CN101955977B - Expression system for nonintegrated, long-time and erasable expression vector - Google Patents
Expression system for nonintegrated, long-time and erasable expression vector Download PDFInfo
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Abstract
The invention provides an expression system for a nonintegrated, long-time and erasable expression vector. The expression vector of the expression system adopts human herpes virus replication origin (Orip), an EBNA-1 expression box is introduced, and both ends of the Orip, both end of EBN-1 or both ends of Orip-EBNA-1 are provided with recombinase recognition sequences. The expression vector is not integrated in a cell genome, can stably express for a long time, can controllably erase vector plasmid by instantaneously inducing (or expressing) recombinase from a hose cell, can be applied to most kinds of cells by using a strong initiator and can distinguish a cell with the vector plasmid from a cell without the vector plasmid by using a real-time marker. The expression system can widely applied to mammal cell expression and biomedicine engineering for realizing cell reprogramming by expressing a foreign gene.
Description
Technical field
The present invention relates to genetic engineering technique, be specifically related to a kind of expression vector and based on the expression system of this expression vector.
Background technology
The method of expression alien gene is varied in mammalian cell.For example, 1) can utilize the common plasmid of transfection (Plasmid DNA) to carry out nonconformable transient expression, also can be through plasmid integration is realized the long-time expression of gene to genome; 2) utilize the virus vector to be packaged as virion,, need and be incorporated in the genome of cell the foreign gene transfered cell and be able to stable expression alien gene chronically like lentiviral vectors (lentiviral vector), retrovirus vector etc.; 3) adenovirus (adenovirus) carrier can be with the foreign gene transfered cell, and with nonconformable form at cell inner expression, express but this method can only realize the short-term of foreign gene, can not realize the expression of long-time (about month).
The long-time expression that can realize foreign gene that has in these methods, but need exogenous origin gene integrator in the genome of cell, thus destroyed the genomic integrity of cell; Though what have can guarantee the complete of cellular genome, the time ratio of expressing is shorter.The more important thing is that these methods all can not control deletion exogenous gene expression carrier artificially, thereby in biomedical engineering, exist very big risk.
Summary of the invention
To above-mentioned deficiency, the present invention provides a kind of expression system of nonconformable, long, adjustable deletion expression vector.
For realizing above-mentioned purpose; The present invention at first provides a kind of expression vector; The replicon of this expression vector adopts herpes virus hominis's replicon (replication origin; Orip), and introduce the EBNA-1 expression cassette, have the recombinase recognition sequence at the two ends of said replicon, the two ends of EBNA-1 or the two ends of Orip-EBNA-1.
There is the expression vector plasmid in the NA of EBNA-1 genes encoding with free chromosomal form, in cell, realize the nonconformity and the long-term expression of foreign gene.
Because the promotor of EBNA-1 gene is its viral original promotor, and this promotor can be activated in the cell of numerous species hardly, thereby can only in few cell such as minority blood system cell, liver cell and myocardial cell, use.For this reason; The present invention replaces with the strong promoter with eukaryotic cell broad spectrum with the promotor of this expression cassette; Such as CMV, PGK, CAG or EF-1 α or the like promotor, make it in various cell, to express, particularly in mammalian cell, express.
The present invention adds the recombinase recognition sequence at the two ends of replicon or EBNA-1, and it can be deleted the dna sequence dna in the recognition sequence under the effect of recombinase, thereby makes not reproducible of plasmid, and plasmid will be by deletion gradually when passage.Described recombinase recognition sequence can adopt the Loxp sequence of Cre/LoxP system or be the frt sequence of FLP/FRT system.Certainly, also can be the fusion sequence of Loxp-frt.
Further, expression vector of the present invention also comprises selection markers, for example drug screening mark or selection markers, perhaps their combination in real time.The drug screening marker gene can be neomycin phosphotransferase (neo), hygromycin B phosphotransferase (hph), xanthine/guanine monophosphate transferring enzyme (gpt), xanthoglobulin phosphotransferase (Hprt), thymidine kinase (tk) or tetracycline Transacetylase (puro) an or the like gene.The selection markers gene can be fluorescence protein gene or non-fluorescent label gene in real time; Fluorescence protein gene can be such as green fluorescent protein gene, yellow fluorescence protein gene and DsRed gene or the like, and non-fluorescent label can be immunohistochemical methods mark or the like.Adopted the combination of neo and GFP selection markers in one embodiment of the present of invention; The selection markers expression cassette of its introducing be by CMV promotor, enhancement type green fluorescent protein gene (EGFP), RES (internal ribosome entrysite, IRES) and the CMV-EGFP-IRES-NEO selectable marker gene that constitutes of neomycin phosphotransferase gene (neo).Utilize the real-time fluorescence mark can the cell differentiation that contain vector plasmid and lost vector plasmid be come.
In an embodiment of the present invention, adopt pBR322, made up A as setting out plasmid) .pBR322-rrs-Orip-rrs-EBNA1-MCS; B) .pBR322-Orip-rrs-EBNA1-rrs-MCS; C.pBR322-rrs-Orip-EBNA1-rrs-MCS; And D) expression vector such as .pBR322-rrs-Orip-EBNA1-rrs-EGFP.
The various recombinant vectorss that contain foreign gene of those skilled in the art's above-mentioned expression vector establishment capable of using are used for the functional study or the various application of gene.Therefore the present invention also comprises the recombinant vectors by above-mentioned expression vector establishment, and the host cell that contains said expression vector or recombinant vectors, for example mammalian cell.
In embodiments of the present invention, made up such as 1) pBR322-rrs-Orip-rrs-EBNA1-OSN; 2) pBR322-rrs-Orip-rrs-EBNA1-MK; 3) .pBR322-Orip-rrs-EBNA1-rrs-OSN; 4) .pBR322-Orip-rrs-EBNA1-rrs-MK; 5) expression vector such as .pBR322-rrs-Orip-EBNA1-rrs-MK .pBR322-rrs-Orip-EBNA1-rrs-OSN and 6).These expression vectors contain such as Oct-4, Sox-2, and Nanog, c-Myc, Klf4 etc. and become the relevant gene of reprogramming of somatic cells, to induce HFF's reprogrammed be multipotential stem cell and keep it genomic complete.
Above-mentioned expression vector and recombinase are constituted expression system, and this expression system passes through above-mentioned expression vector (recombinant vectors) transfection host cell, and through recombinase this expression vector (recombinant vectors) is being deleted in good time." in good time " described here is meant the needs of those skilled in the art according to research, in certain specified phase this recombinant plasmid deleted.Said recombinase can carry out transient expression through the recombinase expression vector, or carries the proteic recombinase such as TAT through adding the N end, utilizes the protein mediated recombinase of TAT to get into cell.
Utilize expression vector according to the invention, recombinant vectors or expression system to can be used for expressing foreign protein; But as express the protein of the toxic or cell death inducing of pair cell; Be used for this proteic mechanism of action of research or toxicity analysis, particularly expression has cytotoxicity or the protein relevant with apoptosis in mammalian cell.Can also express cell reprogrammed albumen be used to change the differentiation state and the cell function of cell, for example will become reprogramming of somatic cells is versatile stem cell.
In addition, for the ease of using, can also for example restriction enzyme, damping fluid etc. be assembled into test kit with expression vector of the present invention, recombinant vectors or expression system and suitable reagent.
In embodiments of the present invention, with above-mentioned recombinant vectors transfection HFF (HFF), the result is illustrated among the people HFF and expresses Oct-4; Sox-2, Nanog and c-Myc, Klf4 gene; Can induce this cell to change multipotential stem cell into; Mediate Cre through TAT and get into stem cell, with this expression vector deletion, obtain the multipotential stem cell that carrier free is incorporated into genome, carrier free existence final the separation.
In the present invention, rrs representes the recombinase recognition sequence, and OSN representes foreign gene Oct4-Sox2-Nanog, and MK representes foreign gene cMyc-Klf4.
Expression vector of the present invention has following advantage:
1) unconformability is in cellular genome;
2) stably express for a long time;
3) can from cell, controllably delete vector plasmid;
4) through using stronger promotor can be applied to the cell of most of kinds;
5) through using real-time mark can the cell differentiation that contain vector plasmid and lost vector plasmid be come.
Expression vector of the present invention can be in the widespread use of following field:
1) but the proteinic mechanism of action and the toxicity analysis of the toxic or cell death inducing of research pair cell.
2) utilize carrier of the present invention in mammalian cell, to have cytotoxicity or the protein expression relevant with apoptosis.
3) utilize the nonconformity of carrier of the present invention and the characteristics of controlled deletion carrier, will become reprogramming of somatic cells is multipotential stem cell.
4) carrier of the present invention is in various research and clinical applications aspect differentiation state, cell type, cell fate and the cell function of expression alien gene change cell.
Description of drawings
Shown in Figure 1 is the plasmid map of pBR322-rrs-Orip-rrs-EBNA1-MCS;
Shown in Figure 2 is the plasmid map of pBR322-Orip-rrs-EBNA1-rrs-MCS;
Shown in Figure 3 is the plasmid map of pBR322-rrs-Orip-EBNA1-rrs-MCS;
Shown in Figure 4 is the plasmid map of pBR322-rrs-Orip-EBNA1-rrs-EGFP;
Shown in Figure 5 is the plasmid map of pBR322-rrs-Orip-rrs-EBNA1-OSN;
Shown in Figure 6 is the plasmid map of pBR322-rrs-Orip-rrs-EBNA1-MK;
Shown in Figure 7 is the plasmid map of pBR322-Orip-rrs-EBNA1-rrs-OSN;
Shown in Figure 8 is the plasmid map of pBR322-Orip-rrs-EBNA1-rrs-MK;
Shown in Figure 9 is the plasmid map of pBR322-rrs-Orip-EBNA1-rrs-OSN;
Shown in Figure 10 is the plasmid map of pBR322-rrs-Orip-EBNA1-rrs-MK;
Figure 11 shows is to use 2 days, 5 days, 10 days, 20 days displaing micro picture of expression vector pBR322-rrs-Orip-EBNA1-rrs-EGFP electric shock transfection HFF cell;
Shown in Figure 12 is the detection of expression of foreign gene in HFF (HFF), and wherein H7 is a human embryo stem cell; HFF is the HFF who buys from ATCC; HFF/OSNMK4d, HFF/OSNMK10d are respectively the HFF cell of cotransfection expression vector pBR322-rrs-Orip-rrs-EBNA1-OSN and pBR322-rrs-Orip-rrs-EBNA1-MK4 days and 10 days;
Figure 13 shows be to use above-mentioned expression vector in the HFF cell expression alien gene in the time of 25 days detection of alkaline phosphatase active.Wherein A, B are depicted as the SEAP positive colony that is produced when foreign gene is expressed 25 days in the HFF cell, and C is the human embryo stem cell positive control, and D is that the HFF cell is as negative control;
Figure 14 shows is to use expression vector of the present invention to change the HFF cell induction evaluation of multipotential stem cell into, and wherein A. uses expression system of the present invention to express Oct4, Sox2, Nanog gene from the multipotential stem cell that the HFF cell obtains; B.HFF cell itself is not expressed Oct4, Sox2, Nanog gene.First row show the cell of expressing Oct4, Sox2, Nanog respectively, and secondary series DAPI dyeing shows all cells, and the 3rd classifies the above two stack as.
What Figure 15 showed is to carry can get into cellular localization in nuclear Cre expression vector, and wherein, TAT comes from HIV virus, can mediate albumen and get into cell, and NLS is a nuclear localization signal.
Figure 16 shows is the expression of Cre albumen in intestinal bacteria and the electrophoresis picture that carries out purifying through the Ni post.
What Figure 17 showed is the HFF cell is induced formation through this expression vector positive multipotential stem cell of GFP and the multipotential stem cell of not expressing GFP after Cre albumen is removed foreign gene.
Figure 18 shows pcr amplification foreign gene from cellular genome, detects the induced multi-potent stem cells that obtains and has or not foreign gene to insert.Having detected the HFF cell of this expression vector of transfection, the HFF cell of not transfection and two strains that obtain respectively is iPS1 and iPS2 through the induced multi-potent stem cells of Cre processing and enlarged culturing.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Under the situation that does not deviate from the present invention's spirit and essence, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.Reference " molecular cloning experiment guide " third editions such as the cultivation of pcr amplification condition, enzyme tangent condition, cell, dyeing or related prods carry out.
The structure of embodiment 1 expression vector
The structure of pBR322-rrs-Orip-rrs-EBNA1-MCS:
1) primer of anamorphic zone recombinase recognition sequence pcr amplification from pCEP4 carrier (available from Invitrogen company) obtains rrs-OriP-rrs (primer sequence: P5 ': GAATTCATAACTTCGTATAATGTATGCTATACGAAGTTATCCT; P3 ': AAGCTTATAACTTCGTATAGCATACATTATACGAAGTTATGCAG), and introduce restriction enzyme site EcoRI and HindIII at two ends, enzyme is cut and is inserted into and obtains PBR322-rrs-OriP-rrs among the PBR322.2) pcr amplification EBNA1 (primer sequence: P5 ': CGAGCTAGCATGTCTGACGAGGGGCCAGGTACAGG from the pCEP4 carrier; P3 ': GGATCCTCTAGAAGATCTCTCGAGTCACTCCTGCCCTTCC); And at 5 ' end introducing NheI; Introduce XhoI-BglII-XbaI-BamHI (calling MCS in the following text) at 3 ' end, be inserted into after cutting with NheI and BamHI enzyme and obtain PBR322-rrs-OriP-rrs-(p) EBNA1-MCS among the PBR322-rrs-OriP-rrs.3) pcr amplification IRES-neo (primer sequence: P5 ': GAATTCAGCTCGCTGATCAGCCCCTCT from pLP-IRESneo (available from Clontech company) carrier; P3 ': TCTAGAGGATCCTCAGAAGAACTCGTC), be inserted into the downstream of EGFP-N1 (available from Clontech company) support C MV-EGFP through EcoRI and XbaI.Pcr amplification CMV-EGFP-IRES-neo (primer sequence: P5 ': TCTAGATAGTTATTAATAGTAATCAAT from above carrier construction; P3 ': TCTAGAGGATCCTCAGAAGAACTCGTC); XbaI enzyme cutting is inserted into .4 among PBR322-rrs-OriP-rrs-(p) EBNA1-MCS) pcr amplification CMV promotor (primer sequence: P5 ': ATCGATATAGTTATTAATAGTAATCAAT from the EGFP-N1 carrier; P3 ': ATCGATGATCTGACGG TTCACTAAAC), after the ClaI enzyme is cut, be inserted in the EBNA1 promotor,, finally obtain PBR322-rrs-OriP-rrs-EBNA1-MCS to change the promotor of EBNA1.
Make up respectively with reference to aforesaid method and to obtain pBR322-Orip-rrs-EBNA1-rrs-MCS, pBR322-rrs-Orip-EBNA1-rrs-MCS.
The structure of embodiment 2 recombinant vectorss
The structure of pBR322-rrs-Orip-EBNA1-rrs-EGFP:
Utilize the BglII restriction enzyme site that the EF1a-EGFP-BGHpolyA in pWPXL (available from the Addgene company) carrier is inserted among the pBR322-rrs-Orip-EBNA1-rrs-MCS.The structure of pBR322-rrs-Orip-rrs-EBNA1-OSN:
1) pcr amplification Oct4 (NM_001159542) ORFs (primer sequence: P5 ': GGATCCATGGCGGGACACCTGGCTTC from human embryo stem cell cDNA; P3 ': GAATTCGTTTGAATGCATGGGAGAGCC), be inserted into the downstream of PSP73 carrier (available from Invitrogen company) EF1a promotor through BamHI/EcoRI.2) Synthetic 2 A sequence (AAAATTGTCGCTCCTGTCAAACAAACTCTTAACTTTGATTTACTCAAACTGGCTGG GGATGTAGAAAGCAATCCAGGTCCA), and have the 2A sequence (primer sequence: P5 ': GAATTCAAAATTGTCGCTCCTG of EcoRI and XhoI restriction enzyme site as template amplification with this; P3 ': CTCGAGTGGACCTGGATTGCT).Pcr amplification Sox2 gene (NM_003106) from human embryo stem cell cDNA equally, (primer sequence: P5 ': CTCGAGATGTACAACATGATGGA; P3 ': ACTAGTCATG TGTGAGAGGGGCAG), 2A PCR product is cut through the EcoRI/XhoI enzyme, and Sox2 PCR product is cut through the XhoI/SpeI enzyme, inserts the PSP73-EF1a-Oct4 downstream simultaneously and obtains carrier PSP73-EF1a-Oct4-2A-Sox2.3) equally with above-mentioned 2A sequence as template, amplification has the 2A sequence (primer sequence: P5 ': ACTAGTAAAATTGTCGCTCCTG of SpeI and SmaI restriction enzyme site; P3 ': CCCGGGTGGACCTGGATTGCT).Pcr amplification Nanog gene (NM_024865) from human embryo stem cell cDNA, upstream primer band SmaI restriction enzyme site downstream band XhoI restriction enzyme site (primer sequence: P5 ': CCCGGGATGAGTGTGGATCCAGC; P3 ': CTCGAGTCACACGTCTTCAGGTTGCATGT); 2A PCR product is cut through the SpeI/SmaI enzyme; Nanog PCR product is cut through the SmaI/XhoI enzyme; With the BGH polyA of band XhoI restriction enzyme site downstream, upper reaches band BglII restriction enzyme site (from obtaining primer sequence: P5 ': CTCGAGCTGTGCCTTCTAGTTGCCAGC available from pcr amplification the pcDNA3.1+ carrier of invitrogen company; P3 ': AGATCTCCATAGAGCCCACCGCATCCCC) insert simultaneously in the above PSP73-EF1a-Oct4-2A-Sox2 carrier and obtain PSP73-EF1a-Oct4-2A-Sox2-2A-Nanog.4) utilize the BglII restriction enzyme site that whole EF1a-Oct4-2A-Sox2-2A-Nanog is inserted in the pBR322-rrs-Orip-rrs-EBNA1-MCS carrier at last.
PBR322-Orip-rrs-EBNA1-rrs-OSN utilizes the BglII restriction enzyme site that whole EF1a-Oct4-2A-Sox2-2A-Nanog is inserted in the pBR322-Orip-rrs-EBNA1-rrs-MCS carrier.
PBR322-rrs-Orip-EBNA1-rrs-OSN utilizes the BglII restriction enzyme site that whole EF1a-Oct4-2A-Sox2-2A-Nanog is inserted in the pBR322-rrs-Orip-EBNA1-rrs-MCS carrier.
The structure of pBR322-rrs-Orip-rrs-EBNA1-MK:
1) pcr amplification C-Myc ORFs (primer sequence: P5 ': GGTACCATGCCCCTCAACGTTAGCTTCACCA from C-Myc (available from Wuhan Sanying Bio-Technology Co., Ltd.) the cDNA clone who buys; P3 ': GGATCCCGCACAAGAGTTCCGTAGCTG), the downstream through KpnI/BamHI is inserted into PSP73 carrier (available from Invitrogen company) EF1a promotor obtain PSP73-EF1a-c-Myc.2) equally with above-mentioned 2A sequence as template, amplification has the 2A sequence (primer sequence: P5 ': GGATCCAAAATTGTCGCTCCTG of BamHI and SalI restriction enzyme site; P3 ': GTCGACTGGACCTGGATTGCT).Pcr amplification Klf4 ORFs (primer sequence: P5 ': GTCGACATGAGGCAGCCACCTGGCGAGTCTG from Klf4 (available from Wuhan Sanying Bio-Technology Co., Ltd.) the cDNA clone who buys; P3 ': AAGCTTTTAAAAATGCCTCTTCATGTGTA).2A PCR product is cut through the BamHI/SalI enzyme, and the Klf4PCR product is cut through the SalI/HindIII enzyme, inserts the PSP73-EF1a-c-Myc downstream simultaneously, obtains PSP73-EF1a-c-Myc-2A-Klf4.3) BGHpolyA pcr amplification from the pcDNA3.1+ carrier obtains primer sequence: P5 ': AAGCTTCTGTGCCTTCTAGTTGCCAGC; P3 ': AGATCTCCATAGAGCCCACCGCATCCCC inserts the PSP73-EF1a-c-Myc-2A-Klf4 downstream through HindIII/BglII.4) utilize the BglII restriction enzyme site that whole EF1a-c-Myc-2A-Klf4 is inserted in the pBR322-rrs-Orip-rrs-EBNA1-MCS carrier at last.PBR322-Orip-rrs-EBNA1-rrs-MK utilizes the BglII restriction enzyme site that whole EF1a-c-Myc-2A-K1f4 is inserted in the pBR322-Orip-rrs-EBNA1-rrs-MCS carrier.
PBR322-rrs-Orip-EBNA1-rrs-MK utilizes the BglII restriction enzyme site that whole EF1a-c-Myc-2A-Klf4 is inserted in the pBR322-rrs-Orip-EBNA1-rrs-MCS carrier.
The expression of embodiment 3 recombinant vectorss in cell
The above-mentioned pBR322-rrs-Orip-EBNA1-rrs-EGFP carrier that builds adopts the method transfection HFF (HFF) of electric shock: 3.5 μ gDNA/1.0 * 10
6Cells uses nuclefector consideration convey appearance in 100 μ l amaxa damping fluids (available from LONZA company, the product article No. is DPI-1002), the U023 program is carried out transfection.After the transfection 48 hours, the G418 (the great Bioisystech Co., Ltd in Shanghai) that adds 100 μ g/ml screens, and observes the expression of GFP always, and the GFP expression amount is along with the prolongation of time reduces, but the sustainable expression of GFP more than month under the G418 screening pressure.
Same method transfection HFF (HFF) more than above-mentioned pBR322-rrs-Orip-rrs-EBNA1-OSN that builds and pBR322-rrs-Orip-rrs-EBNA1-MK carrier adopt.Behind the HFF cell transfecting, collect sample in different time and carry out Western blot detection expression of exogenous gene.Express foreign gene is all crossed and is expressed successfully as a result, and can express for a long time.
Embodiment 4 induce produce multipotential stem cell with and the evaluation of versatility
The G418 that the HFF cell that changes pBR322-rrs-Orip-rrs-EBNA1-OSN and pBR322-rrs-Orip-rrs-EBNA1-MK carrier over to was added 100 μ g/ml after transfection in 48 hours screens; After the drug screening 6 days, the major part not HFF of expression alien gene is killed.Can digest to MEC (MEF) at the HFF of surviving under the G418 condition of 100 μ g/ml, inoculate with 10% the rate of converging.Change the embryonic stem cell substratum in second day in inoculation, change liquid every day.Be cultured to the growth of back about 12 days promptly visible clone's like cells of inoculation.Carry out alkaline phosphatase staining (operating) and confirm tentatively whether clone's like cell possibly be multipotential stem cell according to milipore alkaline phosphatase staining test kit specification sheets, and the ratio of definite SEAP positive colony.Further this induced multi-potent stem cells of enlarged culturing, the method for immunofluorescence dyeing and pcr amplification is identified its surface markers SSEA-3, SSEA-4 and specific gene oct4, sox2, the expression level of nanog.And, confirm its ability to all directions differentiation through identifying the expression of each germinal layer marker gene after the EB differentiation.And further identify that through the mouse of this multipotential stem cell being injected immunodeficient it forms teratomatous ability mouse and confirms its versatility.The result shows: clone's like cell that pBR322-rrs-Orip-rrs-EBNA1-OSN and pBR322-rrs-Orip-rrs-EBNA1-MK carrier transfection HFF cell are produced is the alkaline phosphatase enzyme positive; And after enlarged culturing, confirm its surface markers SSEA-3; SSEA-4 and gene oct4; Sox2; The expression level of nanog is all suitable with human embryo stem cell, explains that pBR322-rrs-Orip-rrs-EBNA1-OSN and pBR322-rrs-Orip-rrs-EBNA1-MK carrier can induce the HFF cell to form multipotential stem cell.Further the EB differentiation detects the marker gene that shows each germinal layer all has expression; This induced multi-potent stem cells is injected the mouse of immunodeficient; Can obviously observe teratomatous formation after two months, explain through this expression vector and induce the multipotential stem cell of generation to have versatility.
The structure and the purifying of embodiment 5Cre expression vector
Pcr amplification TAT-NLS-Cre (primer sequence: P5 ': CATATGGGCATGGGCGCTGCAGGTCG from pTriEx-HTNC (available from Addgene company) carrier; P3 ': GAATTCCTAATCGCCATCTTCCAGC), insert the pet28a prokaryotic expression carrier through NdeI/EcoRI, with the warm expression of 6 * His.In the BL21 cell; Induce 4 hours expression Cre albumen through 1mMIPTG, in the damping fluid that contains the 25mM imidazoles, combine 250mM imidazoles wash-out target protein with the Ni post; The elutriant that will contain target protein according to electrophoresis result is collected and is concentrated, and it is subsequent use to the PBS to change damping fluid.
Embodiment 6Cre removes the exogenous gene expression carrier in the induced multi-potent stem cells
After induced multi-potent stem cells success, in substratum, add purifying good carry the Cre albumen that tat can get into cell, concentration is 10 μ m, acts on 2 hours.Cre handles two days later; Fluorescent microscope is observed the expression of green fluorescent protein down, and the cell of 70-80% is expressing green fluorescent protein not, and then filters out the not cell of expressing green fluorescent protein through the method for fluidic cell sorting; Carry out enlarged culturing, and set up clone.Further carrying out PCR for the induced multi-potent stem cells strain of setting up good no foreign gene insertion detects; Design be positioned at the outer primer sequence of recombination on the expression vector (Oct4p5 ': TGCAGTAGTCGCCGTGAACG, Oct4p3 ': TGGGGCCAGAGGAAAGGACA; Sox2p5 ': TTGGTACCCCAGGCTATGGG, Sox2p3 ': CTGCCCGCGGGACCACACCA; Nanogp5 ': GGAACACTCAGACCTGGTGC, Nanogp3 ': CACCCCACCCCCCAGAATAG; CMycp5 ': TACCAGCAGCAGCAGCAGAG, cMycp3 ': TGGAGGGAGGCGCTGCGTAG; Klf4p5 ': GTGGTGGCGCCCTACAACGG, Klf4p3 ': AAAGGACAGTGGGAGTGGCA), whether have foreign gene to be inserted into cellular genome for the template pcr amplification detects with the cellular genome.Detected result shows not expression alien gene of induced multi-potent stem cells that the no foreign gene of foundation inserts, and does not have the insertion (Figure 18) of foreign gene carrier in its genome.
Sequence table
< 110>Institute of Biophysics, Academia Sinica
< 120>a kind of nonconformable, long, the expression system that can delete expression vector
<130>KHP10112347.9
<160>43
<170>PatentIn?version?3.5
<210>1
<211>43
<212>DNA
< 213>artificial sequence
<400>1
gaattcataa?cttcgtataa?tgtatgctat?acgaagttat?cct 43
<210>2
<211>44
<212>DNA
< 213>artificial sequence
<400>2
aagcttataa?cttcgtatag?catacattat?acgaagttat?gcag?44
<210>3
<211>35
<212>DNA
< 213>artificial sequence
<400>3
cgagctagca?tgtctgacga?ggggccaggt?acagg 35
<210>4
<211>40
<212>DNA
< 213>artificial sequence
<400>4
ggatcctcta?gaagatctct?cgagtcactc?ctgcccttcc 40
<210>5
<211>27
<212>DNA
< 213>artificial sequence
<400>5
gaattcagct?cgctgatcag?cccctct 27
<210>6
<211>27
<212>DNA
< 213>artificial sequence
<400>6
tctagaggat?cctcagaaga?actcgtc 27
<210>7
<211>27
<212>DNA
< 213>artificial sequence
<400>7
tctagatagt?tattaatagt?aatcaat 27
<210>8
<211>27
<212>DNA
< 213>artificial sequence
<400>8
tctagaggat?cctcagaaga?actcgtc 27
<210>9
<211>28
<212>DNA
< 213>artificial sequence
<400>9
atcgatatag?ttattaatag?taatcaat 28
<210>10
<211>26
<212>DNA
< 213>artificial sequence
<400>10
atcgatgatc?tgacggttca?ctaaac 26
<210>11
<211>26
<212>DNA
< 213>artificial sequence
<400>11
ggatccatgg?cgggacacct?ggcttc 26
<210>12
<211>27
<212>DNA
< 213>artificial sequence
<400>12
gaattcgttt?gaatgcatgg?gagagcc 27
<210>13
<211>81
<212>DNA
< 213>artificial sequence
<400>13
aaaattgtcg?ctcctgtcaa?acaaactctt?aactttgatt?tactcaaact?ggctggggat 60
gtagaaagca?atccaggtcc?a 81
<210>14
<211>22
<212>DNA
< 213>artificial sequence
<400>14
gaattcaaaa?ttgtcgctcc?tg 22
<210>15
<211>21
<212>DNA
< 213>artificial sequence
<400>15
ctcgagtgga?cctggattgc?t 21
<210>16
<211>23
<212>DNA
< 213>artificial sequence
<400>16
ctcgagatgt?acaacatgat?gga 23
<210>17
<211>24
<212>DNA
< 213>artificial sequence
<400>17
actagtcatg?tgtgagaggg?gcag 24
<210>18
<211>22
<212>DNA
< 213>artificial sequence
<400>18
actagtaaaa?ttgtcgctcc?tg 22
<210>19
<211>21
<212>DNA
< 213>artificial sequence
<400>19
cccgggtgga?cctggattgc?t 21
<210>20
<211>23
<212>DNA
< 213>artificial sequence
<400>20
cccgggatga?gtgtggatcc?agc 23
<210>21
<211>29
<212>DNA
< 213>artificial sequence
<400>21
ctcgagtcac?acgtcttcag?gttgcatgt 29
<210>22
<211>27
<212>DNA
< 213>artificial sequence
<400>22
ctcgagctgt?gccttctagt?tgccagc 27
<210>23
<211>28
<212>DNA
< 213>artificial sequence
<400>23
agatctccat?agagcccacc?gcatcccc 28
<210>24
<211>31
<212>DNA
< 213>artificial sequence
<400>24
ggtaccatgc?ccctcaacgt?tagcttcacc?a 31
<210>25
<211>27
<212>DNA
< 213>artificial sequence
<400>25
ggatcccgca?caagagttcc?gtagctg 27
<210>26
<211>22
<212>DNA
< 213>artificial sequence
<400>26
ggatccaaaa?ttgtcgctcc?tg 22
<210>27
<211>21
<212>DNA
< 213>artificial sequence
<400>27
gtcgactgga?cctggattgc?t 21
<210>28
<211>31
<212>DNA
< 213>artificial sequence
<400>28
gtcgacatga?ggcagccacc?tggcgagtct?g 31
<210>29
<211>29
<212>DNA
< 213>artificial sequence
<400>29
aagcttttaa?aaatgcctct?tcatgtgta 29
<210>30
<211>27
<212>DNA
< 213>artificial sequence
<400>30
aagcttctgt?gccttctagt?tgccagc 27
<210>31
<211>28
<212>DNA
< 213>artificial sequence
<400>31
agatctccat?agagcccacc?gcatcccc 28
<210>32
<211>26
<212>DNA
< 213>artificial sequence
<400>32
catatgggca?tgggcgctgc?aggtcg 26
<210>33
<211>25
<212>DNA
< 213>artificial sequence
<400>33
gaattcctaa?tcgccatctt?ccagc 25
<210>34
<211>20
<212>DNA
< 213>artificial sequence
<400>34
tgcagtagtc?gccgtgaacg 20
<210>35
<211>20
<212>DNA
< 213>artificial sequence
<400>35
tggggccaga?ggaaaggaca 20
<210>36
<211>20
<212>DNA
< 213>artificial sequence
<400>36
ttggtacccc?aggctatggg 20
<210>37
<211>20
<212>DNA
< 213>artificial sequence
<400>37
ctgcccgcgg?gaccacacca 20
<210>38
<211>20
<212>DNA
< 213>artificial sequence
<400>38
ggaacactca?gacctggtgc 20
<210>39
<211>20
<212>DNA
< 213>artificial sequence
<400>39
caccccaccc?cccagaatag 20
<210>40
<211>20
<212>DNA
< 213>artificial sequence
<400>40
taccagcagc?agcagcagag 20
<210>41
<211>20
<212>DNA
< 213>artificial sequence
<400>41
tggagggagg?cgctgcgtag 20
<210>42
<211>20
<212>DNA
< 213>artificial sequence
<400>42
gtggtggcgc?cctacaacgg 20
<210>43
<211>20
<212>DNA
< 213>artificial sequence
<400>43
aaaggacagt?gggagtggca 20
Claims (13)
1. expression vector; Comprise replicon; It is characterized in that, said replicon behaviour herpes virus replication, this expression vector also has the EBNA-1 expression cassette; Has the recombinase recognition sequence at the two ends of said replicon, the two ends of EBNA-1 or the two ends of replicon-EBNA-1, the LoxP sequence that said recombinase recognition sequence is the Cre/LoxP system or the FRt sequence of FLP/FRT system; Said carrier has the drug screening marker gene.
2. expression vector as claimed in claim 1 is characterized in that, the promotor of said EBNA-1 expression cassette is the strong promoter with eukaryotic cell broad spectrum.
3. expression vector as claimed in claim 2 is characterized in that, said promotor is CMV, PGK, CAG or EF-1 α promotor.
4. expression vector as claimed in claim 3, it also has real-time selection markers gene.
5. expression vector as claimed in claim 1 is characterized in that, said drug screening marker gene is neo, hph, gpt, Hprt, tk or puro gene.
6. expression vector as claimed in claim 4 is characterized in that, described real-time selection markers gene is a fluorescence protein gene.
7. by the recombinant vectors of each said expression vector establishment of claim 1～6.
8. recombinant vectors as claimed in claim 7, it comprises foreign gene OSN or MK, and said OSN representes foreign gene Oct4-Sox2-Nanog, and MK representes foreign gene cMyc-K1f4.
9. the host cell that contains each said expression vector of claim 1～6 or claim 7 or 8 said recombinant vectorss.
10. expression system, it comprises each said expression vector of claim 1～6 or claim 7 or 8 said recombinant vectorss, it also comprises recombinase.
11. each said expression vector of claim 1～6, claim 7 or 8 said recombinant vectorss or the said expression system of claim 10 application in expressing foreign protein.
12. each said expression vector of claim 1～6, claim 7 or 8 said recombinant vectorss or the said expression system of claim 10 in the differentiation state of cell and the application in the cell function, are the one-tenth somatocyte to be rearranged become multipotential stem cell.
13. contain the test kit of each said expression vector of claim 1～6, claim 7 or 8 said recombinant vectorss or the said expression system of claim 10.
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| CN103923920B (en) * | 2014-04-04 | 2016-09-21 | 清华大学 | A kind of strengthen the method for nonconformity gene expression in human cell |
| CN104450785A (en) * | 2014-12-08 | 2015-03-25 | 复旦大学 | Genome editing method using attachment carrier for encoding targeted endonuclease and kit |
| KR20230172000A (en) * | 2021-04-16 | 2023-12-21 | 난징 진스크립트 바이오테크 컴퍼니 리미티드 | Selective marker-free plasmid system and method for producing the same |
-
2010
- 2010-03-25 CN CN2010101413840A patent/CN101955977B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| Christine Magin-Lachmann等.Retrofitting BACs with G418 resistance, luciferase, and oriP and EBNA-1 – new vectors for in vitro and in vivo delivery.《BMC Biotechnology》.2003,全文. * |
| Takashi Nagawa等.Long-term transgene expression and inhibition of HIV-1 replication by a Cre/loxP-EBNA-1/oriP HIV-1-dependent ribozyme vector: Applications for HIV-1 gene therapy.《Journal of RNAi and Gene Silencing》.2006,第2卷(第1期),全文. * |
| Valeri A. Krougliak等.Stabilization of transgenes delivered by recombinant adenovirus vectors through extrachromosomal replication.《THE JOURNAL OF GENE MEDICINE》.2000,第3卷全文. * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105219729A (en) * | 2015-09-28 | 2016-01-06 | 首都医科大学宣武医院 | A kind ofly utilize method of nonconformity plasmid vector induced nerve stem cells and uses thereof |
| CN105219729B (en) * | 2015-09-28 | 2018-09-25 | 首都医科大学宣武医院 | A kind of method and application thereof using nonconformity plasmid vector induced nerve stem cells |
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