CN108531510A

CN108531510A - A kind of application of transgenic zebrafish in the animal model for preparing chronic myelocytic leukemia

Info

Publication number: CN108531510A
Application number: CN201810464900.XA
Authority: CN
Inventors: 张文清; 黄志斌; 许梦畅; 刘伟; 叶志安; 叶茵; 徐进
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2018-05-16
Filing date: 2018-05-16
Publication date: 2018-09-14
Anticipated expiration: 2038-05-16
Also published as: CN108531510B

Abstract

The invention discloses a kind of application of transgenic zebrafish in the animal model for preparing chronic myelocytic leukemia.The transgenic zebrafish is BCR/ABL transgenic zebrafishes, is the transgenic zebrafish of humanized BCR ABL inductions.When the present invention is by the BCR/ABL mRNA microinjections to zebrafish embryo of humanized, it is found that the phenotype of similar chronic myelocytic leukemia occurs in its embryo：Neutrophil leucocyte increases significantly, this suggests that the BCR/ABL albumen of humanized can playing a role in vivo in zebra fish；And be found that for the first time in the present invention transgenic zebrafish childhood human leukemia similar with adulthood appearance phenotype, and it demonstrates and high flux screening can be carried out to the drug for treating leukaemia using it, it has been successfully established the animal model of chronic myelocytic leukemia in the present invention, effective approach can be provided for the research and its drug screening of chronic myelocytic leukemia.

Description

A kind of transgenic zebrafish is in the animal model for preparing chronic myelocytic leukemia Using

Technical field

The invention belongs to biotechnology, more particularly to a kind of transgenic zebrafish is preparing chronic myelocytic leukemia Animal model in application.

Background technology

Chronic leukemia is common chronic myelocytic leukemia (CML) and chronic lymphocytic leukemia (CLL).CML suffers from It can find that BCR/ABL fusions, the CML courses of disease are divided into chronic phase, accelerated period and rapid change period in person leukaemia cell.Same disease Different times, the biological behaviour of tumour cell, clinical manifestation, therapy and prognosis are completely different.Chronic phase, is main Show as a large amount of granulocytes accumulation in marrow, peripheral blood and spleen, mainly connect it is maturescent in, based on metamylocyte, this Phase patient is completely reacted for associated treatments such as hydroxycarbamide, interferon, mild chemotherapy and tyrosine kinase inhibitors, can be quickly Complete incidence graph is obtained, some patientss can receive Allogeneic Hematopoietic Stem Cell Transplantation to reach long-term disease-free survival after alleviating. Chronic phase, continues about 3~5 years or so, and CML patient enters accelerated period, and accelerated period generally continues 4~6 months, and accelerated period is original Cell, immature cell proportion increase, and finally enter rapid change period.Rapid change period conditions of patients further deteriorates, marrow and peripheral blood Middle nonfunctional leucocyte largely gathers, similar to AML's (acute myelocytic leukemia) and ALL (acute lymphoblastic leukemia) Performance.CML can be to medullary system and the Lymphatic System sudden turn of events, and for rapid change period to various therapeutic effect Low Responses, prognosis is bad.

The treatment of CML is a process constantly explored, and experienced radiotherapy, radionuclide therapy, busulfan, hydroxycarbamide, other Drug such as cytarabine, homoharringtonine, indigo red, the use of isoindigo first, interferon and combined chemotherapy, finally tyrosine Kinase inhibitor (Druker BJ, Tamura S, Buchdunger E, Ohno S, Segal GM, Fanning S, Zimmermann J,Lydon NB.Effects of a selective inhibitor of the Abl tyrosine kinase on the growth of Bcr-Abl positive cells.Nature medicine,1996.2(5): P.561-566.) after the use of (TKI), raising significant in efficacy.Nevertheless, bone-marrow transplantation is only can really cure CML at present Unique method, bone-marrow transplantation is to make CML patient's body inhibiting tumour cells using strong chemotherapy, then transplants normal bone marrow To patient's body, but bone-marrow transplantation high cost, general family are difficult to bear, and bone-marrow transplantation is difficult to find that suitable confession Body, this is even more extremely difficult for the relatively nervous China of donor total amount.With the extensive application of TKI, clinically increasingly More CML drug resistance cases occur, and drug resistance has become the major reason of clinically CML treatment failures.

It is such as traditional although there are many CML models of mouse and cell and having obtained greatly breakthrough Journal of Sex Research now The transgene mouse model and transplanting mouse model of BCR/ABL1 and some targeted drugs found from these models.But have CML models there are still deficiency, disease model and the vivo physiological conditions phases of cellular level in Mechanism Study and drug screening Difference farther out, cannot reflect whole physiological functions and be unable to thoroughly evaluating adverse drug reaction and toxic side effect.Mouse model can be with From the effect of whole simulation morbid state and evaluation drug, before the result obtained has highly important clinical value and application Scape.But that there are builds is big, of high cost for the vertebrate animal models such as traditional mouse, the period is long, required sample size is big, can not be advised greatly Mould targeted drug screening etc. clearly disadvantageous place (1, Hariharan IK, Harris AW, Crawford M, Abud H, Webb E,Cory S,Adams JM.A bcr-v-abl oncogene induces lymphomas in transgenic mice.Mol Cell Biol,1989.9(7):p.2798-805.2、Honda H,Fujii T,Takatoku M,Mano H, Witte ON,Yazaki Y,Hirai H.Expression of p210bcr/abl by metallothionein promoter induced T-cell leukemia in transgenic mice.Blood,1995.85(10):p.2853- 61.3、Kelliher MA,McLaughlin J,Witte ON,Rosenberg N.Induction of a chronic myelogenous leukemia-like syndrome in mice with v-abl and BCR/ABL.Proceedings of the National Academy of Sciences of the United States of America,1990.87 (17):p.6649-53.4、Zhang,X.and R.Ren,Bcr-Abl efficiently induces a myeloproliferative disease and production of excess interleukin-3and granulocyte-macrophage colony-stimulating factor in mice:a novel model for chronic myelogenous leukemia.Blood,1998.92(10):p.3829-40.).Up to the present, do not have also Establish that a kind of modeling is fast, and small, feeding cost is low, convenient drug administration, phenotype reads simple vertebrate animal model.

Invention content

The primary purpose of the present invention is that the shortcomings that overcoming the prior art with it is insufficient, a kind of transgenic zebrafish is provided and is being made Application in the animal model of standby chronic myelocytic leukemia (CML).

It is white to chronic granulocyte for screening in preparation that another object of the present invention is to provide a kind of transgenic zebrafishes Application in the animal model of the effective drug of blood disease.

Have to chronic myelocytic leukemia using transgenic zebrafish screening it is still another object of the present invention to provide a kind of The method of the drug of effect.

The purpose of the invention is achieved by the following technical solution：A kind of transgenic zebrafish is preparing the white blood of chronic granulocyte Application in the animal model of sick (CML), wherein the transgenic zebrafish is BCR/ABL transgenic zebrafishes.

The BCR/ABL transgenic zebrafishes are the transgenic zebrafish of humanized's BCR-ABL inductions；Preferably by Following method builds to obtain：By recombinant plasmid pTol2hsp70:BCR/ABL and Tol2 transposases mRNA imports wild type spot jointly In horse fish, BCR/ABL transgenic zebrafishes are obtained.

The recombinant plasmid pTol2hsp70:BCR/ABL builds obtain as follows：

(1) BCR/ABL fusions are connected in the plasmid with Tol2 transposons recognition sites, obtain recombinant plasmid pTol2BCR/ABL；

(2) hsp70 gene promoters are inserted into recombinant plasmid pTol2BCR/ABL, obtain recombinant plasmid pTol2hsp70:BCR/ABL。

BCR/ABL fusions described in step (1) are preferably by restriction enzyme EcoRI from plasmid vector NGFR P210 cut out the BCR/ABL fusions of acquisition.

Plasmid described in step (1) is preferably T2AL200R150G plasmids.

Hsp70 gene promoters described in step (2) are the hsp70 with Cla I, EcoR V and I recognition sites of Age Gene promoter sequence；Its nucleotide sequence such as SEQ ID No：Shown in 1.

It is described by recombinant plasmid pTol2hsp70:BCR/ABL and Tol2 transposases mRNA imports wild type zebra jointly It is realized preferably by following steps in fish：By recombinant plasmid pTol2hsp70:BCR/ABL and Tol2 transposases mRNA passes through aobvious The method of microinjection is imported into 1 cell stage of Zebrafish Embryo in zebrafish embryo.

The Tol2 transposases mRNA is to pass through mMESSAGE mMACHINE systems from pCS2-Tol2 swivel bases zymophore The Tol2 transposases mRNA of in-vitro transcription；Its nucleotide sequence such as SEQ ID No：Shown in 2.

Caused by the chronic myelocytic leukemia is the expression by BCR/ABL fusions.

Chronic myelocytic leukemia remission after the drug therapy to be worked by blocking tyrosine kinase, Or remission after the drug therapy by blocking TGF β R-I accesses.

The drug that the disconnected tyrosine kinase works be preferably in Apoptosis, Dasatinib and Bosutinib extremely Few one kind.

The drug that the blocking TGF β R-I accesses work is preferably LY364947.

A kind of transgenic zebrafish is being prepared for screen the animal to the effective drug of chronic myelocytic leukemia (CML) Application in model, wherein the transgenic zebrafish is BCR/ABL transgenic zebrafishes.

The chronic myelocytic leukemia (CML) is caused by the expression of BCR/ABL fusions.

The chronic myelocytic leukemia (CML) symptom after the drug therapy to be worked by blocking tyrosine kinase Alleviate, or remission after the drug therapy by blocking TGF β R-I accesses.

The drug that the blocking TGF β R-I accesses work is preferably LY364947.

The screening is realized preferably by following steps：

(1) embryo of first day to the two days transgenic zebrafish of after fertilization is handled with the drug candidate of same concentrations With wild type siblings fish embryo：

(2) after treatment the second to five day observation the region embryo tail portion hematopoietic tissue (CHT) blood phenotype, to determine State therapeutic effect of the drug candidate to the chronic myelocytic leukemia (CML).

The blood phenotype includes Sudan black B (SB) stained positive granulocyte count.

A method of using transgenic zebrafish screening to the effective drug of chronic myelocytic leukemia (CML), wherein The transgenic zebrafish is BCR/ABL transgenic zebrafishes.

The chronic myelocytic leukemia is caused by the expression of BCR/ABL fusions.

The drug that the blocking TGF β R-I accesses work is preferably LY364947.

The present general inventive concept is：

(1) embryonic stage carries out whole mount in situ hybridization (WISH) using the gene probe of label different phase myeloid cell Experiment changes to detect detailed medullary system hematopoiesis, and early stage myeloid cell is marked with lcp；The ripe neutral grain of mpo, lyz label is thin Born of the same parents；Mfap4 marks ripe macrophage.Illustrate specificity in the chronic myelocytic leukemia zebra fish of BCR-ABL inductions Meloid progenitor breaks up and the then effect in neutrophil leucocyte and the selection of macrophage destiny.

(2) in adult fish period, we follow kidney, the blood of the BCR-ABL chronic myelocytic leukemia zebra fish induced Flow cytometer (FACS) and cytology point can be used in loop system the case where the quantity, hypotype of myeloid cell, differential period It analyses (March, 1 year), whether the feature for observing leukaemia can occur in these fishes.

(3) according to the marrow blood of model as middle myeloid cell maturity and natural history and clinic are compared in detail, Statistics transgenic zebrafish develops into the ratio of AML respectively.

(4) medicament for treatment of leukemia known to verifies the pharmacology of Leukemia Model：The chronic grain that BCR-ABL is induced is thin Born of the same parents' leukaemia zebra fish hybridizes with wild-type zebrafish AB, and every female fish lays eggs 200~300.Green fluorescence chooses BCR-ABL It is overexpressed fertilized eggs.BCR-ABL overexpression fertilized eggs are put into 96 orifice plates or 384 orifice plates, 3~5 are put per hole.

(5) have clinical drug or choose small molecule libraries (the predominantly clinic with potential biology reactive compound Upper existing CML drugs).Selected different small molecules are added in the hole containing fish-egg and water, only add a kind of medicine per hole Object.After being incubated a period of time, by the methods of SB dyeing large scale analysis, BCR-ABL overexpression fish-eggs can be changed by, which finding, makes Blood develops the compound of phenotype.

The present invention has the following advantages and effects with respect to the prior art：

(1) since the homology of mouse and people is higher, (albumen homology of BCR is that the albumen homology of 93.8%, ABL is 87.8%), so the plasmid and model of many humanizeds are all established on the basis of mouse.And the BCR/ of zebra fish and people The albumen homology of ABL compared with mouse for it is relatively low, respectively 73.8%/69.5%.But in key domain Homology it is still relatively high.So far, chronic myelocytic leukemia zebra fish model was not also reported, the present invention is by people When in the BCR/ABL mRNA microinjections to zebrafish embryo of source property, it is found that the phenotype of similar CML occurs in its embryo：Neutral grain Cell increases significantly, this suggests that the BCR/ABL albumen of humanized can playing a role in vivo in zebra fish.

(2) zebra fish is the idealized model animal for carrying out medicament research and development.Zebra fish is more suitable for doing extensive compared to mouse Drug screening, there are the advantages such as at low cost, efficient.Following advantages may be implemented for drug screening in zebra fish：High throughput, spot Horse fish quantity of laying eggs is more, and 1 day of after fertilization starts dosing, is detected after 2-5 days, during this period yolk can carry on a shoulder pole for embryotrophy and Without feeding so that drug screening can be carried out with 96 orifice plates or 384 orifice plates.Low expense, the expense of zebra fish raising and consumption Well below mouse, average every consumption daily is about the 1/100 of mouse.Convenient drug administration：Can be directly soluble in water by compound, Diffuse to embryo, the 1/100-1/1000 of the amount Gongwei mouse of required compound.The present invention is to cross table in zebra fish body for the first time Up to humanized BCR-ABL, then cause the phenotype of medullary system hematopoiesis exception；And it is found that transgenic zebrafish exists for the first time in the present invention The phenotype of childhood human leukemia similar with adulthood appearance, and demonstrate and drug for treating leukaemia can be carried out using it High flux screening.

(3) present invention is overexpressed BCR-ABL zebra fish by structure, finds there is following phenotype：1. early stage, medullary system ancestral was thin Born of the same parents' disdifferentiation, the apparent hyperplasia of granulocyte；2. granulocytosis in adult fish kidney blood；3. being separated in the middle part of 1 age zebra fish now white Blood disease cell whole body infiltrates, and which part can progress to acute myeloid leukemia (AML)；4. the chemotherapeutics Imatinib of the mankind Equal CML drugs can be obviously improved this abnormal blood change, tested using bone-marrow transplantation, determine the zebra fish disease model It is similar to the therapeutic effect of human diseases.

(4) Tg (hsp70 in the present invention:p210^BCR/ABL) transgenic zebrafish other than there is symptom in embryonic period, embryonic phase, adult Zebra fish kidney marrow (being equivalent to human marrow) is inner also to there is chronic myelocytic leukemia symptom, even occurs similar acute Granulocytic leukemia symptom, it means that can study we obtain one and be changed into the chronic granulocyte of acute stage from chronic phase Leukemia Model, this can not accomplish in mouse and cell model.

(5) the BCR/ABL cDNA gene orders of humanized are connect by the present invention with the hsp70 promoters of thermal starting albumen, Allow people to control the expression quantity of BCR/ABL under different temperatures and time, simulates in different patients different degrees of The case of BCR/ABL expression quantity, furthermore it is also possible to study the different times in disease, the variation of different BCR/ABL with this To the influence of disease process.In addition, the present invention by Tol2 Transposon Systems by hsp70:p210^BCR/ABLIt is integrated into zebra Fish genome, structure can stablize heredity and express p210^BCR/ABLTg (hsp70:p210^BCR/ABL) transgenic zebrafish.And The model that different dosage control can not carried out in the mouse of existing expression humanized BCR/ABL still, can not be better The heterogeneity of clinical patient is simulated, and chooses the best alternatives and carries out drug screening.

Description of the drawings

Fig. 1 is transgenic carrier pTol2hsp70 of the present invention:The arrangement architecture figure of the upper each elements of BCR/ABL；Wherein, " Tol2 " be transposons identify sequence, " hsp70promotor " be heat shock gene hsp70 promoter sequence, " BCR " be from BCR Gene Partial sequences in the BCR/ABL fusions that chronic myelocytic leukemia patient's blood sample obtains, " ABL " are from chronic Abl gene partial sequence in the BCR/ABL fusions that granulocytic leukemia patient's blood sample obtains, " pA " are translation termination signal Identify sequence.

Fig. 2 is wild-type zebrafish (WT) and Tg (hsp70:P210^BCR/ABLBCR/ABL in transgenic zebrafish embryo The result figure of mRNA expressions detection (is divided into four groups of samples, every group of sample is examined according to gene type and Embryo Culture condition 160 pieces of embryos are surveyed)；Wherein, " h " is that embryo carries out heat thermostability；" 28.5 " are persistently incubated for embryo is placed in 28.5 degree of perseverances Warm incubator (" * * " P<0.001；“****”P<0.0001).

Fig. 3 is to utilize in situ hybridization detection Tg (hsp70:p210^BCR/ABL) BCR/ABL genes in transgenic zebrafish embryo Expression quantity result figure (collects transgenic zebrafish and the wild-type zebrafish embryo of different developmental phases, carries out BCR/ABL probes Result after whole embryo hybridization in situ experiment)；Wherein, " h " is that embryo carries out heat thermostability, and " black arrow " indicating positions is HSCs (candidate stem cell) generates position.

Fig. 4 is Tg (hsp70:p210^BCR/ABL) transgenic zebrafish medullary system marker in embryonic hematopoiesis growth course (lcp, lyz, SB and mfap4) unconventionality expression figure；Wherein, A~D, I~J are schemed for lcp, the result of lyz WISH and to all (lower right black box is PBI regional signal enlarged drawings, figure upper right side label to the result of positive signal progress counting statistics in figure Number be the zebrafish embryo number that increases of gene expression than upper total zebrafish embryo number)；It is SB dyeing to scheme E, F, K As a result and to all positive signals carry out counting statistics result (in figure lower right black box be the areas PBI signal enlarged drawing, The number of figure right label is the zebrafish embryo that increases of gene expression than upper total zebrafish embryo number)；Figure G, H, L are The result of mfap4WISH and to all positive signals carry out counting statistics result (lower right black box is respectively in figure Head elevation and PBI regional signal enlarged drawings, the number that right marks in figure are the zebrafish embryo ratio that gene expression increases Upper total zebrafish embryo number)；Statistical method is examined for t, " * " p<0.05；“****”p<0.0001, " h " indicates embryo It carries out heat thermostability.

Fig. 5 is wild-type zebrafish and Tg (hsp70:p210^BCR/ABL) transgenic zebrafish adult fish kidney blood and periphery Blood carries out the result figure of blood film Giemsa staining；Wherein, figure a is that (blood is thin for WT adult fish peripheral blood film Giemsa stainings result Born of the same parents form based on red blood cell, fresh few lymphocyte, myeloid cell and blood platelet)；Figure b is Tg (hsp70:p210^BCR/ABL) turn (blood phenotype is mainly shown as the neutral grain of each stage of development to gene zebra fish adult fish peripheral blood film Giemsa staining result Cytosis)；It is that (all types of cells of development in different stages all account for one to WT adult fish kidney blood film Giemsa stainings result to scheme c Certainty ratio, in the majority with red blood cell and neutrophil leucocyte, lymphocyte takes second place, and other cell types occupy less ratio)；Scheming d is

Tg(hsp70:p210^BCR/ABL) transgenic zebrafish adult fish kidney blood film Giemsa staining result (blood phenotype It is mainly shown as the neutrophilic leukocytosis of each stage of development)；Asterisk is initial cell in figure, before traditional thread binding arrow indicates medullary system Body, triangular arrowheads are immature neutrophil leucocyte, and lightning arrow is the neutrophil leucocyte of maturation.

Fig. 6 is Tg (hsp70:p210^BCR/ABL) transgenic zebrafish progresses to the blood phenotype of AML；Wherein, figure a is WT (haemocyte forms based on red blood cell adult fish peripheral blood film Giemsa staining result, fresh few lymphocyte, myeloid cell and blood Platelet)；Figure b is Tg (hsp70:p210^BCR/ABL) transgenic zebrafish adult fish peripheral blood film Giemsa staining result (blood table Type is mainly shown as that myeloblast increases significantly)；Figure c is WT adult fish kidney blood film Giemsa stainings result (in development The all types of cells of different phase all account for certain proportion, and in the majority with red blood cell and neutrophil leucocyte, lymphocyte takes second place, other classes Type cell occupies less ratio)；Figure d is Tg (hsp70:p210^BCR/ABL) transgenic zebrafish adult fish kidney blood film Jim Sa Coloration result (blood phenotype is mainly shown as that myeloblast increases significantly)；Traditional thread binding arrow indicates that original neutral grain is thin in figure Born of the same parents.

Fig. 7 is Tg (hsp70:p210^BCR/ABL) transgenic zebrafish embryo is to the reaction result figure of TKIs；Wherein, figure A is Placebo Tg (hsp70:p210^BCR/ABL) transgenic zebrafish embryo 5dpf lcp WISH (whole mount in situ hybridization) result；Figure B~D is that TKIs handles Tg (hsp70:p210^BCR/ABL) transgenic zebrafish embryo, 5dpf lcp WISH results；Right side in figure The number of label is lcp⁺Tg (the hsp70 of Leukopenia:p210^BCR/ABL) transgenic zebrafish embryo is than upper total Tg (hsp70: p210^BCR/ABL) transgenic zebrafish embryo's number, lower right corner black box is PBI regional enlarged drawings；In figure " h " indicate into Row heat shock experiment process.

Fig. 8 is Tg (hsp70:p210^BCR/ABL) transgenic zebrafish embryo is to the reaction results of TGF β R-I pathway inhibitors Figure；Wherein, figure A, B is placebo wild type wt zebra fish (A) and Tg (hsp70:p210^BCR/ABL) transgenic zebrafish embryo (B) 5dpf lcp WISH results；Scheme C, D be TGF β R-I pathway inhibitors LY364947 handle wild type wt zebra fish (C) and Tg(hsp70:p210^BCR/ABL) transgenic zebrafish embryo (D) 5dpf lcp WISH results；It is lcp sun in statistics A~D to scheme E Property neutrophil leucocyte asterisk number (" * " p<0.05).

Specific implementation mode

With reference to embodiment, the present invention is described in further detail, and embodiments of the present invention are not limited thereto. Each raw material used in following embodiments can be obtained from commercially available in addition to particularly pointing out.

Term " hpf " used in the present invention refers to the hourage of after fertilization.Term as used herein " dpf " refers to fertilization Number of days afterwards.For example, 3hpf refers to 3 hours of after fertilization, and 8dpf refers to 8 days of after fertilization.

Term " wild type " or " WT " used in the present invention all refer to wild-type zebrafish.

Term " fish (sibling) born of the same parents " used in the present invention refers to by the offspring individuals of same parents.

Embodiment 1

1, material and method：

(1) zebra fish culture

The cultivation of zebra fish such as document (Westerfield M:The zebrafish:guide for the laboratory use of zebrafish(Brachdanio rerio).Edition by Eugene,OR, M.Westerfield, 1993) it is described.Following strain is used in the present invention：AB wild-type zebrafish, BCR-ABL transgenosis spots Horse fish.

(2) transgenic zebrafish is established

The application is using transposon-mediated transgenic technology structure transgenic zebrafish system.BCR/ABL is merged in research Gene order is to cut out to obtain from plasmid vector NGFR P210 by restriction enzyme EcoRI.Wherein, NGFR P210 (Addgene, plasmid#27486) plasmid vector is by entrusting the China Addgene sole agent company Beijing Central Plains to close Poly- Trade Co., Ltd. buys to Addgene companies of the U.S. and obtains (contract number：QC13120106).NGFR after our amplifications P210 plasmid vectors send Invitrogen (Invitrogen^TM| Thermo Fisher Scientific) company's progress DNA base survey Sequence, selection and the BCR/ABL fusion gene sequences obtained in clinically chronic myelocytic leukemia patient blood sample are completely the same Plasmid vector carries out subsequent experimental.

It links system by digestion first BCR/ABL fusions are connected into Tol2 transposons recognition sites (T2AL200R150G plasmids are public in the article that the laboratories Japanese Koichi Kawakami are delivered for T2AL200R150G plasmids It opens：Functional Dissection of the Tol2Transposable Eleme nt Identified the Minimal cis-Sequence and a Highly Repetitive Sequence in th e Subterminal Region Essential for Transposition.), to obtain pTol2BCR/ABL plasmid vectors.Then in order to obtain The transgenic zebrafish system of heredity and conditionity expression BCR/ABL fusion proteins must be stablized, we select only in 38.5 DEG C of temperature The promoter of the hsp70 genes of high expression is just had under the conditions of degree, the promoter of hsp70 genes is by extracting wild-type zebrafish Genome simultaneously clones its sequence, nucleotide sequence such as SEQ ID No：Shown in 1.Start BCR/ABL fusion proteins in zebra fish Expression in vivo.The preceding primer sequence with I recognition sites of restriction enzyme Cla is separately designed for hsp70 promoter sequences (C laⅠ-hsp70-FP：5 '-CCATCGATTCAGGGGTGTCGCTTGGT-3 ') and with V Hes of restriction enzyme EcoR Rear primer sequence (V-Age of EcoR, the I-hsp70-RP of I recognition sites of Age：5’-C CGATATCACCGGTCTGCAGGAAAAAAAAAC-3 ') amplification hsp70 promoter sequences.

System is linked by digestion hsp70 promoter sequences are inserted into above-mentioned pTol2BCR/ABL plasmid vectors, tie Fruit is as shown in Figure 1, obtain pTol2hsp70:BCR/ABL plasmid vectors.(it is from pCS2- finally by Tol2 transposases mRNA Tol2 swivel bases zymophore is by the Tol2 transposase mRNA of mMESSAGE mMACHINE system in-vitro transcriptions, and nucleotide sequence is such as SEQ ID No：Shown in 2) and pTol2hsp70:(dosage of the two can be with for the method for BCR/ABL plasmid vectors microinjection together It adjusts according to actual needs, such as each zebrafish embryo injection 50pg Tol2 transposase mRNA+50pg pTol2hsp70:BCR/ ABL plasmid vectors), it is imported in zebrafish embryo in 1 cell stage of Zebrafish Embryo, it, will under the action of transposase Gene DNA fragment on transgene carrier between two Tol2 recognition site sequences is integrated on the genome of zebra fish, is obtained Tg(hsp70:P210^BCR/ABL) transgenic zebrafish, i.e. BCR/AB L transgenic zebrafishes.

(3) expression of real-time fluorescence quantitative PCR detection BCR/ABL

We utilize expression of the fluorescence quantitative PCR detection BCR/ABL in genetically engineered fish first：First and second group of sample point It Wei not wild type AB strains zebra fish or Tg (hsp70:P210^BCR/ABL) transgenic zebrafish embryo, persistently it is placed in 28.5 degree of perseverances It is incubated 3d in warm incubator；Third and fourth group of sample is respectively wild type AB strains zebra fish or Tg (hsp70:P210^BCR/ABL) turn Gene zebrafish embryo after fertilization for 24 hours, 36h, 48h, 60h and 72h carry out heat thermostability.RNAeasy Kit are utilized later (Qiagen,Maryland,USA；74004) total serum IgE for extracting wild type and genetically engineered fish, then utilizes reverse transcription reagent box (Promega, Madison, USA, M1701) synthesizes cDNA.Real-time fluorescence quantitative PCR utilizes SYBR Green Real-time PC R Master Mix(Applied Biosystems,Austin,USA；4472908) dyestuff is in LightCycler N ano BCR/ABL mrna expression amount detections (bcr-qPCR-FP is carried out on SW1.0 machines：5’-GGCT CTATGGGTTTCTGAATGTC-3’；abl-qPCR-RP：5’-TTTCCTTGGAGTTCCAACG AG-3’).

The results are shown in Figure 2：Tg(hsp70:P210^BCR/ABL) transgenic zebrafish offspring in BCR/ABL mRNA tables Up to amount compared with the offspring of control group wild-type zebrafish, thousands of times are increased.In the transgenic progeny carried out heat thermostability BCR/ABL mrna expression amounts compared with the transgenic progeny that 28.5 degree of constant incubators are persistently incubated and conspicuousness increase High.Further illustrate that the expression of BCR/ABL in the transgenic models that we are established is regulated and controled by the startup of hsp70,38.5 It high can be expressed under the conditions of degree.Such transgenic models contribute to us to study the relationship of BCR/ABL expression quantity and disease.In order to Avoid BCR/ABL excessively high in zebrafish embryo phase expression quantity and influence its normal growth and development or cause its can not survive at Year, we can raise genetically engineered fish consecutive low temperature, then be needed that the time is selected to carry out heat thermostability induction according to research The expression of BCR/ABL, condition are controllable.

(4) expression of in situ hybridization detection BCR/ABL

We are with reference to the mankind and the BCR and abl gene nucleic acid sequence of zebra fish in NCBI and ENSEMBLE databases, design The special BCR/ABL probes of the mankind, nucleotide sequence such as SEQ ID No：Shown in 3.It is used in combination BCR/ABL probes to carry out zebra fish The spatial and temporal expression of BCR/ABL mRNA in embryo's whole mount in situ hybridization experiment detection transgenic zebrafish embryoid body.Wait for Tg (hsp70:P210^BCR/ABL) embryonic development to pf for 24 hours, carries out juvenile fish heat shock experiment, select respectively for 24 hours pf, 32hpf, 50hpf, 80hpf, 110hpf, 140hpf carry out heat shock, collect 36hpf, 3dpf (85hpf) and 5dpf (145hpf) embryo, use BCR/ABL Probe carries out whole embryo hybridization in situ experiment (being control with wild type AB strain zebra fish).

The results are shown in Figure 3：Tg(hsp70:p210^BCR/ABL) Each point in time can all detect in transgenic zebrafish embryo To the expression of BCR/ABL mRNA, expression pattern is institute's wide expression in a organized way；And control group wild type AB strain zebra fish embryos The expression of BCR/ABL mRNA is then can't detect in tire completely.

(5) whole mount in situ hybridization

Collect the Tg (hsp70 of 3dpf (about 78hpf) size:p210^BCR/ABL) transgenic zebrafish embryo, select medullary system thin Born of the same parents' specific marker gene (l-plastin, lyz, mpx) probe (probe please provide the commercial source that can be commercially available) carries out whole Embryo hybridization in situ experiment and Sudan black B (SB) Coloration experiment, detection transient expression p210 in zebra fish body^BCR/ABL, to embryo The influence that myeloid cell is developed in hematopoiesis." l-plastin " marks all myeloid cell monoids, including neutrophil leucocyte and Macrophage.The metamylocyte cell and ripe neutrophil leucocyte of " mpx, lyz " label development relative maturity." SB " is marked Middle children's grain, evening young grain, ripe neutrophil leucocyte.

The results are shown in Figure 4, and p210 is expressed in zebra fish body^BCR/ABL, l-plastin, mpx, lyz, SB label this A little myeloid cell numbers significantly increase.It is known that clinically the major blood phenotype of CML chronic phase patients is exactly that grain system increases It is raw.And the transient expression p210 in zebra fish body^BCR/ABLAlso it can cause increasing for granulocyte, illustrate the p210 of humanized^BCR/ABL Expression can also play similar biological function in human body in zebra fish body.

(6) cytological analysis

In order to avoid p210^BCR/ABLHeight expression leads to Tg (hsp70:p210^BCR/ABL) transgenic zebrafish embryonic death without Can normal development to adult, we are by Tg (hsp70:p210^BCR/ABL) transgenic zebrafish raising in 28.5 DEG C of cyclic culture systems System, waits for that it is developed to 8 months sizes and carries out heat-inducible p210 again^BCR/ABLHeight expression, continuous Heat thermostability 1 month, with subsequent It is continuous normally to raise to 1 year size.Then, we are to 100 Tg (hsp70:p210^BCR/ABL) transgenic zebrafish and same batch processing 55 wild-type zebrafish dissected, collect kidney blood and peripheral blood and carry out blood film Giemsa staining, observe and count Variety classes and the different degrees of haemocyte composition of development in kidney blood and peripheral blood.The separation of hematopoietic cell is as follows：Zebra fish It is anaesthetized with tricaine, peripheral blood (PB) is obtained by the Trace Blood cytopipetle crossed with test tube of hepari from the gill.Kidney blood (KM) is thin Born of the same parents detach from kidney.The PB and KM isolated is used to be resuspended containing 5% FBS, then by the 400 turns of centrifugations in 3 minutes of the cell of resuspension To slide.Finally use Ji's nurse Sa (Merck, Germany；1.09204.0500) fixation washes after five minutes, then with a Switzerland Ji (Merck:1.01424.0500) dyeing washing observation after forty minutes.PB and KM cells are counted according to cellular morphology.

As a result as shown in Fig. 5 and table 1 and 2：There are 76 medullary systems shown as in peripheral blood or marrow blood in 100 fishes Or myeloid precursor increases.And I-VI class can be divided into according to its degree, it is similar with the CML blood pictures of people.And such as Fig. 6 institutes Show：Similar with the CML sudden turn of events processes of people, wherein having 1 to develop into AML rapid change period in 100 transgenic zebrafishes, it is outer to cash In all blood and marrow blood, there are a large amount of progenitor cells.

Table 1.Tg (hsp70:P210BCR/ABL) zebra fish blood phenotypic classification (peripheral blood)

Table 2.Tg (hsp70:P210BCR/ABL) zebra fish blood phenotypic classification (marrow blood)

Embodiment 2

CML can pass through by blocking remission after the drug therapy that works of tyrosine kinase and block tyrosine The drug that kinases works can be Apoptosis (Imatinib), Dasatinib (Dasatinib) or Bosutinib (Bosutinib)；CML can also be by remission after blocking the drug (such as LY364947) of TGF β R-I accesses to treat, specifically Experimental procedure is as follows：

(1) tyrosine kinase inhibitor processing experiment

A. experimental group is Tg (hsp70:p210^BCR/ABL) after 39.5 degree of heat shocks of transgenic zebrafish embryo plus DMSO (diformazans Base sulfoxide, placebo) processing.Control group is to add after 39.5 degree of heat shocks of BCR/ABL transgenic zebrafishes embryo under similarity condition Tyrosine kinase inhibitor drug-treated.

B. Heat thermostability：Collect embryo, respectively after fertilization 10 hours Heat thermostabilities 30min, 22hpf, 34hpf, 46hpf, 58hpf and 70hpf Heat thermostability 2h then switch into 28.5 DEG C of incubators and are normally incubated.

C. with the drug (Apoptosis of 5~100uM concentration：100uM；Dasatinib：10uM；Bosutinib：5uM) or DMSO is to Tg (hsp70:p210^BCR/ABL) transgenic zebrafish embryo progress continuous dipping processing in 48 hours.

D. to the Tg (hsp70 after 5dpf after processing:p210^BCR/ABL) transgenic zebrafish embryo progress lcp in situ hybridizations Experiment is observed the neutrophil phenotype in embryo tail portion hematopoietic tissue region, is controlled the CML with the determination drug candidate Therapeutic effect.Blood phenotype can also include Positive Cell Counts and counting after lcp gene hybridization in situ.Lcp positive granulocyte numbers Purpose reduction is considered as remission after drug therapy.

The results are shown in Figure 7：Versus Placebo group, be added tyrosine kinase inhibitor (Apoptosis, Dasatinib or Bosutinib) lcp positive granulocyte numbers are all considerably reduced, alleviate the symptom of granulocytosis in CML.Prompt we with Clinical drug effect is consistent, and tyrosine kinase inhibitor can not only alleviate the symptom of clinical CML, can similarly alleviate Tg (hsp70:p210^BCR/ABL) the CML phenotypes that increase of transgenic zebrafish granulocyte anomaly.This transgenic zebrafish is treatment The new medicament screen of CML provides a valuable platform.

(2) TGF β R-I pathway inhibitors processing experiment

A. experimental group is Tg (hsp70:p210^BCR/ABL) 39.5 degree of Heat thermostability groups of transgenic zebrafish embryo.Control group For 39.5 degree of Heat thermostability groups of wild type WT zebrafish embryos under similarity condition.

C. with the LY364947 drugs of 5uM concentration or DMSO to Tg (hsp70:p210^BCR/ABL) transgenic zebrafish or open country Raw type embryo carries out continuous dipping and handles for 48 hours.

The results are shown in Figure 8：Relative to placebo (DMSO), TGF β R-I pathway inhibitors (LY364947) are being added Considerably reduce Tg (hsp70:p210^BCR/ABL) lcp positive granulocyte numbers in transgenic zebrafish embryo, it alleviates in CML The symptom of granulocytosis.And wild-type zebrafish group is not substantially change then.TGF β R-I accesses are the pathogenic passes BCR/ABL One of key access, current clinic there is no the drug discovery of related pathways, this prompts us that can utilize Tg (hsp70:p210^BCR ^/ABL) transgenic zebrafish screens new CML drugs.

The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment Limitation, it is other it is any without departing from the spirit and principles of the present invention made by changes, modifications, substitutions, combinations, simplifications, Equivalent substitute mode is should be, is included within the scope of the present invention.

Sequence table

<110>South China Science ＆ Engineering University

<120>A kind of application of transgenic zebrafish in the animal model for preparing chronic myelocytic leukemia

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1520

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>Hsp70 promoter sequences

<400> 1

tcaggggtgt cgcttggtta tttccaaaaa tcaaattaat tttattaaac tattagaacg 60

agcatgtttt gtctatatgc tacagaagat aaaaaataat aggagttaac agttataaaa 120

caacacactt tgtttctatt gattgttgac cacactgggg tctcattaag ttagattaaa 180

gacacactaa ctgggtcaaa agcagcagat tgatttcata gcaccagggt aaactttcta 240

acacttttac ggcaatcata tacattaaaa ttaaatacag accacgactg aacaaggagg 300

atgatctcca atattaaaca aagagacttg tgcctatttc tctgagggta aacatgacct 360

ctcaagttag caagttgttt ttaacactac aaaaatagtt aagactgcaa tcccagaata 420

aagtattggt tttaaccaat caatatagta cagtaaacat ccatttgttt tgttgaaacg 480

ttaaacaaat ctgaccaaag ctattagctt atataaaaca ggtttgcctt ctatgtagct 540

gaaaacacca caggcccgat tttgctactg tgtaaaacat ttcagcaaga tttttttatt 600

gcattttttt ttactgaatc gttcaaacat tttatcattt tagtttgttc attcattgca 660

actggaaaaa caacacatca cacaaccgca catatttcag caataagtac aataaaacac 720

tcaaataaaa aaaaacattt taaatctctt tgtatttttg accgctgttt cgcgtaattt 780

cacggtaaaa ctctggaaat ctccactaca ttcctctcag cggctcctct caatgacagc 840

tgaagaagtg acgcggctgc ctgctgtgtt ttgattggtc gaattcactg gaggcttcca 900

gaacagtgta gagtctgaac gggtgcgcgc tctgctgtat ttaaagggcg aaagagagac 960

cgcagagaaa ctcaaccgaa gagaagcgac ttgacaaaga agaaaagagc agcctgacag 1020

gactttttcc ccgacgaggt gtttattcgc tctatttaag aatctactgt aaggtaagtc 1080

tcaatatatt gtactctatt ggctaatcag aattatatag agattatatg tacttaatgt 1140

caaaaaatca actttgtata tgtaatcttt ttacatgtgg actgcctatg ttcatcttat 1200

tttaggtcta ctagaaaatt atatttcccg ttttcacaat aaggattttt aaaaaaagca 1260

atgaacagac gggcatttac tttatgttgc tgacattatt ttatatgagc ataataacca 1320

taaatactag caaatgtcct aaatgaattt gtgttaatgt tgtctacaaa agaaaattag 1380

cgttttactt gtacaactaa taataacttg gttattaaga gaatttcact tgttgactag 1440

aaaaatcctt tcataatgaa acaattgcac cataaattgt ataaatataa aattaattct 1500

aattgttttt ttttcctgca 1520

<210> 2

<211> 6287

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>PCS2-Tol2 sequences

<400> 2

cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag 60

ctacttgttc tttttgcagg atcccatcga ttcgaattca aggcctctcg aggtcgagtc 120

acatctatta ccacaatgca cagcaccttg acctggaaat tagggaaatt ataacagtca 180

atcagtggaa gaaaatggag gaagtatgtg attcatcagc agctgcgagc agcacagtcc 240

aaaatcagcc acaggatcaa gagcacccgt ggccgtatct tcgcgaattc ttttctttaa 300

gtggtgtaaa taaagattca ttcaagatga aatgtgtcct ctgtctcccg cttaataaag 360

aaatatcggc cttcaaaagt tcgccatcaa acctaaggaa gcatattgag agaatgcacc 420

caaattacct caaaaactac tctaaattga cagcacagaa gagaaagatc gggacctcca 480

cccatgcttc cagcagtaag caactgaaag ttgactcagt tttcccagtc aaacatgtgt 540

ctccagtcac tgtgaacaaa gctatattaa ggtacatcat tcaaggactt catcctttca 600

gcactgttga tctgccatca tttaaagagc tgattagtac actgcagcct ggcatttctg 660

tcattacaag gcctacttta cgctccaaga tagctgaagc tgctctgatc atgaaacaga 720

aagtgactgc tgccatgagt gaagttgaat ggattgcaac cacaacggat tgttggactg 780

cacgtagaaa gtcattcatt ggtgtaactg ctcactggat caaccctgga agtcttgaaa 840

gacattccgc tgcacttgcc tgcaaaagat taatgggctc tcatactttt gaggtactgg 900

ccagtgccat gaatgatatc cactcagagt atgaaatacg tgacaaggtt gtttgcacaa 960

ccacagacag tggttccaac tttatgaagg ctttcagagt ttttggtgtg gaaaacaatg 1020

atatcgagac tgaggcaaga aggtgtgaaa gtgatgacac tgattctgaa ggctgtggtg 1080

agggaagtga tggtgtggaa ttccaagatg cctcacgagt cctggaccaa gacgatggct 1140

tcgaattcca gctaccaaaa catcaaaagt gtgcctgtca cttacttaac ctagtctcaa 1200

gcgttgatgc ccaaaaagct ctctcaaatg aacactacaa gaaactctac agatctgtct 1260

ttggcaaatg ccaagcttta tggaataaaa gcagccgatc ggctctagca gctgaagctg 1320

ttgaatcaga aagccggctt cagcttttaa ggccaaacca aacgcggtgg aattcaactt 1380

ttatggctgt tgacagaatt cttcaaattt gcaaagaagc aggagaaggc gcacttcgga 1440

atatatgcac ctctcttgag gttccaatgt ttaatccagc agaaatgctg ttcttgacag 1500

agtgggccaa cacaatgcgt ccagttgcaa aagtactcga catcttgcaa gcggaaacga 1560

atacacagct ggggtggctg ctgcctagtg tccatcagtt aagcttgaaa cttcagcgac 1620

tccaccattc tctcaggtac tgtgacccac ttgtggatgc cctacaacaa ggaatccaaa 1680

cacgattcaa gcatatgttt gaagatcctg agatcatagc agctgccatc cttctcccta 1740

aatttcggac ctcttggaca aatgatgaaa ccatcataaa acgaggcatg gactacatca 1800

gagtgcatct ggagcctttg gaccacaaga aggaattggc caacagttca tctgatgatg 1860

aagatttttt cgcttctttg aaaccgacaa cacatgaagc cagcaaagag ttggatggat 1920

atctggcctg tgtttcagac accagggagt ctctgctcac gtttcctgct atttgcagcc 1980

tctctatcaa gactaataca cctcttcccg catcggctgc ctgtgagagg cttttcagca 2040

ctgcaggatt gcttttcagc cccaaaagag ctaggcttga cactaacaat tttgagaatc 2100

agcttctact gaagttaaat ctgaggtttt acaactttga gtagcgtgta ctggcattag 2160

attgtctgtc ttatagtttg ataattaaat acaaacagtt ctaaagcagg ataaaacctt 2220

gtatgcattt catttaatgt tttttgagat taaaagctta aacaagaaaa aaaaaaaaaa 2280

aaaaaaaaaa aagtactagt cgagcctcta gaactatagt gagtcgtatt acgtagatcc 2340

agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa 2400

atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa 2460

taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg 2520

ggaggttttt taattcgcgg ccgcggcgcc aatgcattgg gcccggtacc cagcttttgt 2580

tccctttagt gagggttaat tgcgcgcttg gcgtaatcat ggtcatagct gtttcctgtg 2640

tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa 2700

gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct 2760

ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 2820

ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc 2880

gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa 2940

tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 3000

aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 3060

aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 3120

ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 3180

tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc 3240

agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 3300

gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 3360

tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 3420

acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 3480

tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 3540

caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 3600

aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 3660

aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt 3720

ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac 3780

agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc 3840

atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc 3900

cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata 3960

aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc 4020

cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc 4080

aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca 4140

ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa 4200

gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca 4260

ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt 4320

tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt 4380

tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg 4440

ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga 4500

tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc 4560

agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg 4620

acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag 4680

ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg 4740

gttccgcgca catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa 4800

aattcgcgtt aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca 4860

aaatccctta taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga 4920

acaagagtcc actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc 4980

agggcgatgg cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc 5040

gtaaagcact aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc 5100

cggcgaacgt ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg 5160

caagtgtagc ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac 5220

agggcgcgtc ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg 5280

cctcttcgct attacgccag tcgaccatag ccaattcaat atggcgtata tggactcatg 5340

ccaattcaat atggtggatc tggacctgtg ccaattcaat atggcgtata tggactcgtg 5400

ccaattcaat atggtggatc tggaccccag ccaattcaat atggcggact tggcaccatg 5460

ccaattcaat atggcggact tggcactgtg ccaactgggg aggggtctac ttggcacggt 5520

gccaagtttg aggaggggtc ttggccctgt gccaagtccg ccatattgaa ttggcatggt 5580

gccaataatg gcggccatat tggctatatg ccaggatcaa tatataggca atatccaata 5640

tggccctatg ccaatatggc tattggccag gttcaatact atgtattggc cctatgccat 5700

atagtattcc atatatgggt tttcctattg acgtagatag cccctcccaa tgggcggtcc 5760

catataccat atatggggct tcctaatacc gcccatagcc actcccccat tgacgtcaat 5820

ggtctctata tatggtcttt cctattgacg tcatatgggc ggtcctattg acgtatatgg 5880

cgcctccccc attgacgtca attacggtaa atggcccgcc tggctcaatg cccattgacg 5940

tcaataggac cacccaccat tgacgtcaat gggatggctc attgcccatt catatccgtt 6000

ctcacgcccc ctattgacgt caatgacggt aaatggccca cttggcagta catcaatatc 6060

tattaatagt aacttggcaa gtacattact attggaagta cgccagggta cattggcagt 6120

actcccattg acgtcaatgg cggtaaatgg cccgcgatgg ctgccaagta catccccatt 6180

gacgtcaatg gggaggggca atgacgcaaa tgggcgttcc attgacgtaa atgggcggta 6240

ggcgtgccta atgggaggtc tatataagca atgctcgttt agggaac 6287

<210> 3

<211> 1121

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>BCR/ABL probe sequences

<400> 3

cgccacgtct tcctgttcac cgacctgctt ctctgcacca agctcaagaa gcagagcgga 60

ggcaaaacgc agcagtatga ctgcaaatgg tacattccgc tcacggatct cagcttccag 120

atggtggatg aactggaggc agtgcccaac atccccctgg tgcccgatga ggagctggac 180

gctttgaaga tcaagatctc ccagatcaag aatgacatcc agagagagaa gagggcgaac 240

aagggcagca aggctacgga gaggctgaag aagaagctgt cggagcagga gtcactgctg 300

ctgcttatgt ctcccagcat ggccttcagg gtgcacagcc gcaacggcaa gagttacacg 360

ttcctgatct cctctgacta tgagcgtgca gagtggaggg agaacatccg ggagcagcag 420

aagaagtgtt tcagaagctt ctccctgaca tccgtggagc tgcagatgct gaccaactcg 480

tgtgtgaaac tccagactgt ccacagcatt ccgctgacca tcaataagga agatgatgag 540

tctccggggc tctatgggtt tctgaatgtc atcgtccact cagccactgg atttaagcag 600

agttcaaaag cccttcagcg gccagtagca tctgactttg agcctcaggg tctgagtgaa 660

gccgctcgtt ggaactccaa ggaaaacctt ctcgctggac ccagtgaaaa tgaccccaac 720

cttttcgttg cactgtatga ttttgtggcc agtggagata acactctaag cataactaaa 780

ggtgaaaagc tccgggtctt aggctataat cacaatgggg aatggtgtga agcccaaacc 840

aaaaatggcc aaggctgggt cccaagcaac tacatcacgc cagtcaacag tctggagaaa 900

cactcctggt accatgggcc tgtgtcccgc aatgccgctg agtatctgct gagcagcggg 960

atcaatggca gcttcttggt gcgtgagagt gagagcagtc ctggccagag gtccatctcg 1020

ctgagatacg aagggagggt gtaccattac aggatcaaca ctgcttctga tggcaagctc 1080

tacgtctcct ccgagagccg cttcaacacc ctggccgagt t 1121

<210> 4

<211> 26

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>I-hsp70-FP of primer Cla

<400> 4

ccatcgattc aggggtgtcg cttggt 26

<210> 5

<211> 31

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>V-Age of primer EcoR, I-hsp70-RP

<400> 5

ccgatatcac cggtctgcag gaaaaaaaaa c 31

<210> 6

<211> 23

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>Primer bcr-qPCR-FP

<400> 6

ggctctatgg gtttctgaat gtc 23

<210> 7

<211> 21

<212> DNA

<213>Artificial sequence (Artificial Sequence)

<220>

<223>Primer abl-qPCR-RP

<400> 7

tttccttgga gttccaacga g 21

Claims

1. a kind of application of transgenic zebrafish in the animal model for preparing chronic myelocytic leukemia, it is characterised in that：Institute It is BCR/ABL transgenic zebrafishes to state transgenic zebrafish.

2. transgenic zebrafish according to claim 1 answering in the animal model for preparing chronic myelocytic leukemia With, which is characterized in that the BCR/ABL transgenic zebrafishes are built by the following method to be obtained：By recombinant plasmid pTol2hsp70:BCR/ABL and Tol2 transposases mRNA is imported in wild-type zebrafish jointly, obtains BCR/ABL transgenosis spots Horse fish；Wherein,

The recombinant plasmid pTol2hsp70:BCR/ABL builds obtain as follows：

(2) hsp70 gene promoters are inserted into recombinant plasmid pTol2BCR/ABL, obtain recombinant plasmid pTol2hsp70: BCR/ABL。

3. transgenic zebrafish according to claim 2 answering in the animal model for preparing chronic myelocytic leukemia With, it is characterised in that：

Plasmid described in step (1) is T2AL200R150G plasmids；

The nucleotide sequence of hsp70 gene promoters described in step (2) such as SEQ ID No：Shown in 1.

4. transgenic zebrafish according to claim 1 answering in the animal model for preparing chronic myelocytic leukemia With, it is characterised in that：

Caused by the chronic myelocytic leukemia is the expression by BCR/ABL fusions.

5. transgenic zebrafish according to claim 1 answering in the animal model for preparing chronic myelocytic leukemia With, it is characterised in that：

Chronic myelocytic leukemia remission after the drug therapy to be worked by blocking tyrosine kinase, or it is logical Cross block TGF β R-I accesses drug therapy after remission.

6. transgenic zebrafish according to claim 1 answering in the animal model for preparing chronic myelocytic leukemia With, it is characterised in that：

The drug that the disconnected tyrosine kinase works is at least one of Apoptosis, Dasatinib and Bosutinib；

The drug that the blocking TGF β R-I accesses work is LY364947.

7. a kind of transgenic zebrafish is being prepared for screening in the animal model of the effective drug of chronic myelocytic leukemia Application, it is characterised in that：The transgenic zebrafish is BCR/ABL transgenic zebrafishes；

The BCR/ABL transgenic zebrafishes are built by the following method to be obtained：By recombinant plasmid pTol2hsp70:BCR/ ABL and Tol2 transposases mRNA is imported in wild-type zebrafish jointly, obtains BCR/ABL transgenic zebrafishes；Wherein,

The recombinant plasmid pTol2hsp70:BCR/ABL builds obtain as follows：

(2) hsp70 gene promoter sequences are inserted into recombinant plasmid pTol2BCR/ABL, obtain recombinant plasmid pTol2hsp70:BCR/ABL。

8. transgenic zebrafish according to claim 6 is being prepared for screening to the effective medicine of chronic myelocytic leukemia Application in the animal model of object, it is characterised in that：

The chronic myelocytic leukemia is caused by the expression of BCR/ABL fusions.

9. transgenic zebrafish according to claim 6 is being prepared for screening to the effective medicine of chronic myelocytic leukemia Application in the animal model of object, it is characterised in that：

The chronic myelocytic leukemia (CML) symptom after the drug therapy to be worked by blocking tyrosine kinase is slow It solves, or passes through remission after the drug therapy for blocking TGF β R-I accesses.

10. transgenic zebrafish according to claim 6 is effective to chronic myelocytic leukemia for screening in preparation Application in the animal model of drug, it is characterised in that：

The drug that the blocking TGF β R-I accesses work is LY364947.