CN102533744A - Gene expression cassette and recombinant expression vector comprising same - Google Patents
Gene expression cassette and recombinant expression vector comprising same Download PDFInfo
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- CN102533744A CN102533744A CN2011104517117A CN201110451711A CN102533744A CN 102533744 A CN102533744 A CN 102533744A CN 2011104517117 A CN2011104517117 A CN 2011104517117A CN 201110451711 A CN201110451711 A CN 201110451711A CN 102533744 A CN102533744 A CN 102533744A
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Abstract
The invention relates to a gene expression cassette and applications thereof. The gene expression cassette comprises a promoter without cell specificity, one LoxP locus, a terminator, a cell specificity promoter, Cre recombinase gene, a terminator, another LoxP locus, a target gene and a terminator which are connected in sequence, wherein the two LoxP loci are homodromous. Compared with the prior art, the gene expression cassette is capable of realizing high-level expression on the target gene without using a transgenic animal, and simultaneously, is capable of ensuring the selectivity of cells. Moreover, an experiment purpose can be achieved as long as the gene expression cassette is cloned to the expression vector required by an experiment as needed without using multiple expression vectors, thereby, steps and the time of the experiment are saved.
Description
[technical field]
The present invention relates to the genetically engineered field, relate in particular to a kind of expression casette and contain the recombinant expression vector of this expression casette.
[background technology]
Traditional transgenic method mainly adopts virus to carry specific promotor and realizes the expression of goal gene in target cell type; Its main method is to clone proteinic promoter sequence specific expressed in a certain monoid cell, and subclone is realized the specific expressed of gene to the viral transfer vector that carries goal gene then.This method can obtain effect preferably in some cases; But in most cases but can not get the ideal effect; Mainly be that expression efficiency is lower as a rule because of the specificity promoter that is adopted; Simultaneously be difficult to find other alternative specificity promoters again, the expression efficiency that will improve goal gene is so simultaneously taken into account cell selective again becomes urgent problem.
[summary of the invention]
Based on this, be necessary to provide the expression casette that a kind of expression efficiency is higher and cell-specific is stronger.
A kind of expression casette; Comprise the acellular specific promotor, LoxP site, terminator, cell specificity promotor, Cre recombinase gene, terminator, LoxP site, goal gene and the terminator that link to each other successively; Wherein, two LoxP sites are in the same way.
In preferred embodiment, said acellular specific promotor is CMV promotor, EF1-a promotor or PGK promotor.
In preferred embodiment, the gene order of said CMV promotor is the SEQ ID NO:1 in the sequence table.
In preferred embodiment, the gene order in said LoxP site is the SEQ ID NO:2 in the sequence table.
In preferred embodiment, said terminator is a SV40 poly A terminator.
In preferred embodiment, the sequence of said SV40 poly A terminator is the SEQ ID NO:3 in the sequence table.
In preferred embodiment, said Cre recombinase gene sequence is the SEQ ID NO:4 in the sequence table.
The expression casette that comprises above-mentioned characteristic can be used to make up recombinant expression vector, thereby is used for the field such as diagnosis or treatment of disease.Preferably, the expression vector that uses can be plasmid or virus.
Import to certain tissue of living animal when this expression casette after; Transcribing of acellular specific promotor by downstream terminator premature termination, the promotor with cell-specific can be expressed the Cre recombinase in target cell, do not express in the non-target cell; The Cre recombinase can be discerned the LoxP site on the expression casette; With two in the same way the sequence between the LoxP site cut off, thereby opened transcribing of acellular specific promotor, realize efficiently expressing of goal gene.Compare with conventional art, this expression casette need not to use transgenic animal just can realize efficiently expressing of goal gene, guarantees the selectivity of cell simultaneously; And need not to use a plurality of expression vectors, just can reach experiment purpose, saved experimental procedure and time as long as on request expression casette is cloned into the required expression vector of experiment.
[description of drawings]
Fig. 1 is the structural representation of the expression casette of an embodiment;
Fig. 2 is the structural representation of the expression casette of specific embodiment partial design;
Fig. 3 is the technological line figure of pMD18T-CS vector construction;
Fig. 4 cuts qualification result figure for the enzyme of pMD18T-CS carrier;
Fig. 5 is the technological line figure of pMD18T-CCS vector construction;
Fig. 6 cuts qualification result figure for the enzyme of pMD18T-CCS carrier;
Fig. 7 is the technological line figure of pMD18T-CCST vector construction;
Fig. 8 cuts qualification result figure for the enzyme of pMD18T-CCST carrier;
Fig. 9 is the technological line figure of pShuttle-CCS vector construction;
Figure 10 cuts qualification result figure for the enzyme of pShuttle-CCS carrier;
Figure 11 is the technological line figure of pshuttle-CCS-ChR2 vector construction;
Figure 12 cuts qualification result figure for the enzyme of pshuttle-CCS-ChR2 carrier;
Figure 13 is the detected result figure that the adenovirus that contains this expression casette is expressed ChR2 in vivo.
[embodiment]
Mainly combine accompanying drawing and specific embodiment that expression casette and the recombinant expression vector that contains this expression casette are done further detailed explanation below.
As shown in Figure 1; The expression casette of one embodiment; Comprise the acellular specific promotor (Promoter 1), LoxP site, terminator (stop), cell specificity promotor (Promoter2), Cre recombinase gene, terminator (stop), LoxP site, goal gene (Target gene) and the terminator (stop) that connect successively; Wherein, two LoxP sites are in the same way.
The preferred strong promoter of acellular specific promotor of this embodiment, like CMV promotor, EF1-a promotor or PGK promotor etc., wherein the gene order of CMV promotor is the SEQ ID NO:1 in the sequence table.This strong promoter can start the expression of goal gene in the cell (target cell or non-target cell) of numerous types, be used for expressing efficiently goal gene.Can clone corresponding strong promoter sequence to this position according to demand in the practical implementation process.
Cre/LoxP site recombinase system is the strong instrument that carries out genetic expression in vivo and in vitro; The Cre recombinase can be discerned the LoxP site on the dna sequence dna; When two in the same way LoxP sites are arranged in the dna sequence dna; The Cre recombinase can cut off the dna sequence dna between the LoxP site, thereby closes or open expression of gene.Wherein, the gene order in LoxP site is shown in the SEQ ID NO:2 in the sequence table, and Cre recombinase gene sequence is shown in the SEQ ID NO:4 in the sequence table.
Terminator can adopt SV40 polyA commonly used (being called for short pA) terminator sequence, and its sequence is shown in the SEQ ID NO:3 in the sequence table.
Cell specificity promotor can only be expressed the corresponding target gene in the cell (being called for short target cell) of particular type, in non-target cell, then be not activated the function of destination gene expression.Generally speaking, its starting efficiency in specific cells is lower, and the expression efficiency of the goal gene that it is corresponding is also lower.Two promotors are used (being acellular specific promotor and cell specificity promotor) simultaneously; And in conjunction with the Cre/LoxP system; Let the promotor of cell-specific in target cell, express the Cre recombinase, after the Cre recombinase is expressed, will be in target cell with two in the expression cassette in the same way the sequence between the LoxP site cut off; Thereby opened transcribing of strong promoter, realized efficiently expressing of goal gene.
After the expression vector of the expression casette with Fig. 1 structure gets into animal particular organization; The cell specificity promotor that is arranged in the LoxP site will optionally be expressed the Cre recombinase at the target cell monoid, does not then express the Cre recombinase in other cell monoid.Although before cell specificity promotor, be provided with acellular specific promotor; It can start transcribing of downstream sequence in the ordinary course of things; But before the goal gene of this promotor back, be designed with terminator; Thereby premature termination transcribing of it, have only this section terminator sequence disallowable after, the effect of acellular specific promotor competence exertion.After expressing the Cre recombinase in the target cell; In the expression casette two in the same way the LoxP site discerned by the Cre enzyme; Thereby dna sequence dna is therebetween cut off; Significant change takes place in the expression casette that is sheared on dna sequence dna, acellular specific promoter transcription is disengaged by the structure of premature termination, begins efficiently expressing of downstream goal gene.
The expression casette that comprises above-mentioned characteristic can be used to make up recombinant expression vector, thereby is used for the field such as diagnosis or treatment of disease.Preferably, the expression vector that uses can be plasmid or virus.
Explain how to realize that goal gene efficiently expresses in target cell with concrete embodiment below:
Usually be to choose the promotor of TPH at serotonin serotonergic neuron cell inner expression photosensitive protein ChR2 (channelrhodopsin-2) as the promotor of expressing photosensitive protein ChR2; This kind of enzyme is specific to be expressed in the serotonin serotonergic neuron cell because have only; But after being to use this promotor; The expression of goal gene is very faint, is difficult to detect, and almost be nonsensical for experiment.Discovering that the expression level of TPH in serotonin serotonergic neuron cell itself is very low, promptly is that the TPH promotor is a weak promoter, and natural expression efficiency is very low.
The promotor of the acellular characteristic of present embodiment is selected cytomegalovirus promoter (CMV promoter) for use, and cell specificity promotor is selected TPH promotor (TPH promoter) for use.Its concrete construction process is following:
One, experiment material:
1. plasmid and bacterial strain:
PMD 18T-simple and ligase enzyme solution I are available from takara company;
Pshuttle carrier and adenovirus packaging system are available from Stratagene company;
DH5a is available from takara company for intestinal bacteria competence (Escherichia coli).
2. the main related reagent of molecular cloning:
Restriction enzyme Bgl II, EcoR V, Kpn I, Not I, Xba I and Sac I are available from takara company, and Pac I, Pme I, Xma I are available from NEB company.
3. cell strain and laboratory animal:
Human embryonic kidney cell HEK293 is available from U.S. ATCC cell company;
The C57 mouse is available from Guangdong Medical Lab Animal Center.
4. the adenovirus bag changes required reagent:
DMEM is available from GIBCO company;
Foetal calf serum (FBS) is available from GIBCO company;
Pancreatin is available from GIBCO company;
Phosphate buffered saline buffer (PBS) is available from Hyclone company;
Transfection reagent (FuGENE HD transfection reagent), Tissue Culture Dish and transfer pipet are available from coming company.
Two, experimental technique and data:
1.LoxP, the synthetic and clone of dna sequence dna of stop, SV40 ployA and Cre recombinase gene
Utilize gene synthesis technology to synthesize dna sequence dna as shown in Figure 2, this sequence is inserted on the pMD18T-simple carrier, cut through enzyme and identify that this sequence is inserted in the carrier really, and with this carrier called after pMD18T-CS.
The technological line figure of pMD18T-CS vector construction is as shown in Figure 3.The pMD18T-CS enzyme is cut and is identified Bgl II and EcoR V, and it is as shown in Figure 4 that enzyme is cut qualification result, obtains fragment and the fragment of 2.6kb about about 1.7kb, proves to make up successfully that wherein, the Marker that uses in the qualification process is 1Kb DNA ladder.
2.CMV the clone of promoter
Utilizing round pcr, is template with the pCDNA3.1 carrier, and amplification CMV promoter the fragment after the amplification is inserted between the Bgl II and SacI of pMD18T-CS carrier, and with the carrier called after pMD18T-CCS that makes up, building process is as shown in Figure 5.
The pMD18T-CCS enzyme is cut qualification result: as shown in Figure 6; Cut through Bgl II and Sac I enzyme, obtain the fragment of (being the segmental length of CMV promoter) about about 4.3Kb and 600bp, confirm that through order-checking this sequence is correct; Wherein, the Marker that uses in the qualification process is 1Kb DNA ladder.
3.TPH the clone of promoter
Extract the genomic dna of Mouse Neuron cell; With this genomic dna is that template is utilized round pcr amplification TPH promoter; And this sequence is inserted between the Sbf I and Xma I of pMD18T-CCS carrier, building process is as shown in Figure 7, with the carrier called after pMD18T-CCST that makes up.
The pMD18T-CCST enzyme is cut qualification result: as shown in Figure 8, cut through Xma I and Sbf I enzyme, and obtain the fragment about about 4.9Kb and 1KbTPH promotor, this fragment of order-checking confirmation is correctly inserted, and wherein, the Marker that uses in the qualification process is 1Kb DNA ladder.
4. the expression cassette (being pMD18T-CCST) that will contain TPH promoter is cloned on the pshuttle carrier
The method of utilizing PCR is template with pMD18T, and amplification contains the expression cassette of TPH promoter, this fragment is inserted between the Kpn I and Not I of pshuttle carrier, and building process is as shown in Figure 9, and with the carrier called after pshuttle-CCS that makes up.
The pshuttle-CCS enzyme is cut qualification result: shown in figure 10, cut through Kpn I and Not I enzyme, and obtain the fragment about about 3.2Kb and 6.6Kb, confirm that through order-checking this sequence insertion is correct, wherein, the Marker that uses in the qualification process is 1Kb DNA ladder.
5.ChR2 and SV40polyA is cloned on the pshuttle-CCS carrier
Utilize round pcr or gene synthetic mode to obtain ChR2 and SV40polyA sequence, this sequence is inserted between the Xba I and EcoRV of pshuttle-CCS, and the carrier called after pshuttle-CCS-ChR2 building process that makes up is shown in figure 11.
The pshuttle-CCS-ChR2 enzyme is cut qualification result: shown in figure 12; Cut through Xba I and EcoR V enzyme; Obtain the fragment about about 1.3Kb and 9.8Kb, order-checking confirms the correct insertion of this sequence wherein, and the Marker that uses in the qualification process is 1Kb DNA ladder.
6. the carrier that builds above inciting somebody to action utilizes the adenovirus packing technique; The preparation adenovirus utilizes stereotaxic instrument to be expelled to the experiment brain district of mouse packaged virus, and adenovirus can infect the subregion around the injection site; In serotonin serotonergic neuron cell monoid; The TPH promotor can start the poor efficiency of Cre recombinase transcribes, and the Cre recombinase can be expressed in the serotonin serotonergic neuron, then can not express the Cre recombinase in the non-serotonin serotonergic neuron; The structural LoxP of meeting recognition expression box site after the Cre recombinase is expressed; LoxP site intermediary dna sequence dna is sheared, and the terminator sequence in CMV promoter downstream also is sheared simultaneously, thereby has realized photosensitive protein ChR2 efficiently expressing under strong promoter.
The adenovirus vector construct that will have this expression casette and carry the ChR2 gene well after; Utilize this carrier package adenovirus; Packaged adenovirus is utilized three-dimensional locating injection mode, and nuclei of median raphe (DRN) zone of the brain of injection C57 mouse is after four days; Take out the tissue extraction albumen in this nuclei of median raphe zone, immunoblotting detects the expression of ChR2.With the adenovirus carrier that does not have this expression casette is contrast.Detected result is shown in figure 13; Wherein, Lane1: injecting virus C57 not, lane 2: injection contains the virus of expression casette; Lane3: injection only contains the virus of ChR2 gene; Proof ChR2 gene successful expression in the mouse body, and lane 2 band brightness obviously are better than lane 1 and lane 3, explain that the ChR2 gene efficiently expresses in the mouse body.
Import to certain tissue of living animal when this expression casette after; Transcribing of acellular specific promotor by downstream terminator premature termination, the promotor with cell-specific can be expressed the Cre recombinase in target cell, do not express in the non-target cell; The Cre recombinase can be discerned the LoxP site on the expression casette; With two in the same way the sequence between the LoxP site cut off, thereby opened transcribing of acellular specific promotor, realize efficiently expressing of goal gene.Compare with conventional art, this expression casette need not to use transgenic animal just can realize efficiently expressing of goal gene, guarantees the selectivity of cell simultaneously; And need not to use a plurality of expression vectors, just can reach experiment purpose, saved experimental procedure and time as long as on request expression casette is cloned into the required expression vector of experiment.
The above embodiment has only expressed several kinds of embodiments of the present invention, and it describes comparatively concrete and detailed, but can not therefore be interpreted as the restriction to claim of the present invention.Should be pointed out that for the person of ordinary skill of the art under the prerequisite that does not break away from the present invention's design, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with accompanying claims.
Claims (9)
1. expression casette; It is characterized in that; Comprise the acellular specific promotor, LoxP site, terminator, cell specificity promotor, Cre recombinase gene, terminator, LoxP site, goal gene and the terminator that link to each other successively, wherein, two LoxP sites are designed in the same way.
2. expression casette as claimed in claim 1 is characterized in that, said acellular specific promotor is CMV promotor, EF1-a promotor or PGK promotor.
3. expression casette as claimed in claim 2 is characterized in that, the gene order of said CMV promotor is the SEQ ID NO:1 in the sequence table.
4. expression casette as claimed in claim 1 is characterized in that, the gene order in said LoxP site is the SEQ ID NO:2 in the sequence table.
5. expression casette as claimed in claim 1 is characterized in that, said terminator is a SV40 poly A terminator.
6. expression casette as claimed in claim 5 is characterized in that, the sequence of said SV40 poly A terminator is the SEQ ID NO:3 in the sequence table.
7. expression casette as claimed in claim 1 is characterized in that, said Cre recombinase gene sequence is the SEQ ID NO:4 in the sequence table.
8. the recombinant expression vector that contains each said expression casette among the claim 1-7.
9. recombinant expression vector as claimed in claim 8 is characterized in that, said carrier is plasmid or virus.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111088251A (en) * | 2019-12-26 | 2020-05-01 | 云舟生物科技（广州）有限公司 | Gene expression cassette and application thereof in Cre-lox recombination efficiency detection |
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| US20070015195A1 (en) * | 2005-07-18 | 2007-01-18 | Pioneer Hi-Bred International, Inc. | Modified FRT recombination site libraries and methods of use |
| US20090165150A1 (en) * | 2007-12-21 | 2009-06-25 | Yinghui Zhou | Directed complementation with removable gene of interest |
| CN101532020A (en) * | 2008-03-11 | 2009-09-16 | 中国科学院遗传与发育生物学研究所 | Specificity promoter for flowers of plants and screening marker-free conversion vector thereof |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070015195A1 (en) * | 2005-07-18 | 2007-01-18 | Pioneer Hi-Bred International, Inc. | Modified FRT recombination site libraries and methods of use |
| US20090165150A1 (en) * | 2007-12-21 | 2009-06-25 | Yinghui Zhou | Directed complementation with removable gene of interest |
| CN101532020A (en) * | 2008-03-11 | 2009-09-16 | 中国科学院遗传与发育生物学研究所 | Specificity promoter for flowers of plants and screening marker-free conversion vector thereof |
Non-Patent Citations (1)
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| KACZMARCZYK ET AL.: "A single vector containing modified cre recombinase and LOX recombination sequences for inducible tissue-specific amplification of gene expression", 《NUCLEIC ACIDS RESEARCH》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111088251A (en) * | 2019-12-26 | 2020-05-01 | 云舟生物科技（广州）有限公司 | Gene expression cassette and application thereof in Cre-lox recombination efficiency detection |
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