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 PDF

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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
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tol2
abcb4
plasmid
egfp
gene
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CN106070063A (en
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舒莉萍
何志旭
孙琮杰
胡荣英
金璐
何思佳
吴西军
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Guizhou Medical University
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    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027New breeds of vertebrates
    • A01K67/0275Genetically modified vertebrates, e.g. transgenic
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/65Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression using markers
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/66General 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
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/87Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/89Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microinjection
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00Animals characterised by species
    • A01K2227/40Fish
    • AHUMAN NECESSITIES
    • A01AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01KANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00Animals characterised by purpose
    • A01K2267/03Animal model, e.g. for test or diseases
    • A01K2267/0393Animal model comprising a reporter system for screening tests

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

Transgenic zebrafish system and its method for building up with abcb4
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.
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Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1607252A (en) * 2003-10-16 2005-04-20 清华大学 Method for breeding transgenic zebra fish and its special plasmid
CN1607251A (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
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

Family Cites Families (2)

* Cited by examiner, † Cited by third party
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

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1607252A (en) * 2003-10-16 2005-04-20 清华大学 Method for breeding transgenic zebra fish and its special plasmid
CN1607251A (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

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