CN102719465A - Inducible tissue-specific expression vector and purpose thereof - Google Patents

Abstract

The invention belongs to the field of biological technology, relating to an inducible tissue-specific expression vector and a purpose thereof. The inducible tissue-specific expression vector mainly comprises the following structures of: a promoter EF1 alpha, an EGFP fluorescent marker gene, a BGHpoly A tailing transcription termination sequence with two ends linked with same-direction Lox sequences, and an intron sequence with internal enzyme sites introduced. The vector is pEFGLPAL-MIR using a general promoter EF1alpha to drive gene expression; the EGFP fluorescent marker gene is used for indication of the gene expression; two BDHpoly A tailing transcription termination signals linked with same-direction LoxP71 and LoxP66 are inducible by enzyme Cre. The termination signals are followed by an intron sequence, with an miRNA expression precursor is inserted into the intron. The micro RNA expression vector can successfully express specific micro RNA and the micro RNA expression can be determined by various ways. The expression vector is capable of expressing uneasily-silenced micro RNA in an ES cell, so that can be successfully applied in the preparation of transgenic mice.

Description

Inducibility tissue specific expression carrier and uses thereof

Technical field

The invention belongs to biological technical field, relate to inducibility tissue specific expression carrier and uses thereof.Be specifically related to microRNA inducibility tissue specific expression carrier and purposes; Especially mediation, derivable, the tissue-specific miRNA expression vector pEFG LPAL-MIR of a kind of Cre enzyme; Said carrier can use express microRNA and set up inducibility, organizing specific expression transgenic experimental animal model.

Background technology

The research report, the noncoding single stranded RNA molecule of little endogenous that microRNA is made up of 21-25 Nucleotide is to be given birth to through Dicer enzyme processing back by the single stranded RNA precursor of about 70-90 base size with hairpin structure; Said precursor is double-stranded by the microRNA of 22 Nucleotide of RNaseIII Dicer cutting written treaty among cell, and wherein a chain is degraded, and other one is processed to sophisticated microRNA.In the zooblast experiment, microRNA generally combines in 3 '-UTR of target gene mRNA (3 ' end non-coding region) through the partial sequence homology, causes translation to stop, and under situation seldom, causes the degraded of mRNA.The discovery of microRNA has disclosed a kind of new generegulation mechanism, grow and cytodifferentiation aspect rise and have important function and significance.Research discloses, and miRNAs participates in a series of important process in the vital process, as, early development, cell proliferation, apoptosis, necrocytosis, metabolism of fat and cytodifferentiation etc.The very big relation that has of discovering microRNA and numerous disease is arranged, and such as tumour, heart trouble or the like can further instruct the diagnosis of disease.Because popularity and variety that microRNA exists, and much the expression of miRNA has the specificity of tissue and etap expression, therefore points out microRNA to have more extensively various biological function.

The research of diseases related at present need inducibility, organizing specific expression transgenic experimental animal model; But up to now; Shang Weijian has to be specifically designed to and makes up the especially miRNA expression vector of transgenic test mice of transgenic experimental animal model; Common in addition CMV promotor is easy to quilt silence in the ES cell, and these all are that the research of microRNA or the preparation of transgenic experimental animal model have caused very big difficulty.

Summary of the invention

The objective of the invention is to overcome the deficiency of prior art; A kind of inducibility tissue specific expression carrier is provided; Be specifically related to derivable, tissue-specific miRNA expression vector, mediation, derivable, the tissue-specific miRNA expression vector pEFG LPAL-MIR of especially a kind of Cre enzyme.Said carrier can be used for expressing microRNA and set up inducibility, organizing specific expression transgenic experimental animal model.

Further purpose of the present invention provides described expression vector and is being used to express microRNA and is setting up purposes inducibility, in the organizing specific expression transgenic experimental animal model.

The present invention expresses miRNA with the intron mode, and described carrier can also be realized the effective expression of miRNA when not influencing marker gene EGFP expression; This carrier can be used for experiment in vitro and transgenic mice, and described Cre enzyme has good inducibility to the expression of this carrier.

In the expression vector of the present invention, on the basis of pEGFP plasmid, added EF1 alpha promotor.

Particularly; Inducibility tissue specific expression carrier of the present invention; It is characterized in that; Mainly comprise following structure: EF1 α promotor, EGFP fluorescent mark gene, two ends are connected with the BGH polyA tailing transcription termination sequence and the inner intron sequences of introducing restriction enzyme site of LoxP sequence in the same way; Described carrier is pEFG LPAL-MIR, has the structure of sequence 1.

Among the present invention, said carrier uses EF1 alpha generally starting to drive genetic expression; EGFP fluorescent mark gene is used to indicate expression of gene; Be two sections then and be connected with the BDH polyA tailing transcription termination signal of LoxP71 and LoxP66 in the same way, can be by the Cre enzyme induction; Described termination signal thereafter be one section intron sequences, express the miRNA precursor and insert in this intron;

Among the present invention, EF1 α promotor has the higher level expression and is not easy by silence;

Among the present invention, EGFP fluorescent mark gene is convenient to the evaluation and the genetically modified expression of real-time monitored of transgenic mice in the future;

Among the present invention, the Cre enzyme can effectively mediate these two ends and be connected with the excision of the BGH polyA tailing transcription termination sequence of LoxP sequence in the same way, thereby effectively controls whether transcribing of 3 ' sequence;

Among the present invention, intron sequences is transformed, introduced restriction enzyme site, thereby can the miRNA precursor be inserted this site, realize effective shearing of miRNA precursor, do not disturb the EGFP fluorescence gene expression of its front.

In the embodiments of the invention, adopt EF1 α, realize the high expression level of miR424 locus, and for the gene that prevents to be changeed in the transgenic animal in future by silence; Introduced EGFP fluorescent mark albumen, be convenient to identify transgenic mice and monitor genetically modified expression in real time; C) added respectively at the two ends of miRNA precursor intron donor and receptor sequence (can with the miRNA precursor from transcript as intron excision, and be processed to form ripe body), make the tandem expression of miRNA not influence the expression of EGFP; Between EGFP gene and miR424 precursor, inserted one section PolyA tailing transcription termination signal; And respectively add one section LoxP sequence at the tailing signal two ends; Mouse through with myocardium specifically expressing Cre hybridizes, and can the special cyclisation excision in the myocardial cell of this PolyA tailing transcription termination signal be implemented in the expression of F1 for the specific miRNA of special rise in the mouse cardiac muscle; Realize the tissue-specific up-regulated of miRNA, and prevented that expression from causing first-generation transgenic mice dead.

MicroRNA inducibility tissue specific expression carrier of the present invention shows that through experiment in vitro Cre has good inducibility to this miRNA expression vector.

Among the present invention, behind EGFP, added two LoxP71 and LoxP66 sites in the same way, these two LoxP have the site of sudden change, guarantee that other LoxP recombinates on LoxP residual after recombinating with cre and the karyomit(e).The polyA sequence that in the middle of these two LoxP sites, has added BDH makes that microRNA can not express before not recombinating with the cre enzyme, has avoided in the transgenic mice preparation, F1 occurring for the embryonic death that possibly cause because microRNA crosses expression.

Carrier of the present invention has following characteristics:

1) can solve the difficulty that some lethality miRNA transgenic animal is set up: the up-regulated of some miRNA has serious illness; For example miR-1 crosses the rhythm abnormality that causes of expression; So the up-regulated of these miRNA possibly be lethality for animal, so this type miRNA is not easy to obtain the high expression level transgenic mice, and this carrier transgenic mice is not before hybridizing with the Cre mouse; Be not express miRNA, so solved the difficult problem of setting up of lethality miRNA transgenic mice;

2) can realize sense of organization specifically expressing and the expression of etap property of specific miRNA easily: the foundation that routine is set up the tissue specific expression transgenic mice is through the using-system specificity promoter; This mode has its limitation; In case promptly transgenic animal are set up, comparatively mobile is realized the expression of its hetero-organization.And this carrier is set up transgenic mice, can be through selecting tissue specificity and etap specific C re mouse, and mobile raises purpose miRNA at different tissues and different steps.

3) EF1 α promotor is selected for use and intron is expressed the mode of miRNA, also is the assurance that realizes the miRNA effective expression, and the expression that guarantees miRNA can not disturbed the expression of series connection mark;

4) can simplify transgenic animal screening and monitoring in real time with the placed in-line EGFP gene of miRNA.

The present invention has confirmed the carrier transfection HEK293 cell that obtains the expression of microRNA through Fluirescence observation and RT-PCR method.

The expression vector transfection mouse E14 cell of the present invention to making up confirmed the expression of microRNA in ES cells through Fluirescence observation and RT-PCR method.

The present invention prepares transgenic mice with the expression vector that obtains, and at F1 the mouse that has green fluorescence of survival is arranged in generation.

The microRNA expression vector that makes up among the present invention can be successful the special microRNA of expression, and can confirm the expression of microRNA through several different methods.Expression vector among the present invention is in the ES cell, and microRNA expresses and is not easy by silence, can the successful preparation that is applied to transgenic mice.

Description of drawings

Fig. 1 shows the LPAL-mir-363 with pEFG; Behind the pEFG LPAL-mir-363 cre; PEFG LPAL-mir-424 (uses transfection reagent LTX) among the plasmid transfection mouse stem cells E14 behind the pEFG LPAL-mir-424 cre, differentiates plasmid expression through observing green fluorescence behind the 24h.

Fig. 2 shows among the present invention extracting cell RNA behind the cell transfecting 48h, behind tailing method RT-PCR, detects the expression of miR-363 and miR-424, and 18s does confidential reference items, and miRNA can express behind the cre.

Fig. 3 shows that intron primer loxp71 BGH F/loxp66 BGH T PCR specific among the present invention shows that miRNA shears processing back intron and diminishes.

Embodiment

Embodiment 1 makes up pEFG LPAL-MIR

1) inserts EF1 alpha promotor

Design has the primer of corresponding restriction enzyme site according to the pEBG plasmid sequence; With EF1 alpha promoter sequence through the PCR-enzyme cut-ligation is building up in the pEGFP-c1 plasmid; There is not sudden change through order-checking comparison EF1 alpha promoter sequence, and with this plasmid called after pEFG that builds.

2) insert the LoxP-polyA-LoxP sequence

The LoxP71 of each 35bp of chemosynthesis and LoxP66.The polyA1 sequence comes from BGH polyA sequence among the pcDNA3.1 (+).With of the method series connection of 3 sections sequences with overlapping PCR; Order be LoxP71-polyA1-LoxP66, and two LoxP sites keep the cis-position directions, this segment DNA is passed through enzyme cut-ligation is building up in the pEFG carrier; There are not sudden change, called after pEFG LPAL through the order-checking comparison.

3) insert the intron sequence

Intron (intron) sequence comes from the pRL-renilla plasmid; Intron D and intron R are introduced Bgl II successively through overlapping PCR method centre; EcoR V, Sall I restriction enzyme site, and before the DNA that obtains is inserted into pEFGFP plasmid SV40 polyA sequence; The plasmid called after pEFG LPAL-MIR that builds, total length is 6287bp (having the structure like sequence 1).

The application of embodiment 2 pEFG LPAL-MIR

1) makes up pEFG LPAL-mir-363 and pEFG LPAL-mir-424

Mir-363 and mir-424 precursor sequence are obtained through the PCR reaction, and be inserted in the pEFG LPAL-MIR carrier, do not have sudden change through order-checking.

2) plasmid is external recombinates with cre enzyme (NEB)

Reaction system (20ul):

Plasmid 5-10ng

10×Buffer 2ul

Cre 0.5ul

ddH2O to?20ul

37°C 30min，72°C?10min。

Transform the TOP10 competence, 37 ° of C 12h, clone PCR is identified (primer is loxp71 BGH F/loxp66 BGH T), does not have the plasmid for the excision of cyclisation of obvious band, and confirms through order-checking.

3) expression of pEFG LPAL-MIR in mouse embryo stem cell

As shown in Figure 1; With pEFG LPAL-mir-363, behind the pEFG LPAL-mir-363 cre, pEFG LPAL-mir-424; (use transfection reagent LTX) among the plasmid transfection mouse stem cells E14 behind the pEFG LPAL-mir-424 cre, differentiate plasmid expression through observing green fluorescence behind the 24h.

As shown in Figure 2, extracting cell RNA behind the cell transfecting 48h detects the expression of miR-363 and miR-424 behind tailing method RT-PCR, and 18s does confidential reference items, and miRNA can express behind the cre.

As shown in Figure 3, specific intron primer loxp71 BGH F/loxp66 BGH T PCR shows that miRNA shears processing back intron and diminishes.

 

Embodiment 3 preparation transgenic mices

(ApaL I) behind pEFG LPAL-mir-363 and the pEFG LPAL-mir-424 plasmid linearization changed over to the zygote of mouse by ordinary method; This zygote is implanted in the female mice body; The result shows that F1 is accredited as transgenic mice for mouse through egfp expression.

SEQUENCE?LISTING

 

< 110>Fudan University

 

< 120>inducibility tissue specific expression carrier and uses thereof

 

<160> 1

 

<170> PatentIn?version?3.3

 

<210> 1

<211> 6287

<212> DNA

<213> Artificial

 

 

<400> 1

tagttattat?cgacattgat?tattgactag?ttattaatcc?tcggcctctg?agctattcca 60

 

gaagtagtga?ggaggctttt?ttggaggcct?aggcttttgc?aaaaagcttt?gcaaagatgg 120

 

ataaagtttt?aaacagagag?gaatctttgc?agctaatgga?ccttctaggt?cttgaaagga 180

 

gtgggaattg?gctccggtgc?ccgtcagtgg?gcagagcgca?catcgcccac?agtccccgag 240

 

aagttggggg?gaggggtcgg?caattgaacc?ggtgcctaga?gaaggtggcg?cggggtaaac 300

 

tgggaaagtg?atgtcgtgta?ctggctccgc?ctttttcccg?agggtggggg?agaaccgtat 360

 

ataagtgcag?tagtcgccgt?gaacgttctt?tttcgcaacg?ggtttgccgc?cagaacacag 420

 

gtaagtgccg?tgtgtggttc?ccgcgggcct?ggcctcttta?cgggttatgg?cccttgcgtg 480

 

ccttgaatta?cttccacgcc?cctggctgca?gtacgtgatt?cttgatcccg?agcttcgggt 540

 

tggaagtggg?tgggagagtt?cgaggccttg?cgcttaagga?gccccttcgc?ctcgtgcttg 600

 

agttgaggcc?tggcctgggc?gctggggccg?ccgcgtgcga?atctggtggc?accttcgcgc 660

 

ctgtctcgct?gctttcgata?agtctctagc?catttaaaat?ttttgatgac?ctgctgcgac 720

 

gctttttttc?tggcaagata?gtcttgtaaa?tgcgggccaa?gatctgcaca?ctggtatttc 780

 

ggtttttggg?gccgcgggcg?gcgacggggc?ccgtgcgtcc?cagcgcacat?gttcggcgag 840

 

gcggggcctg?cgagcgcggc?caccgagaat?cggacggggg?tagtctcaag?ctggccggcc 900

 

tgctctggtg?cctggcctcg?cgccgccgtg?tatcgccccg?ccctgggcgg?caaggctggc 960

 

ccggtcggca?ccagttgcgt?gagcggaaag?atggccgctt?cccggccctg?ctgcagggag 1020

 

ctcaaaatgg?aggacgcggc?gctcgggaga?gcgggcgggt?gagtcaccca?cacaaaggaa 1080

 

aagggccttt?ccgtcctcag?ccgtcgcttc?atgtgactcc?acggagtacc?gggcgccgtc 1140

 

caggcacctc?gattagttct?cgagcttttg?gagtacgtcg?tctttaggtt?ggggggaggg 1200

 

gttttatgcg?atggagtttc?cccacactga?gtgggtggag?actgaagtta?ggccagcttg 1260

 

gcacttgatg?taattctcct?tggaatttgc?cctttttgag?tttggatctt?ggttcattct 1320

 

caagcctcag?acagtggttc?aaagtttttt?tcttccattt?caggtgtcgt?gaggaattcc 1380

 

tcgactagcg?ctaccggtcg?ccaccatggt?gagcaagggc?gaggagctgt?tcaccggggt 1440

 

ggtgcccatc?ctggtcgagc?tggacggcga?cgtaaacggc?cacaagttca?gcgtgtccgg 1500

 

cgagggcgag?ggcgatgcca?cctacggcaa?gctgaccctg?aagttcatct?gcaccaccgg 1560

 

caagctgccc?gtgccctggc?ccaccctcgt?gaccaccctg?acctacggcg?tgcagtgctt 1620

 

cagccgctac?cccgaccaca?tgaagcagca?cgacttcttc?aagtccgcca?tgcccgaagg 1680

 

ctacgtccag?gagcgcacca?tcttcttcaa?ggacgacggc?aactacaaga?cccgcgccga 1740

 

ggtgaagttc?gagggcgaca?ccctggtgaa?ccgcatcgag?ctgaagggca?tcgacttcaa 1800

 

ggaggacggc?aacatcctgg?ggcacaagct?ggagtacaac?tacaacagcc?acaacgtcta 1860

 

tatcatggcc?gacaagcaga?agaacggcat?caaggtgaac?ttcaagatcc?gccacaacat 1920

 

cgaggacggc?agcgtgcagc?tcgccgacca?ctaccagcag?aacaccccca?tcggcgacgg 1980

 

ccccgtgctg?ctgcccgaca?accactacct?gagcacccag?tccgccctga?gcaaagaccc 2040

 

caacgagaag?cgcgatcaca?tggtcctgct?ggagttcgtg?accgccgccg?ggatcactct 2100

 

cggcatggac?gagctgtaca?agtccggata?gaatagtacc?gttcgtatag?catacattat 2160

 

acgaagttat?catcatcacc?atcaccattg?agtttaaacc?cgctgatcag?cctcgactgt 2220

 

gccttctagt?tgccagccat?ctgttgtttg?cccctccccc?gtgccttcct?tgaccctgga 2280

 

aggtgccact?cccactgtcc?tttcctaata?aaatgaggaa?attgcatcgc?attgtctgag 2340

 

taggtgtcat?tctattctgg?ggggtggggt?ggggcaggac?agcaaggggg?aggattggga 2400

 

agacaatagc?aggcatgctg?gggatgcggt?gggctctatg?gcttctgagg?cggaaagaac 2460

 

cagctggggc?tctagggggt?atccccacgc?gccctgtagc?ggcgcattaa?gcgcggcggg 2520

 

tgtggtggtt?acgcgcagcg?tgaccgctac?acttgccagc?gccctagcgc?ccgctccttt 2580

 

cgctttcttc?ataacttcgt?atagcataca?ttatacgaac?ggtatccgga?ctcagatcct 2640

 

agataacttc?tgacacaaca?gtctcgaact?taagctgcag?aagttggtcg?tgaggcactg 2700

 

ggcaggtaag?tatcaaggtt?acaagacagg?tttaaggaga?ccaatagaaa?ctgggcttgt 2760

 

cgagacagag?aagactcttg?cgtttctgat?aggcaccaag?tagatctgat?atcgtcgacg 2820

 

cactattggt?cttactgaca?tccactttgc?ctttctctcc?acaggtgtcc?actcccagtt 2880

 

caattacagc?tcttaaggct?agagtactta?atacgactca?ctatagctcg?acggtaccgc 2940

 

gggcccgatc?caccggatct?agataactga?tcataatcag?ccataccaca?tttgtagagg 3000

 

ttttacttgc?tttaaaaaac?ctcccacacc?tccccctgaa?cctgaaacat?aaaatgaatg 3060

 

caattgttgt?tgttaacttg?tttattgcag?cttataatgg?ttacaaataa?agcaatagca 3120

 

tcacaaattt?cacaaataaa?gcattttttt?cactgcattc?tagttgtggt?ttgtccaaac 3180

 

tcatcaatgt?atcttaaggc?gtaaattgta?agcgttaata?ttttgttaaa?attcgcgtta 3240

 

aatttttgtt?aaatcagctc?attttttaac?caataggccg?aaatcggcaa?aatcccttat 3300

 

aaatcaaaag?aatagaccga?gatagggttg?agtgttgttc?cagtttggaa?caagagtcca 3360

 

ctattaaaga?acgtggactc?caacgtcaaa?gggcgaaaaa?ccgtctatca?gggcgatggc 3420

 

ccactacgtg?aaccatcacc?ctaatcaagt?tttttggggt?cgaggtgccg?taaagcacta 3480

 

aatcggaacc?ctaaagggag?cccccgattt?agagcttgac?ggggaaagcc?ggcgaacgtg 3540

 

gcgagaaagg?aagggaagaa?agcgaaagga?gcgggcgcta?gggcgctggc?aagtgtagcg 3600

 

gtcacgctgc?gcgtaaccac?cacacccgcc?gcgcttaatg?cgccgctaca?gggcgcgtca 3660

 

ggtggcactt?ttcggggaaa?tgtgcgcgga?acccctattt?gtttattttt?ctaaatacat 3720

 

tcaaatatgt?atccgctcat?gagacaataa?ccctgataaa?tgcttcaata?atattgaaaa 3780

 

aggaagagtc?ctgaggcgga?aagaaccagc?tgtggaatgt?gtgtcagtta?gggtgtggaa 3840

 

agtccccagg?ctccccagca?ggcagaagta?tgcaaagcat?gcatctcaat?tagtcagcaa 3900

 

ccaggtgtgg?aaagtcccca?ggctccccag?caggcagaag?tatgcaaagc?atgcatctca 3960

 

attagtcagc?aaccatagtc?ccgcccctaa?ctccgcccat?cccgccccta?actccgccca 4020

 

gttccgccca?ttctccgccc?catggctgac?taattttttt?tatttatgca?gaggccgagg 4080

 

ccgcctcggc?ctctgagcta?ttccagaagt?agtgaggagg?cttttttgga?ggcctaggct 4140

 

tttgcaaaga?tcgatcaaga?gacaggatga?ggatcgtttc?gcatgattga?acaagatgga 4200

 

ttgcacgcag?gttctccggc?cgcttgggtg?gagaggctat?tcggctatga?ctgggcacaa 4260

 

cagacaatcg?gctgctctga?tgccgccgtg?ttccggctgt?cagcgcaggg?gcgcccggtt 4320

 

ctttttgtca?agaccgacct?gtccggtgcc?ctgaatgaac?tgcaagacga?ggcagcgcgg 4380

 

ctatcgtggc?tggccacgac?gggcgttcct?tgcgcagctg?tgctcgacgt?tgtcactgaa 4440

 

gcgggaaggg?actggctgct?attgggcgaa?gtgccggggc?aggatctcct?gtcatctcac 4500

 

cttgctcctg?ccgagaaagt?atccatcatg?gctgatgcaa?tgcggcggct?gcatacgctt 4560

 

gatccggcta?cctgcccatt?cgaccaccaa?gcgaaacatc?gcatcgagcg?agcacgtact 4620

 

cggatggaag?ccggtcttgt?cgatcaggat?gatctggacg?aagagcatca?ggggctcgcg 4680

 

ccagccgaac?tgttcgccag?gctcaaggcg?agcatgcccg?acggcgagga?tctcgtcgtg 4740

 

acccatggcg?atgcctgctt?gccgaatatc?atggtggaaa?atggccgctt?ttctggattc 4800

 

atcgactgtg?gccggctggg?tgtggcggac?cgctatcagg?acatagcgtt?ggctacccgt 4860

 

gatattgctg?aagagcttgg?cggcgaatgg?gctgaccgct?tcctcgtgct?ttacggtatc 4920

 

gccgctcccg?attcgcagcg?catcgccttc?tatcgccttc?ttgacgagtt?cttctgagcg 4980

 

ggactctggg?gttcgaaatg?accgaccaag?cgacgcccaa?cctgccatca?cgagatttcg 5040

 

attccaccgc?cgccttctat?gaaaggttgg?gcttcggaat?cgttttccgg?gacgccggct 5100

 

ggatgatcct?ccagcgcggg?gatctcatgc?tggagttctt?cgcccaccct?agggggaggc 5160

 

taactgaaac?acggaaggag?acaataccgg?aaggaacccg?cgctatgacg?gcaataaaaa 5220

 

gacagaataa?aacgcacggt?gttgggtcgt?ttgttcataa?acgcggggtt?cggtcccagg 5280

 

gctggcactc?tgtcgatacc?ccaccgagac?cccattgggg?ccaatacgcc?cgcgtttctt 5340

 

ccttttcccc?accccacccc?ccaagttcgg?gtgaaggccc?agggctcgca?gccaacgtcg 5400

 

gggcggcagg?ccctgccata?gcctcaggtt?actcatatat?actttagatt?gatttaaaac 5460

 

ttcattttta?atttaaaagg?atctaggtga?agatcctttt?tgataatctc?atgaccaaaa 5520

 

tcccttaacg?tgagttttcg?ttccactgag?cgtcagaccc?cgtagaaaag?atcaaaggat 5580

 

cttcttgaga?tccttttttt?ctgcgcgtaa?tctgctgctt?gcaaacaaaa?aaaccaccgc 5640

 

taccagcggt?ggtttgtttg?ccggatcaag?agctaccaac?tctttttccg?aaggtaactg 5700

 

gcttcagcag?agcgcagata?ccaaatactg?tccttctagt?gtagccgtag?ttaggccacc 5760

 

acttcaagaa?ctctgtagca?ccgcctacat?acctcgctct?gctaatcctg?ttaccagtgg 5820

 

ctgctgccag?tggcgataag?tcgtgtctta?ccgggttgga?ctcaagacga?tagttaccgg 5880

 

ataaggcgca?gcggtcgggc?tgaacggggg?gttcgtgcac?acagcccagc?ttggagcgaa 5940

 

cgacctacac?cgaactgaga?tacctacagc?gtgagctatg?agaaagcgcc?acgcttcccg 6000

 

aagggagaaa?ggcggacagg?tatccggtaa?gcggcagggt?cggaacagga?gagcgcacga 6060

 

gggagcttcc?agggggaaac?gcctggtatc?tttatagtcc?tgtcgggttt?cgccacctct 6120

 

gacttgagcg?tcgatttttg?tgatgctcgt?caggggggcg?gagcctatgg?aaaaacgcca 6180

 

gcaacgcggc?ctttttacgg?ttcctggcct?tttgctggcc?ttttgctcac?atgttctttc 6240

 

ctgcgttatc?ccctgattct?gtggataacc?gtattaccgc?catgcat 6287

 

Claims (8)

1. inducibility tissue specific expression carrier is characterized in that, mainly comprises following structure: EF1 α promotor, EGFP fluorescent mark gene, BGH polyA tailing transcription termination sequence and intron sequences; Described carrier is pEFG LPAL-MIR, has the structure of sequence 1.

2. by the described inducibility tissue specific expression of claim 1 carrier, it is characterized in that described BGH polyA tailing transcription termination sequence is that two ends are connected with the transcription termination sequence of LoxP sequence in the same way.

3. by the described inducibility tissue specific expression of claim 1 carrier, it is characterized in that its inner restriction enzyme site of introducing of described intron sequences.

4. the inducibility tissue specific expression construction of carrier of claim 1 is characterized in that it comprises step:

1) inserts EF1 alpha promotor

EF1 alpha promoter sequence is inserted in the pEGFP-c1 plasmid, gets plasmid pEFG;

2) insert the LoxP-polyA-LoxP sequence

With the method series connection of LoxP71 and LoxP66 and BGH polyA sequence with overlapping PCR, order be the DNA section of LoxP71-polyA1-LoxP66, be inserted in the pEFG carrier, must plasmid pEFG LPAL;

3) insert the intron sequence

Intron sequences intron D and intron R are through introduce Bgl II successively in the middle of the overlapping PCR method; EcoR V; Sall I restriction enzyme site, and before the DNA that obtains inserted pEFG LPAL plasmid SV40 polyA sequence, make up pEFG LPAL-MIR; Total length is 6287bp, has the structure like sequence 1.

5. by the method for claim 4, it is characterized in that the LoxP71 of said step 2 and LoxP66 are chemosynthesis; Described two LoxP sites are the cis-position direction.

6. the purposes of the inducibility tissue specific expression carrier of claim 1 in improving the effective expression of miRNA.

7. by the purposes of claim 6, the effective expression that it is characterized in that described miRNA is external or the interior effective expression of body.

8. the purposes of the inducibility tissue specific expression carrier of claim 1 in preparation transgenic experimental animal model.

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