CN103352053A

CN103352053A - Exogenous-gene removable slow virus controlled expression carrier system and application

Info

Publication number: CN103352053A
Application number: CN2013102925676A
Authority: CN
Inventors: 付强; 杨润林; 邓黎莉; 彭莹; 丁月娣; 范俊
Original assignee: Jiangsu Institute of Nuclear Medicine
Current assignee: Jiangsu Institute of Nuclear Medicine
Priority date: 2013-07-11
Filing date: 2013-07-11
Publication date: 2013-10-16

Abstract

The invention discloses an exogenous-gene removable slow virus controlled expression carrier system and an application. The system comprises a reaction/regulation plasmid and a packaging plasmid; a construction method is as follows: a loxP sequence is inserted into a pSMPUW 3'LTR through site-specific mutagenesis, an original promoter is replaced by a TRE and a simplest CMV promoter, and an exogenous reporter gene is inserted into an MCS to obtain the reaction plasmid; a CMV-rtTA2S-M2 sequence, an EMCV sequence and a Cre-ER sequence are inserted into the pSMPUW subjected to site-specific mutagenesis to obtain the regulation plasmid. The application is as follows: packaging is conducted to obtain a reaction/regulation slow virus; both the reaction slow virus and the regulation slow virus are cotransfected to a target stem cell, a Dox induction exogenous reporter gene is used for expressing and tracing the image, and finally a 4-OH-tamoxifen is used for inducing and excising the exogenous reporter gene. According to the invention, the exogenous reporter gene is expressed in a controlled manner in vivo and can also be removed immediately to avoid disturbing the normal physiological function of a target cell.

Description

Slow virus controlled expression carrier system and application that a kind of foreign gene is removable

Technical field

The present invention relates to carry the Lentiviral system of exogenous reporter gene, relate in particular to by doxycycline (Dox) regulating and expressing, and the slow virus controlled expression carrier system that the exogenous reporter gene that carries can remove from host chromosome, and the application of this system in stem cell vivo tracking technology.Belong to gene engineering technology field.

Background technology

The expression of crossing of exogenous reporter gene needs one to continue, stablize and efficiently transgenosis and expression system.At present, the expression of most of foreign genes selects the virus expression carrier system to finish.In all kinds of virus expression carrier system: retrovirus (Retrovirus) carrier only can transfection initiatively divide the cell of (actively dividing) usually, and has insertion mutation or copy the ability of virus of future generation, has certain risk; Adenovirus carrier can be guaranteed efficiently expressing of foreign gene, but its immunogenic protein also might obtain expressing, and causes host immune response, in addition, its foreign gene that carries is not integrated into cell chromosome, therefore the expression amount continuous decrease of this foreign gene in the daughter cell of division; And lentiviral vectors can stably be incorporated in the host cell chromosome, can continue long-term expression in the cell of division and non-division, and therefore, slow virus carrier system has become widely one of expression vector of present application conduct.

Now, the gene therapy of the human various diseases (such as Parkinson's disease, thalassemia etc.) take slow virus as carrier has entered clinical trial, development along with the molecular imaging theory and technology, the vivo tracking technology is being played the part of more and more important role in the fundamental research of stem cell (comprising tumour cell) and clinical application research field, its by video picture spike means can non-invasive dynamic monitoring carry stem cell migration, distribution and the survival condition in vivo of the exogenous reporter gene lentiviral vectors of mark, have broad application prospects.

Yet, adopt the lentiviral vectors expression system inevitably exogenous dna fragment (to be comprised that external source is integrated into the stem cell genome, but whether these integrate fragment to existence, the differentiation of stem cell and breed can have a negative impact and it be unclear that, therefore, after the lentiviral vectors expression system was finished its tracking function, it was the optimal selection of avoiding the host cell physiological function disturbed that allogenic gene is removed fully.

Summary of the invention

For the defects that existing Lentiviral system exists, one of purpose of the present invention provides the removable slow virus controlled expression carrier system of a kind of foreign gene.This system comprises expression plasmid and packaging plasmid, and described expression plasmid comprises the reaction plasmid and regulate plasmid, and makes up by the following method respectively and obtain:

(1) structure of reaction plasmid

Rite-directed mutagenesis by lentiviral vectors pSMPUW3 ' LTR U3 area part base is introduced restriction enzyme site, and the loxP sequence is inserted described pSMPUW3 ' LTR, gets mutant plasmid; With the cis-acting elements TRE on the Tet-on system response plasmid pTRE-Tight with simplify the promoter sequence that the CMV promoter sequence replaces described mutant plasmid most, and with the multiple clone site MCS position that exogenous reporter gene inserts described mutant plasmid, get the reaction plasmid; Described exogenous reporter gene is in described TRE and simplifies most under the control of CMV promotor, and by the Dox abduction delivering.In slow virus reverse transcription process, its 3 ' LTR U3 zone will be replicated and transfer to 5 ' LTR, in case the Lentiviral System integration enters host genome, 5 ' LTR and 3 ' LTR be produced a loxP sequence with each.

(2) structure of adjusting plasmid

First rite-directed mutagenesis introducing Asc I restriction enzyme site is carried out in the pSMPUW multiple clone site district of rite-directed mutagenesis in the step (1), be beneficial to the insertion of following CMV-rtTA2S-M2 sequence, internal ribosome entry site EMCV sequence and Cre-ER sequence; CMV-rtTA2S-M2 sequence in the Tet-on system, internal ribosome entry site EMCV sequence and Cre-ER sequence order are coupled together, and the multiple clone site of the pSMPUW of rite-directed mutagenesis zone gets the adjusting plasmid in the inserting step (1); Described rtTA2S-M2 sequence and Cre-ER sequence can be translated on same mRNA chain and express independently of one another, generate rtTA2S-M2 albumen and Cre recombinase estrogen receptor fusion rotein.

Its further technical scheme is:

Described packaging plasmid comprises pRSV-Rev, pCMV-VSV-G, pCgpV.

Described mutational site sequence is ACCGGT, and the sudden change postorder is classified ACGCGT as, introduces Mlu I restriction enzyme site.

Described exogenous reporter gene comprises the expressing gene of fluorescin, kinases, hormone or cytokine receptor.

4-OH-tamoxifen can induce described Cre recombinase estrogen receptor fusion rotein and Hsp90 to remove combination, makes the Cre recombinase excise described exogenous reporter gene and lentiviral vectors sequence between the described loxP sequence.

A further object of the present invention provides the application of above-mentioned slow virus controlled expression systems in the stem cell tracer technique.With described reaction plasmid and regulate plasmid respectively with described packaging plasmid cotransfection packing cell, obtain reacting slow virus and regulate slow virus; With the two cotransfection target stem cell, utilize Dox to induce the expression of described exogenous reporter gene and carry out the video picture spike, utilize at last 4-OH-tamoxifen to induce the exogenous reporter gene that can excise at any time between the described loxP sequence.

The present invention has following beneficial effect:

Defective for Lentiviral system existence in the prior art, the invention provides the removable slow virus controlled expression carrier system of a kind of foreign gene (comprise the reaction plasmid, regulate plasmid and packaging plasmid), its specific implementation process is: abduction delivering---be bonded to the cis-acting elements TRE on the reaction plasmid promotor behind the rtTA2S-M2 formation mixture of doxycycline Dox and adjusting plasmid expression, by the expression of simplifying most CMV promotor unlatching downstream external source reporter gene; Induce excision---under the inducing action of 4-OH-tamoxifen, remove combination by the Cre recombinase estrogen receptor fusion rotein of regulating plasmid expression and Hsp90, make upper exogenous reporter gene and the lentiviral vectors sequence between the loxP sequence of Cre recombinase excision reaction plasmid.The present invention is directed to the particular requirement of stem cell vivo tracking technology, not only realized exogenous reporter gene efficient controlled expression in vivo, after spike is finished, also can immediately remove the exogenous genetic fragment that is integrated into host chromosome, thoroughly avoided integrating fragment to the interference of target cell normal physiological function, can predict, this cover system has important using value in stem cell vivo tracking research field.

Description of drawings

Fig. 1 is the pcr amplification electrophorogram of TREminiCMV in the embodiment of the invention 1, wherein, and swimming lane 1:PCR amplified production, swimming lane 2:1Kb plus DNA Ladder.

Fig. 2 is that pcDNA3.1/TREminiCMV recombinant plasmid double digestion is identified electrophorogram in the embodiment of the invention 1, wherein, and swimming lane 1: double digestion product, swimming lane 2:1Kb plus DNA Ladder.

Fig. 3 is the pcr amplification electrophorogram of EGFP cDNA in the embodiment of the invention 2, wherein, and swimming lane 1:1Kb plus DNA Ladder, swimming lane 2:PCR amplified production.

Fig. 4 is pSMPUW carrier 3 ' LTR U3 zone rite-directed mutagenesis base synoptic diagram in the embodiment of the invention 3.

Fig. 5 is that the Lox sequence connects into the upper Mlu I site of pSMPUW-mut synoptic diagram in the embodiment of the invention 3.

Fig. 6 is that recombinant plasmid pSMPUW-mut/TREminiCMV/loxp double digestion is identified electrophorogram in the embodiment of the invention 4, and wherein, swimming lane 1: enzyme is cut group, and swimming lane 2: enzyme is not cut control group, swimming lane 3:1Kb plus DNA Ladder.

Fig. 7 is that recombinant plasmid pSMPUW-mut/TREminiCMV/loxp/EGFP double digestion is identified electrophorogram in the embodiment of the invention 4, wherein, swimming lane 1:1Kb plus DNA Ladder, swimming lane 2: enzyme is not cut control group, and swimming lane 3: enzyme is cut group.

Fig. 8 is the pcr amplification electrophorogram of CMV-rtTA2S-M2 in the embodiment of the invention 5, wherein, and swimming lane 1:1Kb plus DNA Ladder, swimming lane 2: double digestion product.

Fig. 9 is the pcr amplification electrophorogram of EMCV in the embodiment of the invention 6, wherein, and swimming lane 1:1Kb plus DNA Ladder, swimming lane 2:PCR amplified production.

Figure 10 is the pcr amplification electrophorogram of CRE-ER in the embodiment of the invention 7, wherein, and swimming lane 1:PCR amplified production, swimming lane 2:1Kb plus DNA Ladder.

Figure 11 is pSMPUW-mut rite-directed mutagenesis base synoptic diagram in the embodiment of the invention 8.

Figure 12 is that recombinant plasmid pSMPUW-mut II/CMV-rtTA2S-M2 double digestion is identified electrophorogram in the embodiment of the invention 9, wherein, and swimming lane 1: double digestion product, swimming lane 2:1Kb plus DNA Ladder.

Figure 13 is that recombinant plasmid pSMPUW-mut II/CMV-rtTA2S-M2/EMCV double digestion is identified electrophorogram in the embodiment of the invention 9, wherein, and swimming lane 1:1Kb plus DNA Ladder, swimming lane 2:: double digestion product.

Figure 14 is that recombinant plasmid pSMPUW-mut II/CMV-rtTA2S-M2/EMCV/CRE-ER double digestion is identified electrophorogram in the embodiment of the invention 9, wherein,, swimming lane 1:1Kb plus DNA Ladder, swimming lane 2: enzyme is not cut control group, and swimming lane 3: enzyme is cut group.

Figure 15 is the shows fluorescent microscopy images that Dox induces EGFP to express after reacting slow virus in the embodiment of the invention 10 and regulating slow virus cotransfection 293T, and wherein, left figure: do not add the Dox group, right figure: Dox induces group.

Figure 16 is after reacting slow virus in the embodiment of the invention 10 and regulating slow virus cotransfection 293T, the per-cent synoptic diagram that flow cytometer showed Dox induces EGFP to express, and wherein, Sample1: do not add the Dox group, Sample2:Dox induces group.

Figure 17 is that the 4-OH-tamoxifen pre-treatment is induced the shows fluorescent microscopy images that EGFP expresses to be affected to Dox in the embodiment of the invention 10, wherein, and left figure: do not add the 4-OH-tamoxifen group, right figure: 4-OH-tamoxifen pretreated group.

Figure 18 is the synoptic diagram that flow cytometer showed 4-OH-tamoxifen pre-treatment induces EGFP to express impact on Dox in the embodiment of the invention 10, wherein, and Sample1: do not add the 4-OH-tamoxifen group, the Sample2:4-OH-tamoxifen pretreated group.

Embodiment

Below in conjunction with accompanying drawing, and by embodiment the present invention is specifically described.

The related experiment material of following examples is as follows:

The empty bacterium of bacterial strain and plasmid: JM109 is available from Promega company; PcDNA3.1, pBMN-GFP, pMD19-T are available from Invitrogen company; PCI-EGFP is available from Addgene company; PBMN-CRE-ER and pCI-EGFP-EMCVDsRed are for forming by conventional molecular biology method reconstruction on the basis of pBMN-GFP and pCI-EGFP; PTRE-Tight, pTet-on, pSIMPLE-19 EcoR V/BAP Vector are available from TaKaRa company; PSMPUW, pRSV-Rev, pCMV-VSV-G, pCgpV plasmid are available from CellBioLabs company; Human embryo kidney (HEK) 293T cell, mouse mesenchymal cell are available from ATCC.

Reagent and test kit: plasmid extraction test kit, sepharose reclaim test kit, polybrene, Dox available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; Pyrobest archaeal dna polymerase, rTaq archaeal dna polymerase, T4DNA ligase enzyme, restriction enzyme, sudden change test kit, pSIMPLE-19 EcoR V/BAP Vector are available from precious biotechnology (Dalian) company limited; 1Kb plus DNA Ladder, Lipofectamine2000 are available from Invitrogen company; 4-OH-tamoxifen is available from Sigmaaldrich company.

Other reagent and raw material are commercially available domestic or inward.

All primers by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd on behalf of synthetic; All order-checking services are finished by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

Embodiment 1Tet-response element(TRE) and the clone who simplifies CMV promoter sequence (TREminiCMV) most

Plasmid with among the plasmid extraction test kit extraction JM109/pTRE-Tight take it as template, obtains the TREminiCMV sequence by pcr amplification, and used primer is as follows:

TREminiCMV-For:5 '-GAG GG G ATC CCT TTC GTC TTC ACT C-3 ' (primer sequence is shown in SEQ ID NO:1), the line part is BamH I restriction enzyme enzyme recognition site;

TREminiCMV-Rev:5 '-CGA GAT ATCGAA TTC TCC AGG CGA TC-3 ' (primer sequence is shown in SEQ ID NO:2), the line part is EcoR V restriction enzyme enzyme recognition site;

The system of PCR reaction is: pTRE-Tight1 μ l, and each 1 μ l of the upstream and downstream primer of 10 μ mol/L, the dNTP4 μ l of 2.5mmol/L, 10 * Pyrobest Buffer, 5 μ l, the Pyrobest archaeal dna polymerase 0.5 μ l of 5U/ μ l adds distilled water polishing 50 μ l.

The condition of PCR reaction is: 94 ℃ of denaturations 5 minutes; 94 ℃ of sex change 30 seconds, 55 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, and circulated 35 times; 72 ℃ of final extensions 7 minutes.

As shown in Figure 1, behind the pcr amplification, on 1% agarose gel electrophoresis, obtain the specific band of a treaty 350bp, with 1Kb plus DNA Ladder as standard.

Above-mentioned PCR product is through the recovery that separates, taps rubber of BamH I and EcoR V double digestion rear electrophoresis, is connected to get the pcDNA3.1/TREminiCMV recombinant plasmid with carrier pcDNA3.1 through same double digestion, and transforms the JM109 competence bacterium of made fresh; Select positive colony, plasmid extraction is identified correct (as shown in Figure 2) with BamH I and EcoR V double digestion in a small amount, and order-checking confirms that the aim sequence that obtains meets the requirements.

The clone of embodiment 2 green fluorescent proteins (EGFP) cDNA

Take the pBMN-GFP plasmid as template, obtain EGFP cDNA by pcr amplification, the primer is as follows:

EGFP-For:5 '-CGA GAT ATCATG GTG AGC AAG GGC GAG-3 ' (primer sequence is shown in SEQ ID NO:3), underscore are EcoR V restriction enzyme enzyme recognition site;

EGFP-Rev:5 '-CGC GAC CCT TGG TCT TAC TTG TAC AGC TCG-3 ' (primer sequence is shown in SEQ ID NO:4), underscore are Ahd I restriction enzyme enzyme recognition site;

The system of PCR reaction is: pBMN-GFP1 μ l, and each 1 μ l of the upstream and downstream primer of 10 μ mol/L, the dNTP4 μ l of 2.5mmol/L, 10 * PCR Buffer5 μ l, the rTaq archaeal dna polymerase 0.5 μ l of 5U/ μ l adds distilled water polishing 50 μ l.

The condition of PCR reaction is: 94 ℃ of denaturations 4 minutes; 94 ℃ of sex change 30 seconds, 56 ℃ of annealing 30 seconds, 72 ℃ were extended 1 minute, and circulated 35 times; 72 ℃ of final extensions 10 minutes.

As shown in Figure 3, behind the pcr amplification, on 1% agarose gel electrophoresis, obtain the specific band of a treaty 350bp, with 1Kb plus DNA Ladder as standard; Target segment with in the PCR glue recovery test kit recovery application of sample swimming lane is EGFP cDNA.

Get the EGFP cDNA4 μ l of above-mentioned purifying, pMD19-T Vector carrier 4 μ l, T4DNA ligase1 μ l, 10 * T4Buffer1 μ l, 16 ℃ of reactions positive recombinant that spends the night to get transforms the JM109 competent cell of made fresh again; Transformed bacteria is coated on the LB agar plate that contains penbritin, 37 ℃ of overnight incubation; Picking colony is seeded in the 10ml small test tube that 2ml LB liquid nutrient medium is housed, and 37 ℃ of overnight incubation are extracted plasmid, and enzyme is cut and identified correct rear order-checking, confirms that the aim sequence that obtains meets the requirements.This positive recombinant called after pMD19-T/EGFP, clone bacterium liquid is kept among the LB that contains 20% glycerine, and is frozen in-80 ℃.

The sudden change in embodiment 3LTRU3 zone and the insertion of loxp sequence

To the rite-directed mutagenesis that the ACCGGT in pSMPUW carrier 3 ' LTR U3 zone carries out, the sudden change postorder is classified ACGCGT as, introduces Mlu I restriction enzyme site, and the vector plasmid mutant primer is as follows:

Mut-For:5 '-TTG CTT TAT T TG TAA ACG CGT GCA GCT GCT-3 ' (primer sequence is shown in SEQ ID NO:5);

Mut-Rev:5 '-TAG CAT CAC AAA TTT CAC AAA TAA ATT CCG-3 ' (primer sequence is shown in SEQ ID NO:6);

The system of reaction is: pSMPUW plasmid 0.5 μ l, and each 1 μ l of the upstream and downstream primer of 10 μ mol/L, the dNTP4 μ l of 2.5mmol/L, 10 * Pyrobest Buffer5 μ l, the Pyrobest archaeal dna polymerase 0.5 μ l of 5U/ μ l adds distilled water polishing 50 μ l.

The condition of PCR reaction is: 94 ℃ of denaturations 5 minutes; 94 ℃ of sex change 30 seconds, 60 ℃ of annealing 30 seconds, 72 ℃ were extended 7 minutes and 30 seconds, and circulated 35 times; 72 ℃ of final extensions 5 minutes.

Agarose gel electrophoresis with 1% and the purifying PCR reaction product of tapping rubber, carry out Blunting Kination reaction, reaction system is: purified pcr product 0.5ul, 10 * Blunting Kination buffer2ul, Blunting Kination Enzyme Mix1ul adds water and complements to 20ul; Reaction conditions is: hatched 10 seconds 70 ℃ of enzyme deactivations 10 minutes for 37 ℃.

Above-mentioned Blunting Kination reaction product is done the JM109 competent cell that transforms made fresh after the ligation, coats on the LB agar plate that contains kantlex 37 ℃ of overnight incubation; Picking colony is seeded in the 10ml small test tube that 2ml LB liquid nutrient medium is housed, and overnight incubation is extracted plasmid and order-checking, and the confirmation catastrophe point is suddenlyd change (as shown in Figure 4).PSMPUW plasmid called after pSMPUW-mut after the sudden change.

The strand primer annealing is formed Loxp dna double chain, and primer is as follows:

Loxp-For:5 '-CCG A AC GCG TAT AAC TTC GTA TAG CAT ACA TTA TAC GAA GTT AT A CGC GTT CGC T-3 ' (primer sequence is shown in SEQ ID NO:7);

Loxp-Rev:5 '-AGC GA A CGC GTA TAA CTT CGT ATA ATG TAT GCT ATA CGA AGT TAT ACG CGTTCG G-3 ' (primer sequence is shown in SEQ ID NO:8), above-mentioned underscore sequence can form Mlu I enzyme and cut recognition site;

The condition of primer annealing reaction is: each 20 μ l of the above-mentioned primer of 100 μ mol/L, and mixing post-heating to 95 ℃, slow cooling is to annealing at room temperature.

After enzyme was cut pSMPUW-mut and Loxp dna double chain fragment respectively with Mlu I restriction enzyme, agarose gel electrophoresis was cut glue purification plasmid and fragment; Get plasmid purification pSMPUW-mut0.25ul, Loxp fragment 0.5ul, T4DNA ligase1 μ l, 10 * T4Buffer1 μ l, moisturizing to 10 μ l, 16 ℃ of connections are spent the night, transform the JM109 competent cell of made fresh, coat on the LB agar plate that contains kantlex 37 ℃ of overnight incubation; Picking colony is seeded in the 10ml small test tube that 2ml LB liquid nutrient medium is housed, and overnight incubation is extracted plasmid, and order-checking is identified correct, and the clone who selects the Loxp forward to connect into carries out conservation, and plasmid called after pSMPUW-mut/loxp(as shown in Figure 5).

The structure of embodiment 4 reaction plasmids

With BamH I/EcoR V difference double digestion pcDNA3.1/TREminiCMV and pSMPUW-mut/loxp, enzyme is cut product respectively with 1% agarose gel electrophoresis and tap rubber purifying TREminiCMV and pSMPUW-mut/loxp, purified product carries out ligase enzyme and connects, connect the JM109 competent cell that product transforms made fresh, coat on the LB agar plate that contains kantlex 37 ℃ of overnight incubation; Picking colony is seeded in the 10ml small test tube that 2ml LB liquid nutrient medium is housed, and overnight incubation is extracted plasmid; BamH I/EcoR V double digestion identifies that TREminiCMV connects into pSMPUW-mut/loxp(as shown in Figure 6), recon called after pSMPUW-mut/TREminiCMV/loxp.

With EcoR V/Ahd I difference double digestion pSMPUW-mut/TREminiCMV/loxp and pMD19-T/EGFP, enzyme is cut product respectively with 1% agarose gel electrophoresis and tap rubber purifying EGFP and pSMPUW-mut/TREminiCMV/loxp, purified product carries out ligase enzyme and connects, connect the JM109 competent cell that product transforms made fresh, coat on the LB agar plate that contains kantlex 37 ℃ of overnight incubation; Picking colony is seeded in the 10ml small test tube that 2ml LB liquid nutrient medium is housed, and overnight incubation is extracted plasmid; EcoR V/Ahd I double digestion is identified, confirms that EGFP connects into pSMPUW-mut/TREminiCMV/loxp(as shown in Figure 7), recon called after pSMPUW-mut/TREminiCMV/loxp/EGFP.

Embodiment 5 is with the rtTA2S-M2(CMV-rtTA2S-M2 of CMV promotor) the clone

Take pTet-on as template, adopt PCR method clone CMV-rtTA2S-M2 zone, the PCR primer is as follows:

CMV-rtTA2S-M2-For:5 '-GAG G GG ATC CAT ATT GGC TCA TGT CC-3 ' (primer sequence is shown in SEQ ID NO:9), underscore is Bam HI recognition site;

CMV-rtTA2S-M2-Rev:5 '-ATA GGC GCG CCT TAC TTA GTT ACC-3 ' (primer sequence is shown in SEQ ID NO:10), underscore is Asc I recognition site;

The system of PCR reaction is: pTet-on1 μ l, and each 1 μ l of the upstream and downstream primer of 10 μ mol/L, the dNTP4 μ l of 2.5mmol/L, 10 * Pyrobest Buffer5 μ l, the Pyrobest archaeal dna polymerase 0.5 μ l of 5U/ μ l adds distilled water polishing 50 μ l.

The condition of PCR reaction is: 94 ℃ of denaturations 5 minutes; 94 ℃ of sex change 30 seconds, 52 ℃ of annealing 30 seconds, 72 ℃ were extended 2 minutes and 30 seconds, and circulated 30 times; 72 ℃ of final extensions 5 minutes.

As shown in Figure 8, behind the pcr amplification, on 1% agarose gel electrophoresis, obtain the specific band of a treaty 1480bp, be connected with cloning vector pSIMPLE-19EcoR V/BAP Vector behind the rubber tapping purifying, be converted into JM109 competence bacterium after getting recombinant plasmid pT-CMV-rtTA2S-M2; Select positive colony, check order after the plasmid extraction in a small amount, confirm that the aim sequence that obtains meets the requirements; Clone bacterium liquid is kept among the LB that contains 20% glycerine, and is frozen in-80 ℃.

The clone of embodiment 6 internal ribosome entry site EMCV sequences

Internal ribosome entry site EMCV sequence can form bicistronic mRNA and express, and can carry out the independent translation of two genes on a mRNA.Take pCI-EGFP-EMCVDsRed as template, adopt PCR method clone EMCV sequence, the primer is as follows:

EMCV-For:5 '-ATA GGC GCG CCC GCC CCT CTC CCT C-3 ' (primer sequence is shown in SEQ ID NO:11), the underscore sequence is Asc I recognition site;

EMCV-Rev:5 '-CGC GAC CCT TGG TCT GTG GCC ATA T-3 ' (primer sequence is shown in SEQ ID NO:12), the underscore sequence is Ahd I recognition site;

The system of PCR reaction is: p CI-EGFP-EMCVDsRed1 μ l, each 1 μ l of upstream and downstream trip primer of 10 μ mol/L, the dNTP4 μ l of 2.5mmol/L, 10 * Pyrobest Buffer5 μ l, the Pyrobest archaeal dna polymerase 0.5 μ l of 5U/ μ l adds distilled water polishing 50 μ l.

As shown in Figure 9, behind the pcr amplification, on 1% agarose gel electrophoresis, obtain the specific band of a treaty 604bp, be connected with cloning vector pSIMPLE-19EcoR V/BAP Vector behind the rubber tapping purifying, get recombinant plasmid pT-EMCV, transform the JM109 competence bacterium of made fresh; Select positive colony, check order after the plasmid extraction in a small amount, confirm that the aim sequence that obtains meets the requirements; Gram bacterium liquid is kept among the LB that contains 20% glycerine, and is frozen in-80 ℃.

The clone of embodiment 7 recombinant C RE-ER sequences

Take pBMN-CRE-ER as template, adopt PCR method clone CRE-ER sequence, the primer is as follows:

CRE-ER-For:5 '-CGC GAC CAA GGG TCA TGT CCA ATT TAC-3 ' (primer sequence is shown in SEQ ID NO:13), the underscore sequence is Ahd I recognition site;

CRE-ER-Rev:5 '-CC T TAA TTA ATC AGA CCG TGG C-3 ' (primer sequence is shown in SEQ ID NO:14), the underscore sequence is Pac I recognition site;

The system of PCR reaction is: pBMN-CRE-ER1 μ l, and each 1 μ l of the upstream and downstream primer of 10 μ mol/L, the dNTP4 μ l of 2.5mmol/L, 10 * Pyrobest Buffer5 μ l, the Pyrobest archaeal dna polymerase 0.5 μ l of 5U/ μ l adds distilled water polishing 50 μ l.

As shown in figure 10, behind the pcr amplification, on 1% agarose gel electrophoresis, obtain the specific band of a treaty 2010bp, be connected with cloning vector pSIMPLE-19EcoR V/BAP Vector behind the rubber tapping purifying, get recombinant plasmid pT-CRE-ER, transform the JM109 competence bacterium of made fresh; Select positive colony, check order after the plasmid extraction in a small amount, confirm that the aim sequence that obtains meets the requirements; Bacterium liquid the clone be kept among the LB that contains 20% glycerine, and is frozen in-80 ℃.

Embodiment 8pSMPUW-mut carries out rite-directed mutagenesis, introduces Asc I site in the multiple clone site district

Adopt the method for rite-directed mutagenesis, the GACTCGCC in the upper multiple clone site district of pSMPUW-mut that embodiment 3 is made up suddenlys change, and sports GGCGCGCC, introduces Asc I restriction enzyme site.The vector plasmid mutant primer is as follows:

Mut II-For:5 '-AAT TCA T AC AAA AG G GCG CGC CCC TGC CTT GGG G-3 ' (primer sequence is shown in SEQ ID NO:15);

Mut II-Rev:5 '-ACT CTC AGC TCC GGT CGG GGC GGG TTG-3 ' (primer sequence is shown in SEQ ID NO:16);

The system of reaction is: pSMPUW-mut plasmid 0.5 μ l, and each 1 μ l of the primer of 10 μ mol/L, the dNTP4 μ l of 2.5mmol/L, 10 * Pyrobest Buffer5 μ l, the Pyrobest archaeal dna polymerase 0.5 μ l of 5U/ μ l adds distilled water polishing 50 μ l.

The product of the sepharose rubber tapping purifying PCR reaction with 1%, Blunting Kination reaction, reaction system is: purified pcr product 0.5ul, 10 * Blunting Kination buffer2ul, Blunting Kination Enzyme Mix1ul adds water and complements to 20ul; Reaction conditions is: hatched 10 seconds 70 ℃ of enzyme deactivations 10 minutes for 37 ℃.

Product is done the JM109 competent cell that transforms made fresh after the ligation, coats on the LB agar plate that contains kantlex 37 ℃ of overnight incubation; Picking colony is seeded in the 10ml small test tube that 2ml LB liquid nutrient medium is housed, and overnight incubation is extracted plasmid, order-checking, and the confirmation catastrophe point is suddenlyd change (as shown in figure 11).Plasmid called after after the sudden change is criticized pSMPUW-mut II.

Embodiment 9 regulates the structure of plasmid

CMV-rtTA2S-M2, the EMCV and the CRE-ER that prepare among embodiment 5, embodiment 6 and the embodiment 7 are connected into respectively in the mutant plasmid of embodiment 8 structures adjusted plasmid.Concrete operations are: with BamH I/Asc I difference double digestion pT-CMV-rtTA2S-M2 and pSMPUW-mut II, enzyme is cut product respectively with 1% agarose gel electrophoresis and tap rubber purifying CMV-rtTA2S-M2 and pSMPUW-mut II, purified product carries out ligase enzyme and connects, connect the JM109 competent cell that product transforms made fresh, coat on the LB agar plate that contains kantlex 37 ℃ of overnight incubation; Picking colony is seeded in the 10ml small test tube that 2ml LB liquid nutrient medium is housed, and overnight incubation is extracted plasmid; BamH I/Asc I double digestion identifies that CMV-rtTA2S-M2 connects into pSMPUW-mut II(as shown in figure 12), recon called after pSMPUW-mut II/CMV-rtTA2S-M2.

Asc I/Ahd I is double digestion pSMPUW-mut II/CMV-rtTA2S-M2 and pT-EMCV respectively, enzyme is cut product respectively with 1% agarose gel electrophoresis and tap rubber purifying pSMPUW-mut II/CMV-rtTA2S-M2 and EMCV fragment, purified product carries out ligase enzyme and connects, connect the JM109 competent cell that product transforms made fresh, coat on the LB agar plate that contains kantlex 37 ℃ of overnight incubation.Picking colony is seeded in the 10ml small test tube that 2ml LB liquid nutrient medium is housed, and overnight incubation is extracted plasmid.Asc I/Ahd I double digestion is identified, confirms that the EMCV fragment connects into pSMPUW-mut II/CMV-rtTA2S-M2(as shown in figure 13), recon called after pSMPUW-mut II/CMV-rtTA2S-M2/EMCV.

Ahd I/Pac I is double digestion pSMPUW-mut II/CMV-rtTA2S-M2/EMCV and pT-CRE-ER respectively, enzyme is cut product respectively agarose gel electrophoresis rubber tapping purifying pSMPUW-mut II/CMV-rtTA2S-M2/EMCV and the CRE-ER fragment of row 1%, carrying out ligase enzyme behind the purifying connects, be transformed into the JM109 competent cell of made fresh, be coated on the LB agar plate of kantlex 37 ℃ of overnight incubation; Picking colony is seeded in the 10ml small test tube that 2ml LB liquid nutrient medium is housed, and overnight incubation is extracted plasmid; Ahd I/Pac I double digestion is identified, confirms that the CRE-ER fragment connects into pSMPUW-mut II/CMV-rtTA2S-M2/EMCV(as shown in figure 14), recon called after pSMPUW-mut II/CMV-rtTA2S-M2/EMCV/CRE-ER.

Embodiment 10 regulates the packing of slow virus and reaction slow virus and infects the target stem cell

Employing liposome transfection method will regulate plasmid/reaction plasmid and three packaging plasmids change packing cell by a certain percentage over to, becomes the slow virus particle in the cell internal packing, after the packing, collects the slow virus particle that is discharged in the substratum.Specific embodiments is: transfection bed board the day before yesterday human embryo kidney (HEK) 293T cell, about 0.5～2 * 10 ⁶(6cm dish) cell, second day cell carry out transfection when growing to about 90%-95% degree of converging.

1. the plasmid DNA dilution mixes, the 12 μ g mixing plasmids (mass ratio of pSMPUW-mut II/CMV-rtTA2S-M2/EMCV/CRE-ER ︰ pCMV-VSV-G ︰ pRSV-Rev ︰ pCgpV=3 ︰ 1 ︰ 1 ︰ 1, the mass ratio of pSMPUW-mut/TREminiCMV/loxp/EGFP ︰ pCMV-VSV-G ︰ pRSV-Rev ︰ pCgpV=3 ︰ 1 ︰ 1 ︰ 1) of respectively asking for add serum-free DMEM to 500 μ l.

2.Lipofectamine2000 dilution: get 30 μ l Lipofectamine2000, add serum-free DMEM to 500 μ l, mixing, incubated at room 5 minutes.

3. the plasmid DNA of dilution and the Lipofectamine2000 of dilution are mixed, incubated at room is 20 minutes behind the mixing.

4. cell is changed serum-free DMEM(5ml), said mixture is added drop-wise in the substratum, the cross method mixes, and cultivates 4～6 hours for 37 ℃, changes serum (10% tire ox) DMEM(3ml), respectively collected virus liquid one time, and mixed in 48 hours, 72 hours.Two kinds of slow virus difference called after Lenti-Tet-CRE-ER(regulate slow virus) and Lenti-GFP (reaction slow virus).

5. in 1000rpm centrifugal 5 minutes.

6. supernatant in the collection centrifuge tube is filtered in the new 15ml centrifuge tube with 0.22 μ m strainer.

7. treat that target cell infection spreads 6 orifice plates the day before yesterday: 1 * 10 ⁵/ porocyte is totally four holes.

8. the two-strain liquid after will filtering is pressed 1:1 mixing postoperative infection mouse mesenchymal cell, adds polybrene, and final concentration is 8 μ g/ml.

9. infection second day, 6 orifice plates renew bright DMEM perfect medium, and wherein two holes do not add Dox, and two holes add 1ug/ml Dox and induce EGFP to express, 3 days afterwards respectively Fluirescence observation and flow cytometer showed EGFP express cell per-cent.Fluirescence observation the results are shown in Figure 15, and flow cytometer showed is seen Figure 16.

Interpretation of result: Fluirescence observation shows, Dox induces the group green fluorescent protein to be expressed, do not add the Dox group and have no egfp expression, after adding 1ug/ml Dox induced 3 days, the most of mouse mesenchymal cell expressing green fluorescent protein of fluorescence microscope was expressed; The flow cytometer detection by quantitative shows that Dox induces group that 78.56% stem cell expressing green fluorescent protein is arranged, and not adding the Dox group only is 2.97%.

10.Dox the cell of inducing EGFP to express renews bright DMEM perfect medium, does not add Dox and induces, and cultivates after 3 days, goes down to posterity, and spreads 6 orifice plates: 1 * 10 ⁵/ porocyte is totally four holes, and wherein two holes add 4-OH-tamoxifen, two Kong Bujia, and after 3 days, four holes all add 1ug/ml Dox and induce EGFP to express, 3 days afterwards respectively Fluirescence observation and flow cytometer showed EGFP express cell per-cent.Fluirescence observation the results are shown in Figure 17, and flow cytometer showed is seen Figure 18.

Interpretation of result: Fluirescence observation shows that the 4-OH-tamoxifen pretreated group is after 4-OH-tamoxifen processes 3 days, and green fluorescence protein gene is cut, and stem cell significantly reduces egfp expression; The flow cytometer detection by quantitative shows that 77.28% before the stem cell of expressing green fluorescent protein is processed by 4-OH-tamoxifen reduces to 15.93%.

Above-described only is preferred implementation of the present invention, the invention is not restricted to above embodiment.Be appreciated that other improvement and variation that those skilled in the art directly derive or associate under the prerequisite that does not break away from spirit of the present invention and design, all should think to be included within protection scope of the present invention.

Claims

1. slow virus controlled expression carrier system that foreign gene is removable, this system comprises expression plasmid and packaging plasmid, it is characterized in that: described expression plasmid comprises the reaction plasmid and regulates plasmid, and makes up by the following method respectively and obtain:

(1) structure of reaction plasmid

Rite-directed mutagenesis by lentiviral vectors pSMPUW3 ' LTR U3 area part base is introduced restriction enzyme site, and the loxP sequence is inserted described pSMPUW3 ' LTR, gets mutant plasmid; With the cis-acting elements TRE on the Tet-on system response plasmid pTRE-Tight with simplify the promoter sequence that the CMV promoter sequence replaces described mutant plasmid most, and with the multiple clone site MCS position that exogenous reporter gene inserts described mutant plasmid, get the reaction plasmid; Described exogenous reporter gene is in described TRE and simplifies most under the control of CMV promotor, and by the Dox abduction delivering.

(2) structure of adjusting plasmid

CMV-rtTA2S-M2 sequence in the Tet-on system, internal ribosome entry site EMCV sequence and Cre-ER sequence order are coupled together, and the multiple clone site of the pSMPUW of rite-directed mutagenesis zone gets the adjusting plasmid in the inserting step (1); Described rtTA2S-M2 sequence and Cre-ER sequence can be translated and express independently of one another, generate rtTA2S-M2 albumen and Cre recombinase estrogen receptor fusion rotein.

2. slow virus controlled expression carrier system according to claim 1, it is characterized in that: described packaging plasmid comprises pRSV-Rev, pCMV-VSV-G, pCgpV.

3. described slow virus controlled expression carrier system according to claim 1, it is characterized in that: described mutational site sequence is ACCGGT, the sudden change postorder is classified ACGCGT as, introduces Mlu I restriction enzyme site.

4. described slow virus controlled expression carrier system according to claim 1, it is characterized in that: described exogenous reporter gene comprises the expressing gene of fluorescin, kinases, hormone or cytokine receptor.

5. described slow virus controlled expression carrier system according to claim 1, it is characterized in that: in step (2), first the pSMPUW multiple clone site district of rite-directed mutagenesis in the step (1) carried out rite-directed mutagenesis introducing Asc I restriction enzyme site, be beneficial to the insertion of described CMV-rtTA2S-M2 sequence, internal ribosome entry site EMCV sequence and Cre-ER sequence.

6. described slow virus controlled expression carrier system according to claim 1, it is characterized in that: 4-OH-tamoxifen can induce described Cre recombinase estrogen receptor fusion rotein and Hsp90 to remove combination, makes the Cre recombinase excise described exogenous reporter gene and lentiviral vectors sequence between the described loxP sequence.

7. each described slow virus controlled expression carrier system application in stem cell vivo tracking technology in the claim 1～6, it is characterized in that: with described reaction plasmid and regulate plasmid respectively with described packaging plasmid cotransfection packing cell, obtain reacting slow virus and regulate slow virus; With the two cotransfection target stem cell, utilize Dox to induce described exogenous reporter gene expression and carry out the video picture spike, utilize at last 4-OH-tamoxifen to induce exogenous reporter gene between the described loxP sequence of excision.