CN1203181C - Mouse entactin gene promoter and its tissue-specific enhancer - Google Patents
Mouse entactin gene promoter and its tissue-specific enhancer Download PDFInfo
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- CN1203181C CN1203181C CN 00127624 CN00127624A CN1203181C CN 1203181 C CN1203181 C CN 1203181C CN 00127624 CN00127624 CN 00127624 CN 00127624 A CN00127624 A CN 00127624A CN 1203181 C CN1203181 C CN 1203181C
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Abstract
The present invention provides a mouse entactin promoter element, an enhancer element, a construction object containing the elements, a vector containing the elements and an application thereof. The entactin promoter element and/or the enhancer element of the present invention can be used for guiding the expression of exogenous genes and for researching the function of important molecules in the development process of the nervous system, the establishment of neuropathy animal models and the gene therapy of neuropathy.
Description
The present invention relates to molecular biology and DNA recombinant technology field.More specifically, the present invention relates to the promotor and the tissue-specific enhancer thereof of entactin gene, and their purposes.
Mammalian central nervous system is to be grown by the early stage neurocele of embryo to differentiate.In this complicated atomization, the stage that relates to many nervous tissue specificitys and neurocyte specific gene is expressed.Nidogen (nestin) is as a member of moderate fiber protein family, because of its specific expressed clone in the central nervous system in rat precursor cell, and is used as the tagged molecule of central nervous system neural stem cell and is widely used.
At present, rat and people's entactin gene is all cloned.The result of transgenic animal shows, in first intron of rat entactin gene, the enhancer sequence that exists this gene of regulation and control in muscle tissue, to express, and in second intron, the enhancer sequence that exists the regulation and control entactin gene in central nervous system, to express.People's entactin gene expression regulation also has similar elements to exist.Second intron of entactin gene has been used to instruct some foreign gene specificity expression in nervous tissue.
Yet, before the application, also do not obtain mouse entactin gene sequence and relevant regulating and controlling sequence.
In view of mouse is a kind of important experimental animal, and nidogen is albumen specific expressed in central nervous system, and therefore, this area presses for entactin gene sequence of exploitation mouse and relevant regulating and controlling sequence.
Purpose of the present invention just provides a kind of new mouse entactin gene sequence and relevant regulating and controlling sequence.
Another object of the present invention provides the method for these regulating and controlling sequences of production and the purposes of described regulating and controlling sequence.
In a first aspect of the present invention, a kind of promoter element is provided, this promoter element comprises the nucleotide sequence of entactin gene promoter.Preferably, described promoter element comprises the nucleotide sequence shown in the SEQ ID NO:1.
In a second aspect of the present invention, a kind of enhancer element is provided, it comprises the nucleotide sequence of second intron of mouse entactin gene.Preferably, described enhancer element comprises the nucleotide sequence shown in the SEQ ID NO:2.
In a third aspect of the present invention, provide a kind of and be used for, the described enhancer element of claim 3 that this construction contains foreign gene and is operably connected with this foreign gene at the specific expressed construction of neural stem cell.Preferably, described construction also contains the described promoter element of the claim 1 that is operably connected with this foreign gene.
In a fourth aspect of the present invention, a kind of carrier is provided, this carrier contains above-mentioned promoter element and/or enhancer element.Preferably, described carrier is expression vector and plasmid.
In a fifth aspect of the present invention, provide a kind of in neural stem cell the method for specific expressed foreign gene, it comprises step:
(a) provide a construction, the enhancer element that described construction contains foreign gene and is operably connected with this foreign gene, described enhancer element comprise the nucleotide sequence of second intron of mouse entactin gene,
(b) construction in the step (a) is imported neural stem cell, foreign gene is expressed in neural stem cell.
In a preference of the present invention, construction also contains the entactin gene promoter element that is operably connected with foreign gene in the step (a).
Others of the present invention are because disclosing of the technology of this paper is conspicuous to those skilled in the art.
Following accompanying drawing is used to illustrate specific embodiments of the present invention, and is not used in qualification by the scope of the invention that claims defined.
Fig. 1 has shown the structure of mouse entactin gene.
Fig. 2 is the structure iron of construction pNH84.
Fig. 3 has shown the result who the promoter region of mouse entactin gene is carried out transient transfection assays.
Fig. 4 has shown the synoptic diagram that second intron of mouse entactin that links to each other with luciferase reporter gene is carried out the series disappearance.
Fig. 5 has shown the result who the second intron zone of mouse entactin gene is carried out transient transfection assays.
Fig. 6 has shown that second intron of mouse entactin gene is expressed foreign gene (reporter gene LacZ) in the central nervous system (CNS) of growing.Fig. 6 A has shown the structure of the dna fragmentation that is used to produce transgenic mice.Fig. 6 B and 6C have shown that under the guidance of the promotor of mouse entactin gene and second intron reporter gene lacZ expresses specifically in CNS.
Fig. 7 has shown the nucleotide sequence of the promoter element of mouse entactin gene.
Fig. 8 has shown the nucleotide sequence of the enhancer element (i.e. second intron) of mouse entactin gene.
The inventor obtains mouse entactin gene by screening mouse bacterial artificial chromosome (BAC) genomic library.Make up series disappearance plasmid, (Embryonal Carcinoma, EC) P19 identifies the promotor and the enhanser zone of mouse entactin gene to transfection embryonal carcinoma cell.Utilize the promotor and the tissue-specific enhancer thereof of mouse entactin gene, obtain transgenic mice at the central nervous system specifically expressing reporter gene LacZ that grows.The promotor of mouse entactin gene and enhanser thereof are used in expression alien gene or enhancing expression of exogenous gene in the P19 cell.
As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.
As used herein, term " nidogen promoter element ", " nidogen promoter region " are used interchangeably, the polynucleotide of refer to have the nidogen promoter sequence (SEQ ID NO:1) or its active fragments.
As used herein, term " enhancer element of nidogen ", " nidogen second intron " and " neural system of nidogen is expressed enhanser " etc. are used interchangeably, the polynucleotide of refer to have the nidogen enhancer sequence (SEQ ID NO:2) or its active fragments.
The invention still further relates to the polynucleotide varient of above-mentioned promotor and enhanser.The varient that allelic variant that these polynucleotide varients can be natural generations or non-natural take place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be replacement, disappearance or the insertion of one or more Nucleotide, but can be from not changing the function of this promotor or enhanser in fact.
The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably at least 100 above total lengths to nidogen promoter element and/or enhancer element of Nucleotide.Nucleic acid fragment can be used for the amplification technique (as PCR) of nucleic acid to determine and/or to separate the nucleotide sequence of promotor of the present invention or enhanser.
The Nucleotide full length sequence of people's nidogen promotor of the present invention and enhanser or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence design primer, and with mouse gene group DNA or the artificial chromosome clone that contains mouse chromosome as template, amplification and must relevant sequence.
In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.
In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.At present, can be fully obtain the dna sequence dna of promotor of the present invention or enhanser (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.
Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition promotor of the present invention or enhancer element.
The present invention also relates to comprise the construction or the carrier of promotor of the present invention and/or enhancer element, and transform or the host cell of transduction with described construction or carrier, and the method that produces foreign protein through recombinant technology.
Be applicable to that foreign gene of the present invention is not particularly limited, nearly all foreign gene all can be used for the present invention.The example of representational foreign gene comprises (but being not limited to): BDNF, NT-3, STAT3 and APP etc.It should be noted that foreign gene also comprises the gene from host itself.For example, for some neural inherited disease (for example in neural system, express deficiency or do not express the disease that is caused) because of certain albumen, can separate from patient itself and obtain related gene, then it is linked to each other with nidogen promoter element of the present invention and/or enhancer element, formation contains the construction of foreign gene, then described construction is used for gene therapy.
A kind of example of construction is exactly the foreign gene that links to each other with nidogen promoter element and/or enhancer element.As for the position relation of nidogen promoter element and/or enhancer element and foreign gene, promotor should be positioned at the upstream of foreign gene, and enhanser can be positioned at the upstream or the downstream of foreign gene.
Among the present invention, the nucleotide sequence that contains nidogen promotor and/or enhancer element can preferably be inserted into carrier, for example in the expression vector.Term " carrier " refers to that bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus, mammalian cell virus are as adenovirus, retrovirus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: expression vector, cloning vector for example are applicable to the carrier in protokaryon (as bacteriums such as intestinal bacteria), eucaryon (as yeast), insect cell, vegetable cell, the mammalian cell.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.In expression vector, except containing replication orgin, nidogen promoter element and/or nidogen enhancer element, also can contain marker gene and other translation controlling elementss.
Method well-known to those having ordinary skill in the art can be used to make up and contains nidogen promoter element and/or enhancer element and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a LaboratoryManual, Cold Spring Harbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can be connected with treating expressed exogenous gene effectively, and is synthetic to instruct described foreign gene mRNA.
In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.
Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can express exogenous protein.
Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as mammalian cell.Representative example has: intestinal bacteria, streptomyces; The bacterial cell of Salmonella typhimurium; Fungal cell such as yeast; Vegetable cell; The insect cell of fruit bat S2 or Sf9; The zooblast of CHO, COS, 293 cells or Bowes melanoma cells etc.
Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.The method for transformation of some employings includes, but are not limited to: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.
The transformant that obtains can be cultivated with ordinary method, with the expression alien gene encoded polypeptide.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth and expresses, cultivate.
In one embodiment of the invention, screened nidogen cDNA from 10 days the full embryo cDNA of the mouse library, and obtain the full length sequence of the cDNA 5983bp of mouse entactin.Utilize the PCR screening method of genomic library, obtain bacterial artificial chromosome (BAC) clone of an entactin gene.Do Southern hybridization with mouse entactin cDNA sequence as probe.By comparing the gene structure that nidogen cDNA and genomic dna sequence can draw mouse entactin, promptly this gene is made up of 4 exons and 3 introns.
Compare with rat and people's entactin gene, the position of mouse entactin gene intron and size are all more conservative.At present, the sequence of 5 ' upstream of rat and people's entactin gene, the 1st and the 3rd intron is not seen bibliographical information as yet.The 2nd intron of mouse entactin gene of the present invention and the homology of rat and the 2nd intron of people's entactin gene are respectively 88% and 56%.
The inventor has also further anatomized mouse entactin promoter element and enhancer element.And be determined by experiment the definite sequence of mouse entactin promoter element and enhancer element, and confirmed that the nidogen enhancer element can strengthen expression of exogenous gene specifically in central nervous system.
Nidogen promoter element of the present invention and/or enhancer element have broad application prospects, for example 1) mouse entactin gene promoter can instruct foreign gene to express in multipotential cells such as P19 cell.2) mouse entactin gene second intron can strengthen the expression of foreign gene in multipotential cells such as P19 cell.3) mouse entactin gene second intron is because of the specifically expressing characteristic in its neural stem cell, can be combined in overexpression foreign gene in the neural system that Mammals grows with nidogen promotor or other promotors.Therefore, nidogen promoter element of the present invention and/or enhancer element can be applicable to study the foundation of function of important molecule in the nervous system development process, neuropathy animal model and the gene therapy of neuropathy.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1
The acquisition of mouse entactin cDNA sequence and genome sequence
Utilize antibody (the Jing N.H. of the mouse entactin that we prepare, Kitani H., et al., 1996, ChineseJournal of Physiological Sciences, Vol 12, p1-8), screening mouse neural precursor cDNA library obtains mouse entactin gene cDNA 3 ' end 3.6Kb (M5).Fragment with M5 5 ' end 740bp (2402-3143 position Nucleotide) is screened the 10 days full embryo cDNA of mouse libraries that make up with ordinary method, obtains the full length sequence (mouse entactin gene cDNA GenBank accessionno.AF076623) of cDNA 5983 bp of mouse entactin.
Utilize the PCR screening method of genomic library, obtain bacterial artificial chromosome (BAC) clone of an entactin gene.Dna fragmentation with 5 ' end 1-1203 position and 5427-5983 position in the mouse entactin cDNA sequence is that probe is done Southern hybridization.The inventor has obtained 4 sliceable positive colonies that go out the gene structure of mouse entactin: pNX7, pNB6, pNX5 and pNX9 (Fig. 1).By comparing the gene structure that nidogen cDNA and genomic dna sequence can draw mouse entactin, promptly this gene is formed (Fig. 1) by 4 exons and 3 introns.
Compare with rat and people's entactin gene, the position of mouse entactin gene intron and size are all more conservative.At present, the sequence of 5 ' upstream of rat and people's entactin gene, l and the 3rd intron is not seen bibliographical information as yet.The 2nd intron of mouse entactin gene of the present invention and the homology of rat and the 2nd intron of people's entactin gene are respectively 88% and 56%.
Embodiment 2
The evaluation of nidogen promoter element
Mice embryonic cancer cells P19 is a strain multipotential cell, and this cell has normal male mouse chromosome caryogram, 10
-6The vitamin A acid of mol/L (retinoic acid RA) induces down, but the differentiation of neuralward cell direction.In this course, neurodevelopment Expression of Related Genes pattern has been simulated normal mouse neurodevelopment process substantially.Therefore, the P19 cell is used as a neurodevelopmental experiment in vitro model of research mouse at present.Grow different times and vitamin A acid (Retinoic Acid by analyzing entactin gene at mice embryonic, RA) inductive embryonal carcinoma cell (Embryonal Carcinoma, EC) expression rule in the external Neural Differentiation process of P19, the inventor finds that expression rule and this gene expression pattern in animal body of entactin gene in P19 Neural Differentiation process is similar.
The regulating and controlling sequence of mouse entactin gene is building up on the pGL3 carrier that contains luciferase reporter gene, by liposome LipofectAMINE transfection P19 cell.Utilize the activity of two luciferase reporter gene detection kit (dual-luciferase reporter assay system) examining report gene of Promega company, thereby determine the regulation and control zone of mouse entactin gene.
In order to analyze the promotor characteristic of mouse entactin gene, a series of plasmids have been made up.With pNX7 is template, and the design primer is done the PCR reaction and obtained mouse entactin gene cDNA 5 ' upstream 887 bp and 2, the fragment of 660bp.After advancing T-vector, these two fragment clonings are built into plasmid CLP3 and CLP5.
The primer is during preparation plasmid CLP3:
Forward: 5 '-GAGAACGCGTAGAGCCGCGTAACTTCTTCACT-3 ' (SEQ ID NO:3);
Oppositely: 5 '-GAGACTCGAGGTGGAGCACTAGAGAAGGGAGT-3 ' (SEQ ID NO:4).
Preparation plasmid CLP5 the primer is:
Forward: 5 '-GAGAACGCGTGGCAGGTGGCTCACAACTATCT-3 ' (SEQ ID NO:5);
Oppositely: 5 '-GAGACTCGAGGTGGAGCACTAGAGAAGGGAGT-3 ' (SEQ ID NO:4).
After order-checking was identified, CLP3 and CLP5 were with restriction enzyme Mlu I and Xho I cutting and reclaim 895bp and 2,668bp fragment respectively.These two fragments are connected with the carrier pGL3-Basic that contains reporter gene (available from Promega company) that handles through same two kinds of restriction enzymes, obtain cloning pNH85 and pNH83.
PNX7 reclaims the fragment of 3.2Kb after restriction enzyme SacI and AatII cutting.This fragment is building up in the pNH85 that two kinds of same restriction enzymes are handled, and obtains plasmid pNH84 (Fig. 2).After inserting the multiple clone site of plasmid pBluescript KSII (-) among the pNH84, obtain plasmid pNH81.PNH81 does can obtain plasmid pNH82 and pNH86 from connecting reaction after restriction enzyme Pst I or Apa I cutting.PNH81 does to obtain plasmid pNH87 from connecting reaction after restriction enzyme EcoR V and Pvu II cutting.
Except according to 5 ' to 3 ' direction mouse entactin gene 5 ' upstream sequence is done the series disappearance, the inventor lacks 5 ' upstream near-end 2 respectively on the basis of plasmid pNH84,342bp and 780bp obtain plasmid pNH45 and pNH46.PNH45 is that pNH84 forms from connecting after Nco I enzyme is cut.It comprises mouse entactin gene 5 ' upstream far-end 1, the sequence that 650bp is long.Plasmid pNH84 reclaims 8 after Aat II and Xho I enzyme are cut, the fragment that 007bp is long.This fragment is got plasmid pNH46 continuously certainly after mung-bean nuclease is handled.PNH46 comprises mouse entactin gene 5 ' upstream far-end 3, the sequence that 212bp is long.
The plasmid that obtains is carried out uciferase activity detect, the result as shown in Figure 3.Analytical results shows that the long zone of mouse entactin 5 ' end upstream 344bp promptly has promoter activity (Fig. 3).The concrete nucleotide sequence of mouse entactin promoter element is shown in Fig. 7 and SEQ ID NO:1.
Embodiment 3
The evaluation of nidogen enhancer element
In order to verify whether the 2nd intron of mouse entactin gene has the characteristic of central nervous system specifically expressing, and the inventor is at first in its activity to the reporter gene transcriptional control of vitro detection.
In making up the process of changeing cell series disappearance plasmid, the plasmid pGL3-Promoter to band reporter gene luciferase transforms earlier.PGL3-Promoter is after the XhoI enzyme is cut, and is flat with Klenow enzyme benefit, then from connecting to eliminate the XhoI site.Be the convenient series disappearance plasmid that makes up, the multiple clone site that pBluescript II SK (-) is gone up between SacI to KpnI is building up among the pGL3-Promoter that removes the XhoI site, and to name this plasmid be pGL3-Px '.
The 2nd intron of entactin gene is from clone pNB6.The building process of series disappearance plasmid is referring to Fig. 4.
The reporter gene detected result shows that the zone of the about 800bp length of second introne 3 ' end of mouse entactin gene has enhancement (Fig. 5) to the expression of reporter gene.The concrete nucleotide sequence of mouse entactin enhancer element is shown in Fig. 8 and SEQ ID NO:2.
Embodiment 4
Structure contains the construction of mouse entactin promoter element and/or enhancer element
On the basis in promotor that obtains mouse entactin gene and enhanser zone, make up the dna fragmentation that comprises mouse entactin gene promoter, enhanser and reporter gene lacZ and prepare transgenic mice.Plasmid pNH86 obtains mouse entactin gene promoter 344bp fragment after restriction enzyme Kpn I and Xho I cutting.This fragment with obtain plasmid pNH101 through the carrier p β gal-Basic (available from Clontech company) of Kpn I and Xho I cutting _ be connected.Plasmid pNH92 obtains mouse entactin gene enhanser 753bp fragment after restriction enzyme Bgl II cutting.This fragment with through Bgl II cutting and calf intestinal alkaline phosphatase (CIP) Biology/Leonard G.Davis, W.Michael Kuehl, James F.Battey.-2nd ed.1994.By Appleton ﹠amp; Lange Norwalk, Connecticut, USA.).
Construction as shown in Figure 6A, from 5 ', contain mouse entactin gene 344bp promoter region, 753bp enhanser zone, reporter gene lacZ and carrier sequence successively to 3 ' direction.
Experimental result is shown in Fig. 6 B and C, the reporter gene lacZ expression pattern similar to endogenous entactin gene shows, the promotor of mouse entactin gene has function in animal body, and the long zone of the about 753bp of its second introne 3 ' end (being 753bp in SEQ ID NO:2) then has the ability that instructs foreign gene to express in neural stem cell.
Embodiment 5
Utilize PCR to obtain the mouse entactin promoter element from mouse gene group DNA
Synthetic following primer:
Forward primer: 5 '-GGGCCCAGTTCTGTGCA-3 ' (SEQ ID NO:6),
Reverse primer: 5 '-GTGGAGCACTAGAGAAG-3 ' (SEQ ID NO:7).
With the mouse gene group DNA is template, carries out the PCR reaction in following condition: 94 ℃, and 1min; 45 ℃ of 1min; 72 ℃ of 30sec, totally 35 circulations.Obtained the amplified production of about 350bp.To the amplified production evaluation of checking order, the result shows with the sequence of the mouse entactin promoter element shown in the SEQ ID NO:1 and conforms to.
The mouse entactin promoter element can be used for instructing expression of exogenous gene, especially the expression in mammalian cell.
Embodiment 6
Utilize PCR to obtain the mouse entactin enhancer element from mouse gene group DNA
Synthetic as follows:
Forward primer: 5 '-AGATCTAGGAGAGGAGCT-3 ' (SEQ ID NO:8),
Reverse primer: 5 '-GGATCACTCTTTATTGTG-3 ' (SEQ ID NO:9).
With the mouse gene group DNA is template, carries out the PCR reaction in following condition: 94 ℃, and 1min; 45 ℃ of 1min; 72 ℃ of 1min, totally 35 circulations.Obtained the amplified production of about 750bp.To the amplified production evaluation of checking order, the result shows with the sequence of the mouse entactin enhancer element shown in the SEQ ID NO:2 and conforms to.
Mouse entactin tissue-specific enhancer is used in overexpression foreign gene in the neural system that Mammals grows.This can be used for the animal model studying gene function, set up neuropathy and the gene therapy of neuropathy.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
(1) general information:
(ii) denomination of invention: mouse entactin gene promoter and tissue-specific enhancer thereof
(iii) sequence number: 9
(2) information of SEQ ID NO:1:
(i) sequence signature:
(A) length: 344bp
(B) type: nucleic acid
(C) chain: two strands
(D) topological framework: linearity
(ii) molecule type: DNA
(xi) sequence description: SEQ ID NO:1:
GGGCCCAGTT?CTGTGCATCT?TAGGGTGTTC?TGGGCTGTCT?GGCTGTATCT?CAAGCCTCTT 60
TCGGAAAATC?ACCCGCACCG?GACGGGATCC?CCGCCAGGGC?GAGGCTACAA?TTTGATTCTT 120
CTCTGCTGAG?CTGGGATGAT?GCAGGGACCC?GGGCTGTGTG?TTGCACTGAA?CTCTAAAGGG 180
TTAAGGCCTA?GGGACCGCCC?CTTTTCCGCC?CGGCCGGCGG?GAGTATGAAT?ACCCTCGCTT 240
CAGCTCGCTG?CTGGAATCCT?CCGCTTCCGC?TGGGTCACTG?TCGCCGCTAC?TCCCTTTCTC 300
CCCCTTAAAA?GCTCCAAGGG?CCACTCCCTT?CTCTAGTGCT?CCAC 344
(2) information of SEQ ID NO:2:
(i) sequence signature:
(A) length: 753bp
(B) type: nucleic acid
(C) chain: two strands
(D) topological framework: linearity
(ii) molecule type: DNA
(xi) sequence description: SEQ ID NO:2:
AGATCTAGGA?GAGGAGCTGG?AGGGTAGAAG?TCCAGAGAAG?GGAAGCCAGT?GAGTGTCTAG 60
GAAGGACTGA?ATGCACAAGG?CAGGACACTA?TGAGGACGGA?GGAAAGGCTA?TTTTGAGAAC 120
CAGATGTGCT?CAGAGGCCAT?GAATGGAAAC?AAGGGACTAG?CTCCGAATCC?CTTGTGAACT 180
GATTCCCCTC?ATCTCCTCCC?ATCAGCTGTG?AACCTAAAGA?CCTCTGGCCT?GAAGGAGGAT 240
TTGTGATCCT?AAGATGAGGG?CCTTCGCCCC?AGTCAGTCTT?CTGAGGCGAG?GGGAAGGGTC 300
CAGGCAGCTC?TGAGGAATGT?AAGCACTGGT?GTTTGCGGTC?TGAAAAGGAT?TTGGAGAAGG 360
AGAGCTGAAT?TCATTTGCTT?TTGTCTGTCA?CCAGCTCTGG?GGGCCTAGGA?GAGAGCCATC 420
CCATGGGAAC?AGCCTGAGAA?TTCCCACTTC?CCCTGAGGAG?CCCCTCCTTC?TCAGGCCCTC 480
CAGATGGTAG?TGTGGACAAA?AGGCAATAAT?TAGCATGAGA?ATCGGCCTCC?CTCTCCGAGG 540
ATGAGGTCAT?CGGCCTTGGC?CTTGGGTGGG?GAGGCGGAGA?CTGATCTGAG?GAGTCTGATC 600
GAAGTGTTAG?CAATTCACTT?GGCCCTGCTT?CCGAATGTGG?GAATCTGCGT?GTGGGGTCTC 660
CCTGAGTCTC?AAATATGGGT?TCACCAAGTA?TATATCTGTG?GGTATATGAC?TGTGTGGCTT 720
TCATACGACA?ATGGTCACAA?TAAAGAGTGA?TCC 753
(2) information of SEQ ID NO:3
(i) sequence signature
(A) length: 32 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:3:
GAGAACGCGT?AGAGCCGCGT?AACTTCTTCA?CT 32
(2) information of SEQ ID NO:4
(i) sequence signature
(A) length: 32 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:4:
GAGACTCGAG?GTGGAGCACT?AGAGAAGGGA?GT 32
(2) information of SEQ ID NO:5
(i) sequence signature
(A) length: 32 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:5:
GAGAACGCGT?GGCAGGTGGC?TCACAACTAT?CT 32
(2) information of SEQ ID NO:6
(i) sequence signature
(A) length: 17 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:6:
GGGCCCAGTT?CTGTGCA 17
(2) information of SEQ ID NO:7
(i) sequence signature
(A) length: 17 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:7:
GTGGAGCACT?AGAGAAG 17
(2) information of SEQ ID NO:8
(i) sequence signature
(A) length: 18 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:8:
AGATCTAGGA?GAGGAGCT 18
(2) information of SEQ ID NO:9
(i) sequence signature
(A) length: 18 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: linearity
(ii) molecule type: oligonucleotide
(xi) sequence description: SEQ ID NO:9:
GGATCACTCT?TTATTGTG 18
Claims (6)
1. an enhancer element is characterized in that, it has the nucleotide sequence of second intron of mouse entactin gene, and described nucleotide sequence is shown in SEQ ID NO:2.
2. one kind is used for it is characterized in that at the specific expressed construction of neural stem cell the described enhancer element of claim 1 that it contains foreign gene and is operably connected with this foreign gene.
3. construction as claimed in claim 2 is characterized in that, it also contains the mouse entactin promoter element that is operably connected with this foreign gene, and described promoter element has the nucleotide sequence shown in the SEQ ID NO:1.
4. a carrier is characterized in that, it contains the described enhancer element of claim 1.
5. the method for a specific expressed foreign gene in neural stem cell is characterized in that, it comprises step:
(a) provide a construction, the enhancer element that described construction contains foreign gene and is operably connected with this foreign gene, described enhancer element have the nucleotide sequence of second intron of mouse entactin gene,
(b) construction in the step (a) is imported neural stem cell, foreign gene is expressed in neural stem cell.
6. an isolating nucleic acid is characterized in that, it contains the described enhancer element of claim 1.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2004100474502A CN1328378C (en) | 2000-11-30 | 2000-11-30 | Mouse nidogen gene starter and its tissue specificity enhancer |
| CN 00127624 CN1203181C (en) | 2000-11-30 | 2000-11-30 | Mouse entactin gene promoter and its tissue-specific enhancer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 00127624 CN1203181C (en) | 2000-11-30 | 2000-11-30 | Mouse entactin gene promoter and its tissue-specific enhancer |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100474502A Division CN1328378C (en) | 2000-11-30 | 2000-11-30 | Mouse nidogen gene starter and its tissue specificity enhancer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1355317A CN1355317A (en) | 2002-06-26 |
| CN1203181C true CN1203181C (en) | 2005-05-25 |
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Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 00127624 Expired - Fee Related CN1203181C (en) | 2000-11-30 | 2000-11-30 | Mouse entactin gene promoter and its tissue-specific enhancer |
| CNB2004100474502A Expired - Fee Related CN1328378C (en) | 2000-11-30 | 2000-11-30 | Mouse nidogen gene starter and its tissue specificity enhancer |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2004100474502A Expired - Fee Related CN1328378C (en) | 2000-11-30 | 2000-11-30 | Mouse nidogen gene starter and its tissue specificity enhancer |
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| Country | Link |
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| CN (2) | CN1203181C (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101864419B (en) * | 2010-04-27 | 2012-05-02 | 武汉大学 | Mouse Miwi promoter as well as carrier and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5338839A (en) * | 1988-04-12 | 1994-08-16 | Massachusetts Institute Of Technology | DNA encoding nestin protein |
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- 2000-11-30 CN CNB2004100474502A patent/CN1328378C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1550551A (en) | 2004-12-01 |
| CN1355317A (en) | 2002-06-26 |
| CN1328378C (en) | 2007-07-25 |
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