, carries out in 6 well culture plates10 μmolAdriamycin drug-treated, 20 pieces of every hole, 28 DEG C of perseverances
Temperature saves, and exposure duration is 120 hours.
The zebrafish embryo for collecting 48,72,96,120 hours four phases in drug-treated is expressed green fluorescence and is changed
Carry out Preliminary Identification and assessment.It is accurate to ensure to test, it is repeated 3 times under same experimental conditions.
Certainly, above is specific application example of the invention, and there are other embodiments of the invention, all using equivalent
The technical solution that replacement or equivalent transformation are formed, all falls within protection scope of the presently claimed invention.
Claims (4)
1. a kind of method for building up of the transgenic zebrafish system of band abcb4, it is characterised in that include the following steps:
Step 1: the building of the Tol2 recombinant plasmid of the gene promoter containing abcb4；
Step 2:Tol2 transposase mRNA is transcribed in vitro；
Step 3: by transposons Tol2 transformation system, carrying out embryo's microinjection.
2. the method for building up of the transgenic zebrafish system of band abcb4 according to claim 1, it is characterised in that: in step 1
Abcb4 gene promoter Tol2 construction of recombinant plasmid follows the steps below:
(1) by Calcium Chloride Method, bacillus coli DH 5 alpha is prepared as competence bacteria；
(2) it by transform mode, is transferred into bacillus coli DH 5 alpha competence bacteriaTol2Plasmid, and amoxicillin drug
Resistance screening selects positive colony；
(3) monoclonal for choosing the amicillin resistance positive, moves in LB liquid medium of the 3ml added with ampicillin,
Be placed on constant-temperature table, 190rpm, 37 DEG C shaken cultivation 10 ~ 14 hours；
(4) bacterium solution being incubated overnight is taken out to take by the small pumping kit of Axygen plasmid and propose amplificationpCS2 + Plasmid DNA；
(5) EGFP gene PCR fragment and extraction are obtainedTol2Plasmid carries out respectivelyNheI andEcoR Double digestion；
(6) after digestion, the EGFP gene PCR fragment of digestion and extraction are obtainedTol2Plasmid is tapped rubber back
It receives；
(7) by the warp of recyclingNheI andEcoRWhat the EGFP gene PCR fragment of double digestion and extraction obtainedTol2Plasmid,
Pass through T4 Ligase is attached, and 25 DEG C of connections are overnight；
(8) connection product is transferred to by way of conversion in E.coli DH5 α competence bacteria, and amoxicillin resistance
Positive colony is selected in drug screening；Plasmid DNA is extracted with the small pumping kit of Axgen plasmid after bacterium colony amplification；
(9) Tol2-EGFPThe identification of recombinant plasmid:
(10) by transform mode, it is correct that identification is transferred into E.coli DH5 α competence bacteriaTol2-EGFPPlasmid, and pass through
Amicillin resistance drug screening, selects positive colony；
(11) monoclonal for choosing the amicillin resistance positive, moves to 3ml added with the LB liquid medium of ampicillin
In, be placed on constant-temperature table, 190rpm, 37 DEG C shaken cultivation 10 ~ 14 hours；
(12) it takes out the bacterium solution being incubated overnight and amplification is extracted by the small pumping kit of Axygen plasmidTol2-EGFPPlasmid；
(13) willabcb4What gene promoter PCR product and extraction obtainedTol2-EGFPPlasmid carries out respectivelyXhoI andNheI double digestion；
(14) after digestion, by digestionabcb4What gene promoter PCR product and extraction obtainedTol2-EGFPMatter
Grain is tapped and recovered；
(15) by the warp of recyclingXhoI andNheI double digestionabcb4What gene promoter PCR product and extraction obtainedTol2-EGFPPlasmid passes through T4 Ligase is attached, and 25 DEG C of connections are overnight；
(16) connection product is transferred to by way of conversion in E.coli DH5 α competence bacteria, and amoxicillin resistance
Positive colony is selected in drug screening；
Plasmid DNA is extracted with the small pumping kit of Axgen plasmid after bacterium colony amplification；
(17) Tol2-abcb4:EGFPThe identification of recombinant plasmid.
3. the method for building up of the transgenic zebrafish system of band abcb4 according to claim 1, it is characterised in that: in step 2
Tol2 transposase mRNA in-vitro transcription follows the steps below:
(1) willpCS-TPRecombinant plasmid linearization plasmid DNA after NotI single endonuclease digestion, after the identification of 1% agarose gel electrophoresis, then
Plasmid DNA after being tapped and recovered linearisation；
It (2) will linearisationpCS-TPPlasmid is transcribed in vitro as template using Sp6 polymerase, and through 2 hours, 37 DEG C were reacted
Afterwards, Tol2 transposase mRNA is obtained；
(3) through NucAwayTMAfter Spin Columns purifies adsorption column recycling mRNA, determined through 2% agarose gel electrophoresis
Property analysis, and through One Drop instrument carry out quantitative analysis, will packing mRNA after in -70 DEG C save.
4. the method for building up of the transgenic zebrafish system of band abcb4 according to claim 1, it is characterised in that: in step 3
Transposons Tol2 system embryo's microinjection follows the steps below:
(1) willTol2-abcb4:EGFPRecombinant plasmid dna and Tol2 transposase mRNA are purified and are mixed, on ice for use；
(2) normotrophic wild-type zebrafish one cell stage is chosen, is placed in Ago-Gel injection plate；
(3) zebrafish embryo finished will be put to be placed under stereomicroscope, injection needle draws about 2 μ l of mixed solution；
(4) by microinjection instrument, into the cell of zebra fish one cell stage, with about 0.005 μ l of every piece of embryo injectionTol2-abcb4:EGFPRecombinant plasmid dna and Tol2 transposase mRNA mixed solution；
(5) zebrafish embryo that finishes of injection is raised, used in testing later.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610531446.6A CN106070063B (en) | 2016-07-07 | 2016-07-07 | Transgenic zebrafish system and its method for building up with abcb4 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610531446.6A CN106070063B (en) | 2016-07-07 | 2016-07-07 | Transgenic zebrafish system and its method for building up with abcb4 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106070063A CN106070063A (en) | 2016-11-09 |
| CN106070063B true CN106070063B (en) | 2019-03-05 |
Family
ID=57212487
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610531446.6A Active CN106070063B (en) | 2016-07-07 | 2016-07-07 | Transgenic zebrafish system and its method for building up with abcb4 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106070063B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9968077B2 (en) | 2014-11-07 | 2018-05-15 | Glofish Llc | Blue transgenic fluorescent ornamental fish |
| CN108085336A (en) * | 2016-11-21 | 2018-05-29 | 中国科学院上海生命科学研究院 | EGFP expression vector establishments based on col2a1 promoters and the application in transgenic zebrafish |
| SG11201908897PA (en) * | 2017-03-30 | 2019-10-30 | Glofish Llc | Transgenic rainbow shark |
| CN107058386A (en) * | 2017-04-13 | 2017-08-18 | 厦门大学 | A kind of preparation method of transgenic zebrafish |
| CN108642081B (en) * | 2018-03-29 | 2022-05-13 | 苏州木芮生物科技有限公司 | Construction method of luciferase transgenic zebra fish driven by glucocorticoid response original |
| CN108642082B (en) * | 2018-03-29 | 2022-05-13 | 苏州木芮生物科技有限公司 | Construction method of transgenic zebra fish with overexpression of systemic glucocorticoid receptor gene |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1607251A (en) * | 2003-10-16 | 2005-04-20 | 清华大学 | Method for breeding transgenic zebra fish and its special plasmid |
| CN1607252A (en) * | 2003-10-16 | 2005-04-20 | 清华大学 | Method for breeding transgenic zebra fish and its special plasmid |
| CN101696421A (en) * | 2009-10-27 | 2010-04-21 | 中国科学院水生生物研究所 | Recombination gene for strengthening endurance capacity of fishes to low dissolved oxygen stress and application thereof |
| CN103088066A (en) * | 2013-02-07 | 2013-05-08 | 中国科学院水生生物研究所 | Method for controlling fish reproduction |
| CN103224955A (en) * | 2013-05-09 | 2013-07-31 | 中国科学院水生生物研究所 | Vector for efficiently labeling zebra fish PGC, and preparation method and use of transgenic fish |
| CN103667327A (en) * | 2013-12-06 | 2014-03-26 | 中国科学院苏州生物医学工程技术研究所 | Preparation method of transgenetic zebrafish used for detecting environment pollutants |
| CN103805601A (en) * | 2012-11-12 | 2014-05-21 | 中国科学技术大学 | Method for cultivating transgenic zebra fish marking oligodendrocyte of central nervous system and application thereof |
| 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 |
| CN105274140A (en) * | 2015-10-28 | 2016-01-27 | 厦门大学 | Method for building muscle cell-specific expression mCherry fluorescent protein zebra fish family |
| CN105274144A (en) * | 2015-09-14 | 2016-01-27 | 徐又佳 | Preparation method of zebrafish with hepcidin gene knocked out by use of CRISPR / Cas9 technology |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AUPR064600A0 (en) * | 2000-10-09 | 2000-11-02 | Commonwealth Scientific And Industrial Research Organisation | Genetic control of sex ratio in animal populations |
| AU2002224787A1 (en) * | 2000-10-23 | 2002-05-06 | Mermaid Pharmaceuticals Gmbh | Method for temporally controlling antisense-mediated gene inactivation |
-
2016
- 2016-07-07 CN CN201610531446.6A patent/CN106070063B/en active Active
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1607251A (en) * | 2003-10-16 | 2005-04-20 | 清华大学 | Method for breeding transgenic zebra fish and its special plasmid |
| CN1607252A (en) * | 2003-10-16 | 2005-04-20 | 清华大学 | Method for breeding transgenic zebra fish and its special plasmid |
| CN101696421A (en) * | 2009-10-27 | 2010-04-21 | 中国科学院水生生物研究所 | Recombination gene for strengthening endurance capacity of fishes to low dissolved oxygen stress and application thereof |
| CN103805601A (en) * | 2012-11-12 | 2014-05-21 | 中国科学技术大学 | Method for cultivating transgenic zebra fish marking oligodendrocyte of central nervous system and application thereof |
| CN103088066A (en) * | 2013-02-07 | 2013-05-08 | 中国科学院水生生物研究所 | Method for controlling fish reproduction |
| CN103224955A (en) * | 2013-05-09 | 2013-07-31 | 中国科学院水生生物研究所 | Vector for efficiently labeling zebra fish PGC, and preparation method and use of transgenic fish |
| CN103667327A (en) * | 2013-12-06 | 2014-03-26 | 中国科学院苏州生物医学工程技术研究所 | Preparation method of transgenetic zebrafish used for detecting environment pollutants |
| 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 |
| CN105274144A (en) * | 2015-09-14 | 2016-01-27 | 徐又佳 | Preparation method of zebrafish with hepcidin gene knocked out by use of CRISPR / Cas9 technology |
| CN105274140A (en) * | 2015-10-28 | 2016-01-27 | 厦门大学 | Method for building muscle cell-specific expression mCherry fluorescent protein zebra fish family |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106070063A (en) | 2016-11-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106070063B (en) | Transgenic zebrafish system and its method for building up with abcb4 | |
| Fazio et al. | Zebrafish patient avatars in cancer biology and precision cancer therapy | |
| Paez-Gonzalez et al. | Ank3-dependent SVZ niche assembly is required for the continued production of new neurons | |
| Naert et al. | CRISPR/Cas9 disease models in zebrafish and Xenopus: The genetic renaissance of fish and frogs | |
| CN107109494A (en) | The treatment method and diagnostic method of the mediation type diseases of IL 33 | |
| US20140315992A1 (en) | Genetic Alterations Associated with Autism and the Autistic Phenotype and Methods of Use Thereof for the Diagnosis and Treatment of Autism | |
| KR20190036553A (en) | Treatment of metastatic cancer and model system for metastatic disease | |
| Sun et al. | RETRACTED ARTICLE: Long Noncoding RNA PVT1 Promotes Prostate Cancer Metastasis by Increasing NOP2 Expression via Targeting Tumor Suppressor MicroRNAs | |
| CN102265806B (en) | Anti-Alzheimer transgenic Dosophila melanogaster model and application thereof in drug screening | |
| CN103348012B (en) | A kind of humanized transgenic animals | |
| CN105907837A (en) | Application of transgene zebra fishes to evaluation of drug resistance | |
| CN102747096B (en) | Gene search vector, random gene mutation control method and application thereof | |
| Wang et al. | Genome-wide CRISPR-Cas9 screening and identification of potential genes promoting prostate cancer growth and metastasis | |
| Luo et al. | Stepwise crosstalk between aberrant Nf1, Tp53 and Rb signalling pathways induces gliomagenesis in zebrafish | |
| EP3397051B1 (en) | Fly avatars for cancer and uses thereof | |
| KR101838394B1 (en) | Prediction method for using tissue origin of gliblastoma detecting genitic mutation and animal model for gliblastoma | |
| Chen et al. | Phylogenetic analysis, expression patterns, and transcriptional regulation of human CTEN gene | |
| Schmitt et al. | Functional synapses between small cell lung cancer and glutamatergic neurons | |
| RU2579701C2 (en) | Transgenic animal that differs from human, and its application | |
| CN108138165A (en) | The ovum of birds, the production method of birds and birds | |
| Watson et al. | CIC-DUX4 expression drives the development of small round cell sarcoma in transgenic zebrafish: a new model revealing a role for ETV4 in CIC-mediated sarcomagenesis | |
| KR102133179B1 (en) | IRX1 Knock-out Transgenic Zebrafish Model and Method for Producing Thereof | |
| CN104278026B (en) | The foundation and application of zebra fish candidate stem cell defect model | |
| CN109880850A (en) | A kind of double transgenic zebra fish biosensor and its construction method and application | |
| Bi et al. | Upregulation of DGCR8, a candidate predisposing to schizophrenia in Han Chinese, contributes to phenotypic deficits and neuronal migration delay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |