CN106035233A - Transgenic zebra fish model with g6pd1303-1497 locus deletion and construction method - Google Patents
Transgenic zebra fish model with g6pd1303-1497 locus deletion and construction method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a transgenic zebra fish model with g6pd1303-1497 locus deletion. gatal-g6pdM1303-1497-EGFP-PBSK-Isce I plasmids with g6pd1303-1497 locus deletion of g6pd gene and Isce I enzyme are injected into embryo during a unicell stage. The advantages of the transgenic zebra fish model with g6pd1303-1497 locus deletion are that the g6pd gene and an EGFP gene can be driven at the same time, tracking observation of expression in red blood cells of the mutant type g6pd gene with the 1303-1497 locus deletion can be achieved.
Description
Technical field
The invention belongs to design gene engineering technology field, particularly to a kind of band g6pd1303-1497 site deletion
Transgenic zebrafish model and construction method.
Background technology
Glucose 6 phosphate dehydrogenase deficiency (gluocose-6-phosphate dehydrogenase
Deficiency, is called for short G6PD deficiency disease) it is commonly called as favism, it is the most modal familial acholuric red blood cell enzyme defect
Sick.G6PD gene mutation causes G6PD enzymatic activity to reduce, the main activity being affected enzyme by two kinds of mechanism, i.e. affects G6PD function
The effect of structural area and the dimeric formation of G6PD.G6PD enzyme is the initial limit participating in erythrocyte zymolysis phosphopentose oxidative pathway
Speed enzyme, major function is that catalysis generates important reducing substances NADPH (NADPH), and NADPH is to maintain body
Coenzyme necessary to important antioxidant reduced glutathion (GSH) reducing condition.When body G6PD enzymatic activity lacks,
Can affect the generation of NADPH, NADPH is the anabolic hydrogen donor of body many kinds of substance, maintains reduced glutathion simultaneously
The growing amount of (reducing condition of GSH), thus weaken body reduced glutathion (GSH) and hydrogen peroxide (H2O2) antioxidation
The antioxidant effect of agent, makes the hemoglobin in erythrocyte and erythrocyte membrane easily damage when catalytic oxidation damages.Produce big
The oxygen-derived free radicals of amount, now, erythrocyte membrane cannot resist oxidative attacks, and between erythrocyte membrane protein, polymeric bonds wrecks, and gets involved
Therefore erythrocyte loses deformability, easily occurs broken molten under the impact of blood flow and the squeezing action of blood capillary, Jin Erfa
Raw Acute hemolytic crisis.
The detailed mechanism that at present fall ill with regard to G6PD deficiency disease in the whole world is unclear, needs further research and inquirement.Often
See that Acute hemolytic crisis, favism and congenital aspherical cell that clinical manifestation includes that neonatal jaundice, medicine or infection cause are molten
Hemolytic hemolytic anemia (CNSHA) etc..Now, the remedy measures of G6PD deficiency disease is mainly early screening, it is to avoid risk factor etc. are pre-
Anti-remedy measures.Once the case after making a definite diagnosis takes forbidden drugs, it is to avoid induction is because of disorderly.Hyperbilirubinemia gives blue light
Irradiate, alkalize the symptomatic treatments such as blood, defeated albumin, blood transfusion, and relevant auxiliary treatment.There is not specific treatment yet both at home and abroad
The remedy measures of G6PD deficiency disease, does not has a kind of very good animal experimental model can well realize G6PD gene yet
The research of relation between structure change and function.
Summary of the invention
The technical problem to be solved in the present invention is to provide the transgenic zebrafish of a kind of band g6pd1303-1497 site deletion
Model.
In order to solve above-mentioned technical problem, the technical scheme is that a kind of band g6pd1303-1497 site deletion
Transgenic zebrafish model, by the gata1-g6pd of g6pd genophore 1303-1497 site deletionM1303-1497-EGFP-PBSK-
Isce I plasmid injects the one cell stage of Brachydanio rerio together with Isce I enzyme.
Present invention also offers a kind of transgenic zebrafish model building described band g6pd1303-1497 site deletion
Method, comprise the following steps:
(1) by g6pdM1303-1497-EGFP-PCS2+, gata-1-PBSK-IsceI plasmid enzyme restriction connects structure gata1-
g6pdM1303-1497-EGFP-PBSK-Isce I plasmid；
(2) by described gata1-g6pdM1303-1497-EGFP-PBSK-Isce I plasmid injects by the way of microinjection
In one cell stage zebrafish embryo animal pole blastodisc；
(3) whether express green fluorescence and filter out the embryo of transgenic and hatching is supported to one-tenth by observing 24hpf phase embryo
Fish as F0 for transgenic zebrafish；
(4) with described F0 for transgenic zebrafish and wild type post-coitum produce express green fluorescence embryo hatching also
Support to adult fish as F1 generation transgenic zebrafish；
(5) identify, by observation embryo's EGFP fluorescing matter and Genomic PCR method, the g6pd proceeded toM1303-1497-EGFP base
The expression of cause and the situation of insertion.
Present invention also offers the application of the transgenic zebrafish model of a kind of band g6pd1303-1497 site deletion, institute
During stating transgenic zebrafish model spike observing drug treating during screening treatment G6PD deficiency disease medicine
Erythrocytic change.Described transgenic zebrafish model is the transgenic models proceeding to 1303-1497 site mutation G6PD gene,
447-in 435-499 amino acids sequence, with mankind's G6PD protein sequence in this fragment gene coding Brachydanio rerio G6PD protein sequence
489 amino acids sequence high conservatives.In mankind's G6PD protein sequence, 447-489 amino acids sequence is the bound site of NADP+
Point, contains Chinese's G6PD deficiency disease two G6PD gene mutation sites of common G1376T, G1388A, this section of aminoacid sequence
The integrity of row directly affects G6PD structural intergrity and functional completeness.
Gata-1 be original red be developmental specific transcription factor, labelling in early days erythroid cells.At 24hpf, speckle
The blood circulation of horse fish can detect that when beginning setting up that the erythrocyte of gata-1-EGFP labelling is with circulating.After 48h
Gata-1 expresses and gradually decreases.So utilizing gata-1 can make saltant type as the promoter of the g6pd gene driving sudden change
G6pd gene is specific expressed in erythrocyte.Thus realize " traceable " sight that saltant type g6pd gene is expressed in erythrocyte
Examine.
The present invention comparison by biological information, find the G6PD albumen homology of Brachydanio rerio and people be up to 80% with
On.In mankind's G6PD protein sequence, 447-489 amino acids sequence is the binding site of NADP+ and comprises Chinese G6PD sudden change
Two mutational sites of common G1376T, G1388A, the integrity of this section of aminoacid sequence directly affects G6PD structural intergrity
And functional completeness.In contrast Brachydanio rerio G6PD protein sequence in 435-499 amino acids sequence and mankind's G6PD protein sequence
447-489 amino acids sequence high conservative, builds immediately and knocks out Brachydanio rerio G6PD protein 435-499 aminoacid sequence correspondence
Brachydanio rerio saltant type g6pd of gene order of cDNA sequence 1303-1497, by Protocols in Molecular Biology by saltant type
G6pd gene loads in specific support, utilizes microinjection technique to import in Brachydanio rerio body by purpose plasmid, it is achieved dominant negative
Effect, and then set up the zebra fish model of G6PD deficiency disease, thus further in research Brachydanio rerio the sudden change of g6pd to its G6PD
The impact of enzymatic activity, the structure of G6PD and the change of function cause the detailed mechanism that erythrocyte is impaired, for mankind's G6PD deficiency disease
Detailed mechanism and extensive high-flux medicaments sifting basis is provided.
Compared to traditional G6PD deficiency disease animal model, present invention have the advantage that
1, gata-1 be original red be developmental specific transcription factor, labelling in early days erythroid cells.At 24hpf,
Can detect that when the blood circulation of Brachydanio rerio begins setting up that the erythrocyte of gata-1-EGFP labelling is with circulating.Utilize gata-
The promoter of the 1 g6pd gene suddenlyd change as driving can make saltant type g6pd gene specific expressed in erythrocyte.
2, gata1 simultaneously drives g6pd and EGFP gene, and can realize that saltant type g6pd gene expresses in erythrocyte " can
Spike " observe.
3, zebrafish embryo is transparent with adult fish entire body, and this animal model of structure can spike embryo and fish under the microscope
Internal fluorescent labeling erythrocyte Changing Pattern under drug-induced, the medicine for screening treatment G6PD deficiency disease provides visual
Animal model.
Accompanying drawing explanation
Fig. 1 is gata1-g6pdM1303-1497-EGFP transgenic zebrafish embryo and juvenile fish luciferase expression situation photo.
Detailed description of the invention
Below in conjunction with the accompanying drawings the detailed description of the invention of the present invention is described further.At this it should be noted that for
The explanation of these embodiments is adapted to assist in and understands the present invention, but is not intended that limitation of the invention.Additionally, it is disclosed below
As long as each embodiment of the present invention in involved technical characteristic do not constitute conflict each other and just can be mutually combined.
Embodiment one
The construction method of gene zebra fish model, comprises the steps:
(1) gata1-g6pd is builtM1303-1497-EGFP-PBSK-IsceI recombiant plasmid:
By g6pdM1303-1497-EGFP-PCS2+, two plasmids of gata-1-PBSK-Isce I by following linked system recombinate
Connect:
A.g6pdM1303-1497-EGFP-PCS2+Plasmid 3 μ L, gata-1-PBSK-Isce I plasmid 5 μ L, 10 × T4DNA
Ligase Buffer 1 μ L, T4DNA Ligase 1 μ L, cumulative volume 10 μ L.Fully after mixing, 4 DEG C of preservations.
B. by the gata1-g6pd after restructuringM1303-1497-EGFP-PBSK-Isce I Plastid transformation and screening monoclonal bacterium
Fall:
1) by 1 competence escherichia coli suspension, thaw on ice；
2) purpose connecting product pipettor and joins in competence escherichia coli suspension, pressure-vaccum is to mixing gently, in
Place 30min on ice；
3) mixed competence escherichia coli suspension is placed in 42 DEG C of water-bath thermal shock 90s, places on ice rapidly
90s；
4) 300 μ l LB fluid mediums, 37 DEG C of constant-temperature shaking culture 45min, rotating speed 160rpm are added；
5) take the competence escherichia coli suspension after 20 μ l and 80 μ l cultivate and be uniformly applied to the LB solid culture containing Amp resistance
Base, in incubator, 37 DEG C overnight.
6) positive single bacterium colony of similar round is chosen, with the LB liquid training being mixed into after pipettor picking containing 1ul/ml Amp
Supporting in base, 37 DEG C of calorstats, rotating speed 140rpm cultivates more than 12h.
C. recombinate gata1-g6pdM1303-1497The plasmid extraction of-EGFP-PBSK-Isce I plasmid: plasmid extraction uses city
The plasmid sold is little takes out test kit.
D. recombinate gata1-g6pdM1303-1497Three enzyme action of-EGFP-PBSK-Isce I plasmid are identified:
With BamHI, EcoRI, XhoI to gata1-g6pdM1303-1497-EGFP-PBSK-Isce I recombiant plasmid carries out three
Enzyme action is identified:
Take gata1-g6pdM1303-1497-EGFP-PBSK-Isce I 2 μ L, adds BamHI, each 1 μ L of EcoRI, XhoI enzyme, adds
10 × TangoTM buffer 2 μ L, 2d.H2O 3 μ L, cumulative volume 10 μ L, enzyme action 3 hours in 37 DEG C, by digestion products warp
1% agarose gel electrophoresis is identified.
(2) purpose plasmid gata1-g6pdM1303-1497-EGFP-PBSK-IsceI imports in Brachydanio rerio body:
A. the Tuebingen wild-type lines zebrafish embryo of the Brachydanio rerio i.e. one cell stage of 0.75hpf phase is collected, at body
Choose the embryo that form is complete under visor, clean up with egg water.Embryo is placed on microinjection mould, by embryo
Animal pole alignment microinjection direction；
B. by following injection system by gata1-g6pdM1303-1497-EGFP-PBSK-Isce I plasmid microinjection enters list
In the animal pole of cell stage zebrafish embryo: gata1-g6pdM1303-1497-EGFP-PBSK-Isce I plasmid (198.04ng/ μ
L) 0.2 μ L, ISceI 0.4 μ L, 5X ISceI buffer 0.1 μ L, 2d.H2O 1.3 μ L, cumulative volume 2 μ L.
C. gata1-g6pd will be injectedM1303-1497Zebrafish embryo egg after-EGFP-PBSK-Isce 1 plasmid
Water raises, and is developed to 9hpf, at the expression of fluorophor formula Microscopic observation green fluorescent protein, filters out and can express
The embryo of green fluorescence, when fetal development to 24hpf, filters out the embryo of expressing green fluorescent protein in blood circulation, makees
Raise to period of sexual maturity for F0 generation；
(3) F1 generation Tg:gata1-g6pdM1303-1497-EGFP transgenic strain Brachydanio rerio egfp expression situation
Observation, see Fig. 1:
By the F0 of raising to period of sexual maturity for Tg:gata1-g6pdM1303-1497-EGFP transgenic strain Brachydanio rerio with
Tuebingen wild-type lines Brachydanio rerio copulation, collect offspring embryo, with egg water raise, respectively at 11hpf, 24hpf,
36hpf, 48hpf grow phase, at the expression of fluorescence microscopy Microscopic observation green fluorescent protein, select the blood of offspring embryo
Liquid circulation expressing green fluorescent protein as F1 generation Tg:gata1-g6pdM1303-1497-EGFP transgenic strain Brachydanio rerio is raised
Support, simultaneously Preliminary screening go out correspondence F0 for Tg:gata1-g6pdM1303-1497-EGFP transgenic strain Brachydanio rerio.
(4) Genomic PCR method identifies F1 generation Tg:gata1-g6pdM1303-1497-EGFP transgenic strain Brachydanio rerio genome
The genes of interest of middle insertion:
A. by F0 to be sieved for Tg:gata1-g6pdM1303-1497-EGFP transgenic strain Brachydanio rerio is wild with Tuebingen
Raw type strain Brachydanio rerio copulation, collecting blood circulation can be with the offspring embryo of expressing green fluorescent protein, extraction genomic DNA:
1) collect blood circulation and can randomly select 2 pieces with the offspring embryo being developed to 36hpf of expressing green fluorescent protein
Embryo is transferred in 1.5ml EPP pipe, exhausts egg water, adds 100 μ L Lysis buffer, in PCR instrument, 98 DEG C,
Digestion 10min；
2) adding proteinK (10mg/ml) 10 μ L, in water-bath, 55 DEG C of digestion are overnight；
3) embryo digested overnight is placed in PCR instrument, 98 DEG C, 10min；
4) adding isopyknic chloroform in EPP pipe, 12000rpm is centrifuged 10min, sucts clearly to new 1.5mlEPP pipe
In；
5) step (4) is repeated；
6) adding the NaAc of cumulative volume 1/10, add the isopropanol that equal-volume is commensurability, light 5min, 12000rpm are centrifugal up and down
15min, inhales and abandons supernatant；
7) adding the 70% dehydrated alcohol .2d.H2O.1mL/tube prepared in advance, 750g is centrifuged 5min；
8) step (7) is repeated；
9) uncap under room temperature in superclean bench dry 20min；And measure Genome DNA content with One drop.
B. using the genomic DNA that extracted as template, progeny transgenic Brachydanio rerio is identified by the amplification of following amplification system
Middle g6pdM1303-1497Genetic fragment:
KOD Plus 10 × Buffer, 1.25 μ L；2mM dNTPs, 1.25 μ L；25mM MgSO4,1 μ L；50μmol/μl
Primer forward, 0.25 μ L；50 μm ol/ μ l Primer are reverse, 0.25 μ L；
Genomic DNA, 5 μ L；2d.H2O, 3.25 μ L；KOD-Plus (1.0U/ μ l), 0.25 μ L.
Cumulative volume 12.5 μ L.Being sequentially added into the mixing of above-mentioned sample brief centrifugation in PCR pipe, PCR reaction condition is as follows:
Pcr amplification product through 1% agarose gel electrophoresis detected magnitude, voltage 100V, time 30min, buffer 1 ×
TAE, identifies F1 generation Tg:gata1-g6pdM1303-1497-EGFP transgenic strain Brachydanio rerio genome amplifies exogenous importing
Purpose saltant type g6pdM1303-1497Gene.
Embodiment two
The application of a kind of transgenic zebrafish model, uses embodiment 1 gained transgenic zebrafish model to control for screening
Erythrocytic change during drug treating is observed in spike during treatment G6PD deficiency disease medicine.This transgenic zebrafish mould
Type is the transgenic models proceeding to 1303-1497 site mutation G6PD gene, this fragment gene coding Brachydanio rerio G6PD protein sequence
Middle 435-499 amino acids sequence, with 447-489 amino acids sequence high conservative in mankind's G6PD protein sequence.The mankind
In G6PD protein sequence, 447-489 amino acids sequence is the binding site of NADP+, contains Chinese's G6PD deficiency disease common
Two G6PD gene mutation sites of G1376T, G1388A, it is complete that the integrity of this section of aminoacid sequence directly affects G6PD structure
Whole property and functional completeness.gata1-g6pdM1303-1497-EGFP transgenic zebrafish embryo and juvenile fish luciferase expression situation are shown in figure
1, zebrafish embryo is transparent with adult fish entire body, and this animal model of structure can fluorescence in spike embryo and fish body under the microscope
Tagged blood cells Changing Pattern under drug-induced, for the animal mould that the medicine offer of screening treatment G6PD deficiency disease is visual
Type.
Above in association with accompanying drawing, embodiments of the present invention are explained in detail, but the invention is not restricted to described enforcement
Mode.For a person skilled in the art, in the case of without departing from the principle of the invention and spirit, to these embodiments
Carry out multiple change, revise, replace and modification, still fall within protection scope of the present invention.
Claims (3)
1. the transgenic zebrafish model of a band g6pd1303-1497 site deletion, it is characterised in that by g6pd genophore
The gata1-g6pd of 1303-1497 site deletionM1303-1497-EGFP-PBSK-Isce I plasmid injects speckle together with Isce I enzyme
The one cell stage of horse fish.
2. a method for the transgenic zebrafish model of the band g6pd1303-1497 site deletion described in building, its feature exists
In, comprise the following steps:
(1) by g6pdM1303-1497-EGFP-PCS2+, gata-1-PBSK-IsceI plasmid enzyme restriction connects structure gata1-
g6pdM1303-1497-EGFP-PBSK-Isce I plasmid；
(2) by described gata1-g6pdM1303-1497-EGFP-PBSK-Isce I plasmid injects slender by the way of microinjection
In born of the same parents' phase zebrafish embryo animal pole blastodisc；
(3) whether express green fluorescence and filter out the embryo of transgenic and hatching is supported to adult fish and made by observing 24hpf phase embryo
For F0 for transgenic zebrafish；
(4) with described F0 for transgenic zebrafish and wild type post-coitum produce express the embryo hatching of green fluorescence and support to
Adult fish is as F1 generation transgenic zebrafish；
(5) identify, by observation embryo's EGFP fluorescing matter and Genomic PCR method, the g6pd proceeded toM1303-1497The table of-EGFP gene
Reach and insertion situation.
3. the application of the transgenic zebrafish model of a band g6pd1303-1497 site deletion, it is characterised in that described in turn base
Erythrocyte during drug treating is observed because zebra fish model is used for screening spike during treatment G6PD deficiency disease medicine
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