CN103740756B - The non-viral free carrier that a kind of controllable is deleted and construction process thereof - Google Patents
The non-viral free carrier that a kind of controllable is deleted and construction process thereof Download PDFInfo
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- CN103740756B CN103740756B CN201310729099.4A CN201310729099A CN103740756B CN 103740756 B CN103740756 B CN 103740756B CN 201310729099 A CN201310729099 A CN 201310729099A CN 103740756 B CN103740756 B CN 103740756B
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Abstract
The invention provides the non-viral free carrier that a kind of controllable is deleted, described free carrier is that Cre/loxP site differential recombination enzyme system and S/MAR unit construction are formed a kind of controlled free carrier, the HuIFN-β that described S/MAR element derives from.Described free carrier can artificial adjustment pEPI-1 carrier from mammalian cell by complete deletion, this carrier is applied to the preparation of people iPS cell, the safety issue of iPS cell for cell therapy can be solved, this carrier also can the iPS cells produce of other species and the transdifferentiation investigation and application of cell simultaneously, operating process is simple, and efficiency is high.
Description
Technical field
The present invention relates to genetically engineered field, specifically, relate to non-viral free carrier and the construction process thereof of a kind of controllable deletion.
Background technology
Free episomal carrier (episomal vector) is that a class to be free on outside karyomit(e) and can to stablize and parasitizes endonuclear genetic material.This carrier major advantage be foreign gene can in cell long-term expression, do not need vector integration to arrive cellular genome, avoid the radom insertion of carrier to cause native gene to undergo mutation risk.For many years scientist make great efforts to develop can the free carrier (episomal vector) of self-replacation in mammalian cell always.The free carrier of early stage self-replacation is mainly derived from herpes virus hominis (EBV) and SV40 virus vector, their defect is the expression needing trans-acting proteins, EBV carrier needs to express simplexvirus nuclear antigen 1(EBNV-1), SV40 carrier needs to express large T antigen, and the expression for a long time of these viral proteins can cause cell carcinoma wind transmission danger.PEPI-1 is the free carrier being widely studied non-viral self-replacation at present, its core parts are section of DNA sequence (the chromosomal scaffold/matrixattachment region held from β human interferon gene 5 ', S/MAR), S/MAR can give plasmid vector self-replacation and adjoint cell fission in mammalian cell and be genetic to the characteristic of daughter cell.What S/MAR exercised its function demand fulfillment has two conditions: (1) it must be positioned at 5 ' end of gene, (2) it must be transcribed.With the free additional carrier of viral source unlike, pEPI-1 carrier without any need for foreign protein auxiliary, the gene order security of people from source is very high, is highly suitable for gene therapy application.
Under many circumstances, we more wish can when no longer needing genetic expression, and intracellular expression vector also can by permanent removal.Especially inducing human embryo sample stem cell (iPS) is applied in cell therapy, and research shows the existence of the inducible factor (Oct4, sox2, cMyc, klf4) of the external source in iPS cell, increases the risk that iPS is converted into cancer cells in cell therapy.In order to ensure the safety of iPS cell therapy, must by the exogenous factor complete deletion of iPS detail.
Summary of the invention
In order to solve problems of the prior art, the object of this invention is to provide non-viral free carrier and the application thereof of a kind of controllable deletion.
In order to realize the object of the invention, the non-viral free carrier that first the present invention provides a kind of controllable to delete, described free carrier is that Cre/loxP site differential recombination enzyme system and S/MAR unit construction are formed a kind of controlled free carrier, the HuIFN-β that described S/MAR element derives from.
Further, in described free carrier, S/MAR sequence upstream and downstream respectively inserts a loxp sequence in the same way.
Further, the Core Feature region of described free carrier comprises from 5 ' end to 3 ' end: the EF1 α promotor of people, and loxp sequence, derives from the S/MAR element of HuIFN-β, loxp sequence, the neomycin resistance gene of CAG promoters driven and the expression cassette sequence of TK gene.
As preferably, between two loxp sequences, insert fluorescent protein coding sequence, mark as indication.
Wherein, described fluorescent protein coding sequence is EGFP encoding sequence,
Present invention also offers the zooblast of aforementioned free carrier transfection.
The zooblast of described free carrier transfection, when not needing carrier to exist, adding can the Cre enzyme of permeates cell membranes, and as TAT-Cre, give 12-24h, adding Ganc drug screening afterwards can complete deletion carrier.
As preferably, described zooblast behaviour iPS cell (inductive pluripotent stem cells).
The method of free carrier transfected with human iPS cell is, aforementioned free carrier is inserted four factor expression boxes of induction people iPS cell, and transfected with human somatocyte G418 screens induction people iPS cell line, obtains people iPS cell.
Present invention also offers the application of aforementioned free carrier in iPS cells produce and cells transdifferentiate.
The invention provides non-viral free carrier (episomalvector) method of a kind of controllable deletion and the application of this carrier.Its principle is that Cre/loxP site differential recombination enzyme system and S/MAR unit construction are formed a kind of controlled free carrier.The wherein HuIFN-β that derives from of S/MAR, when carrier exist S/MAR sequence and can transcribed time, carrier can self-replacation in cell, and pass to daughter cell with cell fission, and namely carrier can form genetic stability without the need to being incorporated into cellular genome.Respectively insert a loxp sequence in the same way in S/MAR sequence upstream and downstream during design vector of the present invention, the site specific recombination utilizing Cre enzyme to mediate can delete S/MAR, makes carrier cannot form episome again, loses from cell.Use has the Cre enzyme of cell-penetrating ability (holding the small peptide of fusing penetrating cytolemma at the N of Cre albumen), such as TAT-Cre, by selecting to add this Cre enzyme in cell culture medium, control genetic stability or the loss of carrier, concrete operation method is as embodiment 2.Because the albumen such as TAT-Cre cannot realize the cell-penetrating of 100%, causing having can residual support in part cell.We insert a TK in the carrier and bear screening-gene, and cell culture medium adds Ganc medicine and the cell remaining carrier can be killed.In a word, we can select whether to add Cre enzyme and Ganc, carry out the genetic stability of free carrier in regulating cell or lose completely.
Beneficial effect of the present invention is:
The invention provides the non-viral free carrier that a kind of controllable is deleted, by containing free additional carrier Cre/loxp system and S/MAR being bonded controllable and deleting, can artificial adjustment pEPI-1 carrier from mammalian cell by complete deletion, this carrier is applied to the preparation of people iPS cell, the safety issue of iPS cell for cell therapy can be solved, this carrier also can the iPS cells produce of other species and the transdifferentiation investigation and application of cell simultaneously, operating process is simple, and efficiency is high.
Accompanying drawing explanation
Fig. 1 is the structure schema of free carrier of the present invention.
Fig. 2 is the non-viral free carrier that controllable of the present invention is deleted.
The luciferase expression figure that Fig. 3 is free carrier Successful transfection cell of the present invention and is deleted by regulation and control.
Fig. 4 is the free carrier of inducing the controllable of iPS to delete in the embodiment of the present invention 3.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.
Carrier design in following examples is completed by this seminar, and the carrier of use all comes from Agricultural biotechnologies National Key Laboratory of China Agricultural University, and oligoDNA is synthesized by the raw work in Shanghai, and sequencing is completed by Beijing Hua Da.Taq enzyme, T4DNA ligase enzyme, restriction endonuclease all from Beijing NEB company, the equal available from Sigma of somatic cell clone agents useful for same.The normal experiment operation stepss such as enzyme is cut, connect, reclaim, transform, pcr amplification refer to " molecular cloning (third edition) ".
Embodiment 1 is dissociated the structure of carrier
Vector construction step is as follows, builds flow process as Fig. 1;
1.NheI and AgeI double digestion pEPI-EGFP carrier, inserts first loxp sequence, obtains intermediate carrier 1,
2.SalI and BmHI double digestion intermediate carrier 1, inserts the loxp sequence that second direction is identical, obtains intermediate carrier 2,
3.PciI and NheI double digestion intermediate carrier 2, deletes CMV promoter, inserts people EF1 α promotor and replaces CMV promoter, obtain intermediate carrier 3,
4.BsaI and MluI double digestion, deletes the neomycin resistance gene of intermediate carrier 2, inserts ammonia benzyl resistant gene, obtains intermediate carrier 4,
5.AscI and SpeI double digestion pPB-CAG-TKiNeo carrier obtains object fragment, inserts AscI and SpeI double digestion intermediate carrier 4, is configured to final pEPI-EF1 α-EGFP-CAG-TkiNeo(as shown in Figure 2).
Embodiment 2 dissociate carrier cell transfecting and regulation and control delete
1. spend intracellular toxin plasmid extraction kit, extract pEPI-EF1 α-EGFP-CAG-TKiNeo plasmid, be dissolved into ultrapure water and reach 1ug/ul to concentration, careful absorption 3ug plasmid and 5 × 10
6cell fully mixes rear electrotransfection, and after electrotransfection, plating cells is to 5 10cm Tissue Culture Dishs, cultivates and adds G418 to concentration 500ug/ml after 2 days, and the cell clone of green fluorescence is expressed in screening for 10 days afterwards as seen, as Fig. 3.
Due to EGFP genetic expression eGFP, so cell clone can express green fluorescence, demonstrate pEPI-EF1 α-EGFP-CAG-TKiNeo plasmid Successful transfection in cell.
2. add TAT-Cre albumen and cultivate 24 hours to concentration 400ng/ml, then add 0.2umol/Lganc screening to green fluorescence completely dissolve, as Fig. 3.
Extract cellular genome PCR to detect, in result showed cell, plasmid is deleted completely.Experimental result shows to delete pEPI-EF1 α-EGFP-CAG-TkiNeo by adding TAT-Cre albumen in cell, can be deleted the cell of pEPI-EF1 α-EGFP-CAG-TKiNeo after adding Ganc screening completely.
Embodiment 3 induces that iPS's delete free vector construction
AscI enzyme cuts pEPI-EF1 α-EGFP-CAG-TkiNeo, and glue reclaims linear carrier, alkaline phosphatase treatment linear carrier, and removing linear carrier 5 ' phosphoric acid, take pSTEM as template,
Primers F: aat
gGCGCGCCtCGTGAGGCTCCGGTGCC;
With primer R:aat
gGCGCGCCcTTACAATTTACGCCTTAAGATACATTG(glissade part is AscI restriction enzyme site; the little protection base being written as the required interpolation of PCR primer direct enzyme cutting); amplification is to object fragment; AscI enzyme links with linearized vector after cutting; transform; screening obtains object clone, and cloning vector collection of illustrative plates as shown in Figure 4.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (4)
1. the non-viral free carrier of a controllable deletion, it is characterized in that, described free carrier is that Cre/loxP site differential recombination enzyme system and S/MAR unit construction are formed a kind of controlled free carrier, the HuIFN-β that described S/MAR element derives from;
In described free carrier, S/MAR sequence upstream and downstream respectively inserts a loxp sequence in the same way;
The Core Feature region of described free carrier comprises from 5 ' end to 3 ' end: the EF1 α promotor of people, loxp sequence, derive from the S/MAR element of HuIFN-β, loxp sequence, the neomycin resistance gene of CAG promoters driven and the expression cassette sequence of TK gene.
2. free carrier according to claim 1, is characterized in that, between two loxp sequences, insert fluorescent protein coding sequence, marks as indication.
3. free carrier according to claim 2, is characterized in that, described fluorescent protein coding sequence is EGFP encoding sequence.
4. the application of free carrier in iPS cells produce and cells transdifferentiate described in claim 1-3 any one.
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| CN105002213A (en) * | 2014-04-24 | 2015-10-28 | 中国科学院上海巴斯德研究所 | Unconformable deletable novel non-viral vector, and construction method and use thereof |
| CN105087617A (en) * | 2014-05-07 | 2015-11-25 | 中国农业科学院油料作物研究所 | Recombinant vector of 36bplox site sequence as well as construction method and application of recombinant vector |
| WO2019219649A1 (en) * | 2018-05-14 | 2019-11-21 | Vivet Therapeutics | Gene therapy vectors comprising s/mar sequences |
| CN110004181B (en) * | 2019-03-20 | 2022-10-11 | 东北农业大学 | Additional CRISPR/Cas9 expression vector and construction method and application thereof |
| CN113025651B (en) * | 2021-03-31 | 2023-03-24 | 重庆医科大学 | Novel application of drug screening cell model, triciribine and structural analogue of targeted HBV core promoter |
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