CN102002500B - Xenopus leavis dmrt5 gene nervous specificity regulating and controlling element and construction method for carrier thereof - Google Patents

Abstract

The invention discloses a Xenopus leavis dmrt5 gene nervous specificity regulating and controlling element and a construction method for carrier thereof. In the method, a Xenopus leavis dmrt5 starter keeping zone is acquired by using the bioinformatics and molecular biological technique and is guided into PMD18-T carrier by using the purifying and connecting technique, the Dmrt5 starter keeping zone is directionally cloned onto a pCS2-IS-EGFP carrier by using Enzyme digestion technique so as to acquire a pCS2-IS-Dmrt5-EGFP carrier for transgenosis, wherein the EGFP expression is directly regulated and controlled by the Dmrt5 gene. The invention settles a base for over-expression or knockdown of gene tissue specificity of a certain gene. At the same time, the invention provides a powerful tool for monitoring the harmful matters in water which can cause the brain neurodevelopment to deform.

Description

Africa xenopus dmrt5 gene nerve-specific controlling element and construction of carrier thereof

Technical field

The present invention relates to biological field, relate in particular to a kind of clone of the cis-regulating element that nervous specific expresses and construction process of transgene carrier thereof controlled.

Background technology

Cis-regulating element is meant and can combines with specific transcription factor around the gene and influence the dna sequence dna that it is transcribed, for example functional element such as promotor, enhanser, attenuator.Cis-regulating element can the specificity regulatory gene spatial and temporal expression, so the specificity of cis-regulating element has determined specific expressed at tissue and cell levels of its institute's regulatory gene.

Dmrt5 gene in Xenopus laevis (Xenopus laevis) genome contains two exons and an intron, and mRNA length is 2592nt, contains DM territory conservative in the Dmrt gene family, also has DMA and two conservative regions of DMB.At present, also very limited for the research of Dmrt5, genetic expressions such as the in situ hybridization experiment of zebra fish, mouse isotype animal embryo shows that Dmrt5 is tissue specific expression in neural system and sexual gland mainly.For the rule of the tissue specific expression of studying Dmrt5 and their function; Adopt information biology, comparative genomics and molecular biological means; One segment length of having cloned first exon 5 ' end upper reaches of Dmrt5 gene is the potential promoter region of 3371bp; And utilize microinjection technique, the plasmid that will contain this promoter region and enhanced green fluorescence protein (EGFP) is expelled in the Xenopus laevis zygote, observes the transient expression of EGFP.In order further to identify the function of this promotor control Dmrt5 tissue specific expression; And further find can the controlling gene tissue specific expression functional element; The high-efficient transgenic method of I-SceI mediation is taked in this experiment; This promoter region and EGFP be inserted into set up transgenic Xenopus laevis strain in the Xenopus laevis genome and identify functional element wherein, hang down and lay a good foundation for implementing tissue-specific gene overexpression and clpp gene.

I-SceI is a kind of restriction endonuclease of yeast (Saccharomyces cerevisiae) plastosome intron coding; Discern the recognition site of a 18bp (TAGGGATAACAGGGTAAT); Introduce the double-strand break (Monteilhet of DNA; Perrin et al.1990), produce the outstanding end of a 4bp.Because the recognition site of I-SceI has 18 bp,, in common vertebrates genome, generally all there is not the recognition site of I-SceI so in 7 * 1010 stochastic sequence, only occur once.Because I-SceI can introduce the double-strand break of DNA, usually be used to researchs such as genome reparation, homologous recombination and gene replacement.Recently; Develop the transgenic technology that a kind of I-SceI mediation; Can be used for head and build animal analysis and set up the transgenic animal strain, carrying out the low experiment of gene expression analysis, ectopic expression and clpp gene, to gene regulatory network particularly the cis regulation and control study.

A latent defect of I-SceI method is the genetically modified expression of inlaying, if the time of injection too late or the too high this situation of culture temperature of embryonal vaccination (Ogino et al.2006a) sometimes can take place.All injections must be accomplished in 45 minutes after the feritilization of ovum, because the time window that the DNA of injection is inserted in the host genome is limited in the early stage of a cell stage.After the injection, the embryo must cultivate in the minimum temperature that can tolerate (X.tropicalis22 ℃, X.laevis 12-13 ℃) and know that they reach two cells or four cell stage, are used to stimulate genetically modified insertion.The forfeiture of I-SceI enzymic activity, such as surpassing some months-20 ℃ of storages, also can cause transgenic to be inlayed expressing or even transgenic fall flat.

Summary of the invention

The objective of the invention is to deficiency, a kind of clone of Africa xenopus dmrt5 promotor and the construction process of transgene carrier thereof are provided to prior art.

The objective of the invention is to realize through following technical scheme:

A kind of Africa xenopus dmrt5 gene nerve-specific controlling element, the nucleotide sequence of this controlling element is shown in the SEQ ID No.1.

The construction process of a kind of transgene carrier pCS2-IS-Dmrt5-EGFP of the above Africa xenopus dmrt5 gene nerve-specific controlling element may further comprise the steps:

(1) adopt information biology, searching a segment length of finding first exon 5 ' end upper reaches of Dmrt5 gene is the potential promoter region of 3371bp.Extract the Africa xenopus genomic dna, and design is carried out pcr amplification with genomic dna as template to the primer sequence of controlling element.

(2) the good controlling element sequence fragment that will increase is connected into PMD18-T, makes up the PMD18-T-Dmrt5 plasmid, and the fragment that obtains with sequence verification is correct controlling element.

(3) make up transgene carrier pCS2-IS-Dmrt5-EGFP.

Further, in the said step (1), said amplification Xenopus laevis Dmrt5 promotor conserved regions sequence the primer P5 is shown in SEQID NO.2, and amplification Xenopus laevis Dmrt5 promotor conserved regions sequence the primer P3 is shown in SEQ ID NO.3.The reaction system of said amplification amplification purpose promoter fragment is: 10 * Buffer, 15 μ l, dNTP (2.5mM) 1.75 μ l, p5 (10 μ M) 2 μ l, p3 (10 μ M) 2 μ l, genomic dna 1 μ l, ROCHE Taq 0.75 μ l, pH2O 37.5 μ l.

Further; In the said step (2); The PCR product of said step (1) is used with after PMD18-T is connected; Be transformed into intestinal bacteria, conversion fluid is applied on the LB agar plate of IPTG and 20 μ g/mL penbritins of the X-gal that contains 10 μ l 2% (M/V), 4 μ l100mM again, and 37 ℃ of incubated overnight obtain the bacterium mono-clonal.

Further; In the said step (3); With the PMD18-T-Dmrt5 plasmid and the original pCS2-IS-CMV-EGFP plasmid that utilizes molecular biology method to make up in this laboratory that said step (2) obtains, cut with Sal I and the two enzyme enzymes of HindIII respectively, after the promoter fragment that reclaims is connected, transforms with carrier segments; Use the I-SceI enzyme to cut and identify that resulting clone is correct clone, qualification result shows and has successfully made up the pCS2-IS-Dmrt5-EGFP plasmid.

The invention has the beneficial effects as follows:

1. clone and obtain Africa xenopus Dmrt5 promotor conserved regions sequence in the world first;

2. use information biology and Protocols in Molecular Biology in the world first, made up the pCS2-IS-Dmrt5-egfp transgene carrier;

3. the first in the world transgenic strain of Africa xenopus Dmrt5 promotor of having built;

The present invention utilize I-SceI mediation the high-efficient transgenic technical evaluation function of Dmrt5 expression regulation element, for the work of hanging down of specific gene overexpression of subsequent implementation neural system tissue and clpp gene is laid a good foundation;

5. the present invention can utilize the transgenic Xenopus laevis to carry out more convenient, responsive to the objectionable impurities in the water surrounding and keep watch on efficiently, monitors, and prevents the pollution of the environment to the disadvantageous effect of health of people.

Description of drawings

Fig. 1 extracts the electrophorogram that detects behind the Africa xenopus genome, and among the figure, M is the big tick marks of dna molecular, and the 1st, the genome band;

Fig. 2 is the electrophorogram of pcr amplification Dmrt5 promotor, and among the figure, M is the big tick marks of dna molecular, the 1st, and the band of PCR product;

Fig. 3 is a pCS2-IS-CMV-EGFP plasmid structural representation;

Fig. 4 is that pCS2-IS-CMV-EGFP and two kinds of plasmid plasmids of pCS2-IS-Dmrt5-EGFP I-secI enzyme are cut the product electrophorogram; Among the figure; No. 1 band is that the pCS2-IS-CMV-EGFP enzyme is cut the result, and the enzyme of No. 2 band indication PCS2-IS-Dmrt5-EGFP is cut .M1 as a result, and M2 is marker;

Fig. 5 is a pCS2-IS-Dmrt5-EGFP plasmid structural representation.

Embodiment

The present invention uses information biology and Protocols in Molecular Biology to obtain the conservative section of Africa xenopus Dmrt5 promotor, imports in the PMD18-T carrier through technology such as purifying, connections; Then; The utilization enzyme incision technology; To the pCS2-IS-EGFP carrier, acquisition can be used for the pCS2-IS-Dmrt5-EGFP carrier of transgeneic procedure with the conservative section directed cloning of Dmrt5 promotor, and wherein the expression of EGFP receives the direct cis regulation and control of Dmrt5 promotor.After carrying out the transgenic test, we head has built the transgenic Africa xenopus that Dmrt5 promoter regulation EGFP expresses, and fluorescent signal nervous specific property is very high.

The clone of Africa xenopus dmrt5 promotor of the present invention and the construction process of transgene carrier thereof may further comprise the steps:

1. the amplification of the conservative section of Africa xenopus Dmrt5 promotor

Getting tadpole is material, adds lysis buffer, homogenized; Remove RNA for 65 ℃ with RNase, remove albumen with phenol-chloroform again, the careful supernatant that takes out in centrifugal back adds chloroform again and takes out impurity such as unnecessary albumen in supernatant; After centrifugal supernatant is taken out; Add isopyknic isopropanol precipitating DNA again, behind the centrifugal collecting precipitation supernatant is carefully discarded, with twice of 70% ethanol washing and precipitating; After drying precipitated; With 100 μ l ultrapure water dissolution precipitations, with spectrophotometer measurement DNA concentration with purity, and the integrated degree through genomic dna that electrophoresis detection is carried.

For the rule of the tissue specific expression of studying Dmrt5 and their function, adopt information biology, comparative genomics means, search the sequence of this promotor and be used for the promoter fragment that increases according to two primers of sequences Design.Sequence between two primers is that a segment length at first exon 5 ' end upper reaches of Dmrt5 gene is potential promoter regions of 3371bp.

Wherein, Africa xenopus Dmrt5 promotor conserved regions sequence is shown in SEQ ID NO.1; Amplification Xenopus laevis Dmrt5 promotor conserved regions sequence the primer P5 is shown in SEQ ID NO.2, and amplification Xenopus laevis Dmrt5 promotor conserved regions sequence the primer P3 is shown in SEQ ID NO.3.

With extracted genome is template, with above designed primer amplification purpose promoter fragment: 10 * Buffer15 μ l, dNTP (2.5mM) 1.75 μ l, p5 (10 μ M) 2 μ l, p3 (10 μ M) 2 μ l, genomic dna 1 μ l, ROCHE Taq 0.75 μ l, pH2O 37.5 μ l.After the PCR reaction finishes, run glue with painted 0.8% sepharose of EB and detect, get DNA sample 5 μ l, electrophoresis detection obtains the purpose size strip.

2.PMD18-T-Dmrt5 the structure of promotor conserved regions carrier

Reclaim test kit through gel and cut glue recovery purified pcr product.The DNA of purpose band is downcut the sepharose that contains target DNA again through electrophoretic separation under uv lamp; Behind heating for dissolving, adsorption column absorption, desalting and purifying, will prepare pipe and place clean 1.5ml centrifuge tube; Prepare the film centre at DNA and add 50 μ l ultrapure waters; Room temperature leaves standstill several minutes and fully dissolves, centrifugal eluted dna.With concentration and the purity of spectrophotometric determination DNA, and electrophoresis confirms to cut the effect that glue reclaims.

The T-Vector of conservative fragments connects and transforms.With glue reclaim behind the purifying the purpose fragment with carry out the T carrier after the PMD18-T carrier mixes by the certain mol proportion example and be connected, 16 ℃ of connections are spent the night; To connect product again and change in the chemoreception attitude, the bacterium liquid of getting after part transforms is evenly coated on the ammonia benzyl agar plate that contains X-gal and IPTG, carries out blue hickie screening.

Picking list bacterium colony is in the liquid nutrient medium that contains Amp from the above-mentioned flat board, and enlarged culturing is after 3 hours, does template with bacterium liquid and carries out bacterium liquid PCR and detect the positive colony bacterium colony.After positive colony bacterium liquid protected kind again enlarged culturing be used for the extracting plasmid, carry out enzyme and cut detection, come tentatively to judge that institute's DCRP is correct according to the plasmid enzyme restriction collection of illustrative plates.After the sequence verification sequence is correct, successfully obtain PMD18-T-Dmrt5 promotor conserved regions plasmid.

3. the structure of transgene carrier pCS2-IS-Dmrt5-EGFP plasmid

Plasmid and the original pCS2-IS-CMV-EGFP plasmid that utilizes molecular biology method to make up in this laboratory according to the PMD18-T-Dmrt5 promotor of above structure; Cut with Sal I and the two enzyme enzymes of Hind III respectively; After the rubber tapping recovery; Obtain promoter fragment and carrier segments respectively; Through ligation promoter fragment is inserted on the transgene carrier skeleton then, will connects product through conversion and change in the bacterium, the picking clone cuts through bacterium liquid PCR and to plasmid I-SceI enzyme and identifies that resulting clone clones for correct again.Qualification result shows and has successfully made up the pCS2-IS-Dmrt5-EGFP plasmid.

Test through the I-SceI transgenic below and prove result of the present invention.

1, the concrete operation steps of I-SceI transgenic test

During DNA that extraction is used to inject, use the ddH2O dissolving DNA, measure DNA concentration and purity, newly dilute with ddH2O during per injection, guarantee fresh with ultraviolet spectrophotometer.Through test of many times, the injection volume of pCS2-IS-Dmrt5-EGFP plasmid is in 120pg DNA, 1.2 * 10 ^-2During U I-Sce I/ embryo, can obtain best transgenic effect.

In the preliminary experiment,, the amount that adds plasmid and enzyme in the system is groped, seek the righttest injection volume with pCS2-IS-CMV--EGFP positive control plasmid.120pg DNA, 1.2 * 10 ^-2U I-Sce I/ embryo is the righttest injection volume that obtains according to preliminary experiment.Preferably every annotations and comments are penetrated and are all used fresh enzyme to cut product, to guarantee higher transgene efficiency.

After the injection, the embryo was cultivated 2 hours in 13 ℃ of incubators, take out then and place at room temperature.Up to fetal development to the 9 after dates, the embryo to be transferred in the little plastic culture dish that 0.3 * MBS is housed, each ware is put 20-25 embryo, 18 ℃ of overnight cultures.Note, also want the some zygote synchronized culture that do not have injection of picking as contrast.

The developmental state of regularly observing the embryo is observed the green fluorescence phenotype, and is taken pictures under fluorescent microscope.When growing the tadpole that can move about to the later stage, available an amount of MS-222 anaesthetizes to observe and takes pictures.Observe a week continuously, after phenotype is basicly stable, can feed green alga, the transgenic tadpole is formed the adult frog to tadpole.

2. test-results

2.1 the detection of Dmrt5 promotor control EGFP expression plasmid pCS2-IS-Dmrt5-EGFP

The Dmrt5-EGFP embryo whole body expression EGFP that the microinjection of pCS2-IS-Dmrt5-EGFP plasmid obtains explains that the embryo of injection pCS2-IS-Dmrt5-EGFP plasmid can express the EGFP that is started by the Dmrt5 promotor, and promptly the structure of this plasmid is correct.

2.2 EGFP is specific expressed in neural system for the control of Dmrt5 promoter region

Operate like 4 transfer gene injection conditions; And the embryo who successfully integrates IS-Dmrt5-EGFP carried out the observation of successive green fluorescence, find this Dmrt5 promoter region can controlling gene specific expressed at eye, midbrain, hindbrain and whole neurocele.Through careful maintenance, genetically modified embryo survival has arrived the period of the abnormal back frog, has obtained genetically modified head and has built Xenopus laevis.

Following examples only are used to the present invention is described and are not used in the restriction scope of the invention.

Embodiment one

1. material reagent and instrument

1.1 material reagent

Ex-Taq PCR reaction kit, pMD18-T support agent box, restriction enzyme and damping fluid thereof (Sal I, Sac I, EcoR I, Pst I, BamH I, HindIII, Ava I) and T ₄Dna ligase is all available from Japanese TAKARA company; Africa xenopus (Xenopus), culture in this laboratory; Glycerine, penbritin, IPTG, X-Gal, agarose, agar powder, ethidium bromide, Ficoll 400, Proteinase K, phenol-chloroform chloroform are given birth to worker Bioisystech Co., Ltd available from Shanghai; Virahol, absolute ethyl alcohol etc. are available from the traditional Chinese medicines biochemical reagents; ROCHE-Taq PCR reaction kit is available from Switzerland Roche Holding Ag; DNA glue reclaims test kit, plasmid extraction test kit, PCR cleaning agents box all available from Axygen company; The pCS2:IS:CMV:EGFP plasmid comes from this laboratory; Tryptone, yeast soak powder available from OXOID company or the like.

1.2 key instrument

Refrigerated centrifuge (3K15) is the SIGMA Company products; The little whizzer of room temperature (centrifuge 5418) is the eppendorf Company products; The pure water appearance is a Heal Force Company products, and bacterium constant temperature shaking table and constant incubator are Shanghai rich news Company products, and LongGene PCR system (MGL96G) is available from Hangzhou Lang Ji Science and Technology Ltd.; The horizontal strip electrophoresis appearance is the Bio-Rad Company products; Stereoscopic microscope capable of taking pictures (SZX7) is available from OLYMPUS, and the fluor stereomicroscope is available from Nikon company, and gel imaging appearance (Tanon 2500) is available from China's day ability; Microinjection instrument (PLI-100) is available from HARVARD company, and PH meter (PB-10), high-precision electronic balance (BS124S) are available from sartorius company.

2. primer design

Amplify amplification region, to guarantee to obtain required complete fragment as far as possible.Use the design of primers instrument design specific primers of Vector NTI software, further analyze the interaction between forward and reverse primer with Primer 5.0 then.For the rule of the tissue specific expression of studying Dmrt5 and their function; Adopt information biology, comparative genomics means, the sequence between two primers is that a segment length at first exon 5 ' end upper reaches of Dmrt5 gene is potential promoter regions of 3371bp.Primer information is seen SEQ ID NO.2 and SEQ ID NO.3 in detail.

3. the amplification of the conservative section of Africa xenopus Dmrt5 promotor

3.1 the extracting of Xenopus laevis genomic dna

1) gets two of tadpoles, put into the Eppendorf tube of 1.5ml, add the lysis buffer of 500 μ l, homogenized;

2) RNase of the 10mg/mL of adding 4.5 μ l puts into 65 ℃ water-bath temperature and bathes 15-30min, removes RNA, the centrifugal 5min of 14000rpm;

3) supernatant is transferred in the new Eppendorf tube, added 500 μ l phenol-chloroforms.Mix, make the upper strata become white emulsion, the centrifugal 5min of 14000rpm;

4) repeat previous action 2-4 time;

5) supernatant is transferred in the new Eppendorf tube, added 500 μ l chloroforms, mix the centrifugal 5min of 12000rpm;

6) supernatant is transferred in the new Eppendorf tube, added the Virahol of 0.6 times of supernatant volume, visible linear DNA is separated out 4 ℃ of frozen centrifugation 15min of 12000rpm;

7) linear DNA is sucked in the new Eppendorf tube, add 70% ethanol of 500 μ l, the centrifugal 15min of 12000rpm;

8) visible drops of exhaustion all in the Eppendorf tube add 70% ethanol of 500 μ l, put upside down for several times, make deposition floating, 4 ℃ of centrifugal 5min;

9) repeat previous action 1-2 time;

The visible drop of in the exhaustion Eppendorf tube all, drying at room temperature is to there not being the ethanol smell.The pH2O dissolving DNA deposition that adds 100 μ l.Measure DNA concentration, get 1 μ l about 20min of electrophoresis in the sepharose of EB painted 0.8%, observe band, confirm that genome is the above fragment of 20Kb.With spectrophotometric instrumentation light absorption value 260/280,260/230 all more than 1.8.

3.2PCR amplification purpose promoter fragment

Through PCR reaction amplification purpose fragment 2 (ROCHE-Taq), reaction system is following:

10×Buffer1 5μl

dNTP(2.5mM) 1.75μl

p5(10μM) 2μl

p3(10μM) 2μl

Genomic dna 1 μ l

ROCHE?Taq 0.75μl

pH2O 37.5μl

TV 50 μ l

The PCR reaction conditions is:

94℃?2min；

68℃7?min；END

After the PCR reaction finishes, run glue with painted 0.8% sepharose of EB and detect, get DNA sample 5 μ l, the about 20min of electrophoresis.The sequence that amplification obtains is seen SEQ.ID NO1.

The structure of 4PMD18-T-Dmrt5 promotor conserved regions carrier

Cut glue recovery purified pcr product 4.1 reclaim test kit through the AxyPrep dna gel:

1) under uv lamp, downcut the sepharose that contains target DNA, paper towel exhausts gel surface liquid and chopping.Calculated for gel weight (writing down 1.5ml centrifuge tube weight in advance), this weight is as a gel volume (like 100mg=100 μ l volume).

2) the Buffer DE-A of 3 gel volumes of adding mixes the back in 75 ℃ of heating, is interrupted mixing (every 2-3min), melts (about 6-8min) fully until gel piece.

3) add the Buffer DE-B of 0.5 Buffer DE-A volume, mix;

4) mixed solution absorption 3) is transferred to DNA preparation pipe (placing the 2ml centrifuge tube), the centrifugal 1min of 12,000 * g.Abandon filtrating.

5) will prepare pipe and put back centrifuge tube, and add 0.5ml Buffer W1, the centrifugal 30s of 12,000 * g abandons filtrating.

6) will prepare pipe and put back centrifuge tube, and add 0.7ml Buffer W2, the centrifugal 30s of 12,000 * g abandons filtrating.

7) wash the centrifugal 1min of 12,000 * g with 0.7ml Buffer W2 again with same method.

8) pipe be will prepare and 2ml centrifuge tube, the centrifugal 1min of 12,000 * g placed.

9) will prepare pipe and place clean 1.5ml centrifuge tube, and prepare the film centre at DNA and add 25-30 μ l water or Eluent, room temperature leaves standstill 1min.The centrifugal 1min eluted dna of 12,000 * g.

With concentration and the purity of spectrophotometric determination DNA, and electrophoresis confirms to cut the effect that glue reclaims.

4.2 the T-Vector of conservative fragments connects, conversion, coated plate and positive colony are identified:

1) connects

By following reaction system fragment being carried out the T carrier connects:

T-vector 1μl

Insert-DNA (being that PCR cuts glue recovery product) 4 μ l

Solution?I 5μl

TV 10 μ l

16 ℃ of connections are spent the night;

2) conversion and coated plate

1. X-gal and IPTG evenly are applied to agar plate and in Eppendorf tube, add the X-gal of 10 μ l 2% and the IPTG of 4 μ l 100mM, are applied to after mixing that to be positioned over 37 ℃ of incubators on the prefabricated LB nutrient agar flat board that contains 20 μ g/mL penbritins for use;

2. transform

A) take out the TG1 competence bacterium 150 μ l of preparation in advance, the T carrier that adds 5 μ l connects product, leaves standstill 30min in the ice;

B) 42 ℃ of water-bath heat shock 90s;

C) put into ice immediately, ice bath 2min;

D) the SOC substratum of 37 ℃ of preheatings of adding 1ml mixes gently;

E) 37 ℃, 50-80rpm shaking culture recovery 1h;

F) gently get rid of with the palm whizzer, inhale and to remove supernatant, during remaining about 200 μ l liquid nutrient mediums resuspended (available liquid-transfering gun is blown and beaten gently, and also available finger flicks at the bottom of the Eppendorf tube);

G) resuspended bacterium liquid is evenly coated on the flat board that 1. step prepare;

H) place 37 ℃ to cultivate about 1hour;

I) to be dried to there not being the bacterium flow moving, be inverted dull and stereotyped about 10hour of cultivation.

3) positive colony is identified

1. from the above-mentioned flat board 4 single bacterium colonies of white of picking in the liquid nutrient medium that contains Amp, 280rpm, 37 ℃ of concussion overnight cultures (about 10hour); Get 500 μ l bacterium liquid to the 1.5ml centrifuge tube, the guarantor who adds about 150 μ l plants and uses glycerine, mixes, and places-80 ℃ to preserve bacterial classification;

2. through AxyPrep DNA small volume of reagent box a small amount of extracting plasmid

A) get the bacterium liquid of 5ml overnight cultures in the LB substratum, the centrifugal 1min of 12,000 * g abandons most supernatant.

B) added the Buffer S1 suspension bacterial precipitation of RNase A with 250 μ l, suspending needs evenly should not leave little bacterium piece.

C) add 250 μ l Buffer S2, gentle also spinning upside down fully mixed 4-6 time, makes the abundant cracking of thalline, until forming bright solution.This step should not surpass 5min.

D) add 350 μ l Buffer S3, gentle also spinning upside down fully mixed the centrifugal 10min of 12,000 * g 6-8 time.

E) draw the centrifugal supernatant in the step 4 and transfer to DNA preparation pipe (placing the 2ml centrifuge tube), the centrifugal 1min of 12,000 * g.

F) will prepare pipe and put back centrifuge tube, and add 500 μ l Buffer W1, the centrifugal 1min of 12,000 * g abandons filtrating.

G) will prepare pipe and put back centrifuge tube, and add 700 μ l Buffer W2, the centrifugal 1min of 12,000 * g abandons filtrating; With same method more once with 700 μ l Buffer W2 washing.Abandon filtrating.

H) will prepare pipe and put back in the 2ml centrifuge tube the centrifugal 1min of 12,000 * g.

I) will prepare pipe and move in the new 1.5ml centrifuge tube, and prepare the film centre at DNA and add 60-80 μ l water or Eluent, room temperature leaves standstill 1min.The centrifugal 1min of 12,000 * g.

Use the spectrophotometric determination plasmid DNA concentration.

3. enzyme is cut evaluation

Get extractive plasmid and do enzyme and cut evaluation, use restriction endonuclease EcoR I single endonuclease digestion, reaction system is following:

pH2O 15.5μl

Plasmid 2 μ l

Takara?10×H?Buffer 2μl

Restriction endonuclease EcoR I 0.5 μ l

TV 20 μ l

After 37 ℃ of enzymes are cut 3h, run glue with painted 0.8% sepharose of EB and detect, get enzyme and cut product 10 μ l, the about 20min of electrophoresis.Successfully obtain PMD18-T-Dmrt5 promotor conserved regions plasmid.

The structure of 5 transgene carrier pCS2-IS-Dmrt5-EGFP plasmids

According to the plasmid and the pCS2-IS-CMV-EGFP plasmid of the existing PMD18-T-Dmrt5 promotor in laboratory, use Sal I and HindIII double digestion respectively, adopt 37 ℃ of enzymes of following reaction system to cut and spend the night:

pH2O 2.5μl

Plasmid (each about 12 μ g) 35 μ l (the PMD18-T-Dmrt5 promoter plasmid and the pCS2-IS-CMV-EGFP plasmid that have conservative fragments)

TAKARA?10×Buffer?K 7.5μl

Restriction endonuclease HindIII 2.5 μ l

Restriction endonuclease Sal I 2.5 μ l

TV 50 μ l

Soft mixing, and instantaneous centrifugal.

After after enzyme cuts; Use the AxyPrep dna gel to reclaim test kit and cut glue recovery pCS2-IS-EGFP fragment and Dmrt5 promoter fragment; Reclaim segmental concentration and purity with spectrophotometer measurement, carry out ligation then, use 16 ℃ of connections of following reaction conditions to spend the night:

Dmrt5 promoter fragment 5 μ l

PCS2-IS-EGFP carrier framework 2 μ l

10×T4ligase?buffer 2.5μl

T4Ligase 1μl

pH2O 14.5μl

TV 25 μ l

Conversion, coated plate, choose clone and the amplification plasmid (concrete grammar is as implied above),

After a small amount of bacterium liquid glycerol adding guarantor kind, collect the about 5ml of remaining bacterium liquid, carry out a small amount of plasmid extraction; Plasmid extraction back well is that enzyme is cut evaluation with I-SceI 10 μ l corpusculums, and qualification result shows and successfully made up the pCS2-IS-Dmrt5-EGFP plasmid.

6I-SceI transgenic test operation step

In test, the injection volume of pCS2-IS-Dmrt5-EGFP plasmid is in 120pg DNA, 1.2 * 10 ^-2During the UI-SceI/ embryo, can obtain best transgenic effect.During DNA that extraction is used to inject, use the ddH2O dissolving DNA, measure DNA concentration with ultraviolet spectrophotometer, be diluted to 100ng/ μ l with ddH2O, per injection is diluted again, guarantees fresh.

When leaving standstill, set up the endonuclease reaction of an I-SceI digestion transgene carrier.In the preliminary experiment,, the amount that adds plasmid and enzyme in the system is groped, seek the righttest injection volume with pCS2-IS-CMV--EGFP positive control plasmid.Be the righttest injection volume (120pg DNA, 1.2 * 10 that obtain according to preliminary experiment below ^-2The UI-SceI/ embryo) enzyme of setting up is cut system:

10×Buffer 1μL

DNA(100ng/μL) 2μL

I-SceI(10U/μL) 2μL

ddH2O 5μL

Total 10μL

The 10 μ L enzymes system of cutting is incubated 30min at 37 ℃, the endonuclease reaction time to after immediately enzyme is cut product and is placed on ice, injection as early as possible, preferably every annotations and comments are penetrated and are all used fresh enzyme to cut product, to guarantee transgene efficiency.

Treat the zygote upset, the animal pole of black with 0.1 * MBS sucking-off, adds 2%cysteine solution striping with suction pipe up, softly shakes until the outer field cutose of ovum and removes, and ovum can contact with each other.Outwell cysteine solution immediately, softly wash 4-5 time, pour in the clean glass culture dish with 0.1 * MBS.

Examine under a microscope, the ovum of the measured cell stage of matter of picking fertilization is in the injection grid ware that fills 4%Ficoll (among 0.3 * MBS), and 50 left and right sides embryos of every ware line up row with headband with ovum, are convenient to injection.To microinjection instrument dress pin, in pin, draw the reaction mixture of 2-3 μ L simultaneously, be adjusted to each embryo's injection 6nl solution.

For the zygote of X.laevis, the optimal injection amount that preliminary experiment is groped is 120pg DNA, 1.2 * 10 ^-2The UI-SceI/ embryo.Injection of solution is arrived the intersection of animal pole and vegetative pole one side near animal pole, be expelled under the cortex.For guaranteeing that enzyme cuts the fresh of product, should inject as early as possible, leave standstill 5min after having injected.

After the injection, the embryo was cultivated 2 hours in 13 ℃ of incubators, take out then and place at room temperature.Up to fetal development to the 9 after dates, the embryo to be transferred in the little plastic culture dish that 0.3 * MBS is housed, each ware is put 20-25 embryo, 18 ℃ of overnight cultures.Note, also want the some zygote synchronized culture that do not have injection of picking as contrast.

The developmental state of regularly observing the embryo is observed the green fluorescence phenotype under fluorescent microscope, and take pictures (unified exposure 20s).When growing the tadpole that can move about to the later stage, the MS-222 of available 50mg/L anaesthetizes to observe and takes pictures.Observe a week continuously, after phenotype is basicly stable, can feed green alga, the transgenic tadpole is formed the adult frog to tadpole.

7 test-results

7.1Dmrt5 the detection of promotor control EGFP expression plasmid pCS2-IS-Dmrt5-EGFP

The plasmid detected result shows the Dmrt5-EGFP embryo whole body expression EGFP that the microinjection of pCS2-IS-Dmrt5-EGFP plasmid obtains, and control group is the embryo who does not inject, and has no luciferase expression.The embryo that injection pCS2-IS-Dmrt5-EGFP plasmid is described can express the EGFP that is started by the Dmrt5 promotor, and promptly the structure of this plasmid is correct.

7.2Dmrt5 EGFP is specific expressed in neural system in promoter region control

Operate according to 6 transfer gene injection conditions, and to successfully integrating observation, the tracking that the segmental embryo of IS-Dmrt5-EGFP has carried out the successive green fluorescence.When fetal development to the in the time of five days, genetically modified embryo has very strong luciferase expression at eye, midbrain, hindbrain and whole neurocele, explains that the Dmrt5-EGFP transgenic fragment can control green fluorescent protein in the stable expression of neural system.Culture through careful, genetically modified embryo survival has arrived the period of the abnormal back frog, has obtained genetically modified head and has built Xenopus laevis.

In sum, the present invention finds first and proves that the Dmrt5 promotor is the tissue specificity controlling element of taking advantage of a situation, this Dmrt5 promoter region can controlling gene specific expressed at eye, midbrain, hindbrain and whole neurocele.Simultaneously, transgenic experiments has proved that also the method for structure pCS2-IS-Dmrt5-EGFP of the present invention is practicable.

Claims (1)

1. an Africa xenopus dmrt5 gene nerve-specific controlling element is characterized in that the nucleotide sequence of this controlling element is shown in the SEQ ID No.1.

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