CN1370240A - Regulation system of expression using nuclear PPAR receptors - Google Patents
Regulation system of expression using nuclear PPAR receptors Download PDFInfo
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- CN1370240A CN1370240A CN00811928A CN00811928A CN1370240A CN 1370240 A CN1370240 A CN 1370240A CN 00811928 A CN00811928 A CN 00811928A CN 00811928 A CN00811928 A CN 00811928A CN 1370240 A CN1370240 A CN 1370240A
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Abstract
The invention concerns novel methods and compositions for the pharmacological regulation of the expression of transgenes. More particularly, it concerns a composition comprising: (a) a first element comprising a nucleic acid of interest under the control of an inducible promoter comprising an element containing a response element to a PPAR and a minimal transcriptional receptor; and (b) a second element comprising a nucleic acid coding for a PPAR under the control of a transcriptional promoter, for simultaneous, separate or prolonged use. The invention concerns the use of said compositions and methods in the experimental, clinical, therapeutic or diagnostic fields.
Description
The present invention relates to field of biology.Particularly, the present invention relates to genetic expression and regulate the field, more specifically, the present invention describes the new system of development and development pharmacological modulation transgene expression.The present invention activates transgenosis based on end user source construct especially and transcribes.Therefore, the present invention for example describes in muscle cell, in test tube, in vitro or novel composition, construct and the method that can effectively regulate expression of nucleic acid in vivo.It is varied to come from application scenario of the present invention, for example is seen in experiment, clinical, treatment or diagnosis aspect.
Control transgene expression level and time all are absolutely necessary for many application.So aspect gene therapy, successful treatment may require given dose by transgenosis synthetic albumen.Similarly, use in the time of for example can separating the inducible expression system of vegetative period and production phase, can improve the production of recombinant protein in vitro.Making up transgenic animal, research gene action or proteic bioavailability etc. all is to belong to suitable control of heredity to express and bring the situation of many improvement.
Prior art has dreamed up different manual transcription regulatory gene, and the xenobiotic molecule that they are connected with transgenosis transcripting starting subsequence activates.
The VP16 trans-activation domain that suppresses son and hsv (HSV) by intestinal bacteria Lac constructs at first these regulatory gene of explanation.These regulatory gene have two versions, and one can be activated with sec.-propyl b-D-thiogalactoside (IPTG), and another is by IPTG inactivation (Baim S. and colleague thereof, " Proc.Natl.Acad.Sci.U.S.A. ", 88, (1991) 5072-5076; Labow M. and colleague thereof, " Mol.Cell.Biol. ", 10 (1990) 3343-3356).
The VP16 trans-activation domain that suppresses son and HSV by intestinal bacteria Tet constructs another system.These regulatory gene also have two versions, and an available tsiklomitsin or derivatives thereof activates, and another can be by these same molecular inactivations (Gossen M. and Bujard H., " Proc.Natl.Acad.Sci.U.S.A. ", 89, (1992) 5547-5551; Gossen M. and colleague thereof, " science ", 268 (1995) 1766-1769).
The VP16 trans-activation domain of people's receptors ligand sequence and HSV constructs another system on the sequence of the GAL4 protein D NA of S.cerevisiae and the Progesterone, this version can be with activating (Wang Y. and colleague thereof as RU486 Progesterone analogue, " Proc.Natl.Acad.Sci.U.S.A. ", 91, (1994) 8180-8184).The VP16 trans-activation domain of also describing fruit bat ecdysone receptor and HSV merges, and activates (No D. and colleague thereof, " Proc.Natl.Acad.Sci.U.S.A. ", 93, (1996) 3346-3351) with moulting hormone and this steroid hormone analogue.Another system's utilization promotes the ability of some some immunosuppression molecule of cell protein cybotactic (Ciclosporin A, rapamycin and derivative thereof).At this moment, the transcriptional regulatory gene is made of two kinds of protein subunits, first kind can be formed by chimeric DNA sequence and proteic three replica fusion of FKBP people, and second kind of p65 subunit trans-activation domain fusion by proteic rapamycin sequence of FRAP people and people NFkB forms.This transcriptional regulatory gene can be with the rapamycin of two kinds of subunit's dimerizations being activated (Rivera V and colleague thereof, " Nat.Med ", 2 (1996) 1028-1032).
Though these systems can reach gratifying adjusting level in some tissue, they still have some to restrict the deficiency of its working conditions.Therefore, these transcriptional regulatory genes are foreign protein in human body.They in fact are to be made of the protein fragments from bacterium, virus, yeast or insect, or protein region be the people source the time, they are produced by people's outside sequence and engage.These protein regions therefore can the inducing cell toxin immune response, so make the cell of under allosome transcriptional regulatory Gene Handling, expressing target gene be damaged, stopped transgene expression like this.This situation can force takes the repetitive administration therapeutic gene, is formed in defective particularly serious when relating to traumatology department's behavior like this, and this treatment is always not effective, and particularly the carrier at therapeutic gene is when injection causes immunoreactive virus for the first time.In addition, not always satisfactory with the viewed expression level of regulation system of prior art.
Therefore, need a kind of improved expression regulation system, this system is compatible with in vivo using, and also can use in different tissues, and guarantee to have very high expression level under state of activation.The present invention is directed to these problems and propose a solution.
The present invention in fact relates to the regulation system that son is activated in the end user source.This system should be able to avoid administering therapeutic gene repeatedly.
The present invention describes the improve inducible expression system of a kind of use PPAR nuclear receptor (acceptor of peroxide activator enzyme body hyperplasia) as the transcriptional regulatory gene especially.In application WO 98/21349, once described in liver special efficacy expression system and used PPRE.Now, improvement of the present invention system can produce transcriptional regulatory gene (therefore people source PPAR albumen belong to non-basically foreign protein in human body), the evoked promoter of control transgene expression by minimal promoter with PPAR (PPRE) reaction become to be grouped into.System of the present invention, no matter in vitro still in vivo, particularly in muscle, all available PPAR special efficacy part activates.In addition, the transgene expression level that reaches after activation is similar to strong promoter expression level such as the hCMV-IE promotor.
System of the present invention therefore induce in a large number, patience (especially in vivo using), power and utilize and all have many advantages aspect the condition.
Therefore, the invention describes the novel constructs that is used to implement and use this system, particularly promoter region, expression cassette and plasmid.The present invention also describes the new PPAR construct that can improve genetic expression control and the combination of these different constructs.The present invention shows that also these method and compositions can control and adjusting be expressed in a large number in vitro and in vivo.The invention still further relates to the cell that for example comprises construct of the present invention, and the screening method of PPAR ligand compound.
More specifically, first purpose of the present invention is composition, and said composition comprises:
A) first composition, it is included in the purpose nucleic acid under the evoked promoter control, and this evoked promoter comprises composition and the minimum transcripting promoter to the PPAR reaction, and
B) second composition, it is included in the transcripting promoter control nucleic acid of coding PPAR down, so that side by side, use them respectively or with the certain hour compartment of terrain.
In mode more particularly, the present composition also comprises:
(c) PPAR part also is for simultaneously, uses them respectively or with the certain hour compartment of terrain.
Advantageously, these compositions also comprise composition (d), and it is included in the transcripting promoter control nucleic acid of coding X class retinene derivative acceptor (RXR) down.
Its purpose is side by side, show respectively or with the expression that use the certain hour compartment of terrain and can prepare, regulate and use in succession respectively composition (a) and (b), (c) and/or (d) in case of necessity respectively, so that can control the purpose expression of nucleic acids.Typically, prepared composition (a) and (b) and optional (d) and it is packaging together, compound (c) then is separated packing, and with (a) and (b) and optional (d) be used at interval with certain hour, these heterogeneities make up in cell, tissue, organ etc. and obtain desired expression regulating effect.
To this, in the specific implementations of the present composition, add composition (a) and (b) and (d) that choose wantonly by different genetic constructs.
At another of the present composition specific and preferred embodiment in, composition (a) and (b) and optional (d) be integrated in the same genetic constructs.So, the invention describes the compound genetic constructs that can express purpose product and PPAR.These constructs are particularly advantageous, because they have comprised is alone regulating the required whole genetic constitutions of expression purpose nucleic acid.
One or more genetic constructs can be natural and/or various different sources, particularly plasmid episome, karyomit(e), virus, phage etc.Preferably, genetic constructs is plasmid or virus vector.
Have composition (a) or plasmid (b) respectively in order to illustrate, for example can enumerate JxnS-TK-pGL3, JxnAS-TK-pGL3, DR1xnS-TK-pGL3, DR1xnAS-TK-pGL3, JxnAS-CMV-pGL3, pSG5-hPPARg2g2 or Jx10AS-CMV-EF-pGL3 plasmid, these will be described in detail later.
Composition (a) and plasmid (b) have wherein been gathered in order to illustrate, for example can enumerate Jx5AS-TK-Luc-bPPARg2, SV-g2-J10-C-pGL3, hPPARg2-CMV-Jx5AS-TK-pGL3 or hPPARg2-CMV-Jx10AS-CMV-pGL3 plasmid, these will be described in detail later.
As the virus vector example, can enumerate recombinant adenovirus especially, the reorganization retrovirus, AAV, simplexvirus, vaccine virus etc., its preparation can be carried out according to those skilled in the art's currently known methods.
The configuration and the structure of genetic constructs below will be described in more detail at this paper.
To this, as noted earlier, composition (a) comprises inducible promoters, and it comprises at least:
-to the composition of PPAR reaction, and
-minimum transcripting promoter.
Composition (PPRE, " the sub-reacted constituent of peroxysome hyperplasia ") to the PPAR reaction is the nucleic acid district of fixedly PPAR, and the PPAR chain can transmit signal near nucleic acid district then.Therefore composition to the PPAR reaction is the nucleic acid district that can connect PPAR.In order to implement the present invention, the composition of PPAR reaction is more particularly comprised the site of one or more PPAR chains.Some chain sites were so once described in the prior art, for example as in the promotor of different people (for example AII lipophorin gene).Some sites can also be artificial constructed like this, and test their PPRE character, and are as described below.
In a kind of specific implementations, the composition that PPAR is reacted comprises one or more TCAACCTTTACCCTGGTAG sequences (SEQ ID NO:1) site, or the functional variant of this sequence.SEQID NO:1 sequence is corresponding to the J district of people apoAII promotor (Nucleotide-734 to-716).
In another kind of specific implementations, the composition that PPAR is reacted comprises one or more AGGTCAAAGGTCA sequences (SEQ ID NO:5) site, or the functional variant of this sequence.SEQ ID NO:5 sequence is corresponding to the DR1 consensus.
The functional variant of term is meant and keeps above-mentioned PPRE character, promptly particularly connects all modification sequences of the ability of PPAR.These modifications can be included in interpolation, sudden change, disappearance and/or the replacement of the one or more Nucleotide in the sequence of studying.Can adopt molecular biological ordinary method, for example take place especially or be total to composition sequence by people in synthesizer more practically to add these modifications by orthomutation.Usually, variant reservation at least 50% marks the residual sequence of homing sequence.More preferably, these variants have being less than the modification that 5 Nucleotide exert an influence in research sequence.The variant that obtains like this carries out their PPRE activity test then.These character can be confirmed in different ways, especially:
(i) allow the test sequence contact with X class retinene derivative acceptor (RXR), preferably in acellular test, contact, and (for example hysteresis by moving on gel) detects mixture formation with PPAR;
(ii) in the expression cassette that comprises minimal promoter and reporter gene, insert the test sequence, this box added in the cell, and in the presence of or do not exist under the condition of PPAR and PPAR part, detect reporter gene expression (measuring dosage if desired);
(iii) adopt any other technology well known by persons skilled in the art, these technology make may prove for example interaction between the nucleic acid and albumen.
The for example above-mentioned (ii) middle activity of measuring preferably equals the activity 50% with SEQ ID NO:1 or 5 sequence bit point measurements at least, more preferably equals at least at 75% o'clock, and variant can be considered to the functional variant of meaning of the present invention.The functional variant in the PPAR chain site on the meaning of the present invention was for example described in following document: people such as Juge-Aubry, " journal of biological chemistry " (J.Biol.Chem.), 272 (1997) 25252 and people such as Nakshatri, " NAR " 26 (1998) 2491, these documents are incorporated in this paper as a reference.
With three RXR α, RXR β and RXR γ genes encoding X class retinene derivative acceptor (RXR), it separates with sequence and once obtained describing (people (1990) " nature " 345 such as Mangelsdorf DJ, 224-229; People (1992) such as Mangelsdorf DJ, " gene development " 6,329-344).Preferably, composition (d) coding people's RXR α.
About the effect of PPAR/RXR allos dimerization, two examination results open to discussion: Mangelsdorf DJ and Evans RM (1995), " cell " 83,841-850 and Wilson TM and Wahli W (1997), " the general viewpoint in the chemicobiology " (Current Opinion in Chemical Biology), 1,235-241.People (1998) such as Schulman IG, at " molecule and cytobiology " (Molecular andCellular Biology), 18, the transactivation by PPAR γ/RXR α heterodimer described in the article among the 3483-3494.
Use composition (d) to produce synergy to the activity of composition (b).
Just as noted earlier, in the present composition, the composition that PPAR is reacted can comprise a plurality of PPAR chains site.Can relate to the repetition of same loci or the combination of different loci, it is preferred that same loci repeats.More preferably, reacted constituent comprises nearly 30 chain sites, preferably 3-20 chain site, more preferably 5-15 chain site.Preferred implementation of the present invention is the construct in a kind of 10-15 of comprising chain site, the result who lists among the embodiment in fact show such some constructs particularly in muscle cell induce and expression level aspect favourable character.
For composition (a) in the thing combined according to the invention is made evoked promoter, the composition that PPAR is reacted combines with the transcripting promoter of minimum.Minimal promoter is the transcripting promoter that the basis activity is low or do not have basis activity and its basic activity to exist (activated PPAR and PPRE interaction between component) can increase down at transcription activator.Therefore, minimal promoter can be a weak type promotor naturally in mammalian cell, promptly produces nontoxicity and expresses and/or insufficient expression promoter for obtaining remarkable biological action.Advantageously, minimal promoter is to use natural promotor to have the prepared construct of disappearance of the nonessential region of transcriptional activity by one or more.Therefore, preferably relate to a kind of promotor that comprises the TATA box basically, the size of its box is generally less than 160 Nucleotide, and is the center with the initiator codon of transcribing.Minimal promoter can also be with strong or weak cell virus promotor for example as promotor, the promotor of desmin gene, the promotor of vimentin dna, the light chain of myosin or the preparations such as promotor of heavy chain gene of the promotor immediately of the promotor of thymidine kinase (TK) gene of simplexvirus, CMV, PGK promotor, muscle creatine kinase (MCK) gene promoter, the different isotype genes of skeletal muscle Actin muscle.The particular instance of minimal promoter for example-54 to+48 Nucleotide of available CMV or-105 to+56 Nucleotide of TK promotor is represented.Should be appreciated that those skilled in the art can make up all variants that obtained these promotors or similar construct by other promotors, and can be used in the scope of the invention.
Minimal promoter (Pmin), composition (PPRE) and purpose nucleic acid (AN) that PPAR is reacted functionally are configured in the composition (a), that is to say that its activity is regulated by the PPRE composition so that make minimal promoter control purpose expression of nucleic acids.Usually, these districts are therefore according to following arranged in order, at 5 '-3 ' direction: PPRE-Pmin-AN.Yet those skilled in the art can not deviate from the functional configuration of considering any other under the condition of the present invention.
In addition, above-mentioned different functional zone can directly connect each other or the Nucleotide that the control characteristic of composition (a) promotor do not produced tangible disadvantageous effect separately.Some Nucleotide can be to be neutral residue in function aspects like this, for example the neutral residue that is obtained by clone's step (PCR end, restriction site etc.).These Nucleotide also may have biological property, can give characteristic or the performance (management gene amplification, specific tissue amplicon, silencer, intron, splice site etc.) of system of the present invention to improve.To this, in specific implementations of the present invention, evoked promoter also comprises amplification region.Purpose expression of nucleic acid level advantageously can improve in such district.According to following synoptic diagram (5 '-3 '): PPRE-Pmin-AN, such amplification region (E) preferably is positioned at 3 ' of this promotor of being between minimal promoter and the purpose nucleic acid.
On the other hand, in construct of the present invention, minimal promoter and to the composition of PPAR reaction direction (promptly with transcriptional orientation) equally, or (promptly compare, the composition of PPAR reaction be in the other direction) existence in the opposite direction with transcribing of being undertaken by the Pmin promotor.As what illustrate among the embodiment, these two kinds of embodiments can effectively control expression adjusting in vitro as in vivo.
As mentioned above, the composition of the present composition (b) comprises at least:
The nucleic acid of-coding PPAR,
-under the control of second transcripting promoter.
PPAR belongs to the superfamily of nuclear hormone receptor, and merger becomes three different groups, PPAR α, PPAR δ (being also called NCU-1 or PPAR β) and PPAR γ.Once described in the literature the separation of many people PPAR and sequence (specifically referring to Sher T. and colleague thereof, " biological chemistry ", 32 (1993) 5598-5604; Mukher jee R. and colleague thereof, " J.Steroid Biochem.Molec.Biol. ", 51 (1994) 157-166; Fajas L. and colleague thereof, " journal of biological chemistry ", 272 (1997) 18779-18789; Mukher jee R. and colleague thereof, " journal of biological chemistry ", 272 (1997) 8071-8076; Schmidt A. and colleague thereof, " Mol.Endocrinol. " 6 (1992) 1634-1641).WO99/05161 is described as patent application, also once clones PPAR γ promotor recently.
In optimal way of the present invention, the nucleic acid encoding of coding PPAR people's PPAR, PPAR α or PPAR γ specifically.The result who lists among these embodiment in fact shows, uses these molecules can guarantee the system of the present invention gentle high expression level of economizing on water to a high-profile, particularly in muscle cell.
According to first embodiment, relate to the PPAR α or the PPAR γ of its natural form, promptly compare with natural molecule, do not modify primary formation.
According to another kind of embodiment, relate to the PPAR of modification, it comprises a plurality of part chains site.
To this, the present invention describes and relates to simultaneously the PPAR of all any modifications, and it comprises a plurality of part chains site.More particularly, relate to PPAR α or PPAR γ, also more preferably relate to PPAR γ.Preferably, the PPAR of the present invention's modification comprises 2-5 part chain site, more preferably 2-4 chain site.Relate more particularly to PPAR, it is included in 2-5 the replica in part chain E and F district.PPAR albumen comprises different districts: comprise the A/B N-petiolarea in the trans-activation district of non-dependence part, and as the C district of DNA (DBD) sequence, as the D district in knuckle joint district, and the E/F district, it comprises the trans-activation district that relies on part.The E/F district also be referred to as part sequence (LBD) (specifically referring to Schoon jans K. and colleague thereof, " Acta Biochimica et Biophysica Sinica " (Biochim.Biophys.Acta), 1302 (1996) 93-109).The limit in E/F district changes to another PPAR from a PPAR.As an example, for the isotype of the end user's of institute PPAR γ 2, the E/F district expands to 505 amino acids from 284 amino acids.The present invention shows now might make up the modification PPAR that comprises a plurality of repetition E and F district, and the PPAR of these modifications is functional, has the inducibility by the improvement of PPAR part.Therefore some constructs represent a kind of embodiment and specific purpose of the present invention like this.
The typical PPAR example of modifying according to the present invention is the PPAR γ that comprises 2 part chain sites (promptly two E and F district).List PPAR γ 2 γ 2 protein sequences among the SEQ ID NO:24.
SEQ?ID?NO：24
MGETLGDSPIDPESDSFTDTLSANISQEMTMVDTEMPFWPTNFGISSVDLSVMEDHSHSFDI
KPFTTVDFSSISTPHYEDIPFTRTDPVVADYKYDLKLQEYQSAIKVEPASPPYYSEKTQLYN
KPHEEPSNSLMAIECRVCGDKASGFHYGVHACEGCKGFFRRTIRLKLIYDRCDLNCRIHKKS
RNKCQYCRFQKCLAVGMSHNAIRFGRMPQAEKEKLLAEISSDIDQLNPESADLRALAKHLYD
SYIKSFPLTKAKARAILTGKTTDKSPFVIYDMNSLMMGEDKIKFKHITPLQEQSKEVAIRIF
QGCQFRSVEAVQEITEYAKSIPGFVNLDLNDQVTLLKYGVHEIIYTMLASLMNKDGVLISEG
QGFMTREFLKSLRKPFGDFMEPKFEFAVKFNALELDDSDLAIFIAVIILSGDRPGLLNVKPI
EDIQDNLLQALELQLKLNHPESSQLFAKLLQKMTDLRQIVTEHVQLLQVIKKTETDMSLHPL
LQEIYKDLYAWAILTGKTTDKSPFVIYDMNSLMMGEDKIKFKHITPLQEQSKEVAIRIFQGC
QFRSVEAVQEITEYAKSIPGFVNLDLNDQVTLLKYGVHEIIYTMLASLMNKDGVLISEGQGF
MTREFLKSLRKPFGDFMEPKFEFAVKFNALELDDSDLAIFIAVIILSGDRPGLLNVKPIEDI
QDNLLQALELQLKLNHPESSQLFAKLLQKMTDLRQIVTEHVQLLQVIKKTETDMSLHPLLQE
IYKDLY
The invention still further relates to all variants (activation capability in the presence of the PPAR part as BRL49653, comprises the promotor of PPRE sequence) that keep the active SEQ ID of PPAR pattern NO:24 sequence.These variants can be understood that all mutant, deletant and/or comprise the polypeptide of one or more complementary residues.Preferably, a kind of variant keeps at least 80% ID NO:24 sequence residue.
In addition, the invention still further relates to all nucleic acid of a kind of like this PPAR of modification of coding.Can relate to DNA (particularly cDNA or synthetic or semisynthetic DNA) or RNA.This DNA can make up (synthetic, as to connect storehouse screening etc.) according to molecular biology ordinary method well known by persons skilled in the art.Advantageously relate to the sequence that comprises coding SEQ ID NO:24 sequences polypeptide, or with the sequence hydridization of coding SEQ ID NO:24 polypeptide, and coding has all nucleic acid of PPAR pattern active polypeptide.In addition, this DNA can comprise for example promotor and/or transcription terminator.
Second transcripting promoter of the expression of nucleic acid of control coding PPAR can be in mammalian cell, particularly any strong or weak, the ubiquity in people's cell or optionally, functional promotor that constitute or that regulate.Can relate to domestic animal cell promotor (being the gene promoter of mammalian genes promotor, particularly people), natural or synthetic, simple or complicated virus, bacterium, insect, plant promoter etc.The promotor example that is applicable to this composition (b) is viral promotors (the direct promotor of SV40 virus particularly, CMV virus is promotor directly, the long terminal repeat of retrovirus, simplexvirus TK promotor) or promotor (the PGK promotor of cell, albumin, EF1 α, or in muscle the gene of strongly expressed, as: the creatine kinase of flesh (MCK) gene promoter, the promotor of the different isotype genes of skeletal muscle Actin muscle, the promotor of desmin gene, vimentin dna promotor, the light chain of myosin or the promotor of heavy chain gene).In addition, promotor can be modified by adding one or more enhancing subareas, enhancing subarea as 2 introns of beta globin gene, the enhanser of the enhanser EF1 α of the very early gene of CMV virus, one or more silencers, give and organize wholesomeness (for example to organize the isolating district of promotor by special efficacy, as: the creatine kinase of flesh (MCK) gene promoter, the promotor of the different isomerization reformation body gene of skeletal muscle Actin muscle, the promotor of desmin gene, vimentin dna promotor, the light chain of myosin or the promotor of heavy chain gene) or the district of adjustable characteristic, or for example modify by the disappearance with active nonessential region.Some promotors can be used for being expressed in the RXR that comprises in the composition (d) like this.
The preferred embodiment of second promotor is a viral promotors, particularly SV40 virus early promoter and direct promotor of CMV or their derivative.
In addition, in specific embodiment, when composition (a) and (b) and optional (d) when being integrated in the same genetic constructs, the evoked promoter of (composition (b)) second transcripting promoter and composition (a), and the promotor of optional ingredients (d) can be gathered so that only generate the common promoter region, particularly two-way common promoter region will be as will explaining in detail hereinafter.
To this, another object of the present invention is to comprise as previously defined composition (a) and (b) and the carrier of optional (d).
According to the first embodiment of the present invention, composition (a) and (b), and optional (d) gets identical direction in carrier.For example with SV-g2-J10-C-pGL3 plasmid explanation a kind of like this embodiment (Figure 17).
According to another embodiment of the invention, composition (a) and (b), and optional (d) opposed orientation in carrier.For example with a kind of like this embodiment of plasmid explanation of representing among Figure 16,18 and 19.More preferably, in this embodiment, the transcripting promoter of the evoked promoter of composition (a) and composition (b) is integrated in the carrier so that form adjustable bidirectional promoter.For example with a kind of like this embodiment of plasmid explanation of representing in Figure 18 and 19.
To this, specific purpose of the present invention is a kind of carrier, it is characterized in that it comprises the nucleic acid of first coding PPAR in 5 '-3 ' direction, first minimum transcripting promoter of described first expression of nucleic acid of control, one or more compositions to PPAR reaction, second minimum transcripting promoter and be in described second minimum transcripting promoter control second nucleic acid of coding purpose product down.
This structure pattern is favourable, because its allows in same plasmid altogether-express two nucleic acid and regulates this expression of increasing of two nucleic acid by PPAR and part thereof.
Usually, in the presence of PPAR part (composition (c)), the purpose expression of nucleic acids is activated in the present composition.To this, the PPAR according to using can use dissimilar parts, natural or synthetic part.
Therefore, the sub-part of the activation of PPAR α for example is fiber hydrochlorate (fibrate), for example Carboxymethylcellulose and analogue thereof.As the Carboxymethylcellulose analogue, can enumerate gemfibrozil (" atherosclerosis ", 114 (1) (1995) 61) especially, bezafibrate (" hepatology ", 21 (1995) 1025), Win-35833 (" BCE﹠amp; M ", 9 (4) (1995) 825), clofibrate (" safety of medicine " (Drug Safety), 11 (1994) 301), fenofibrate (fenofibrate monograph, the clinical communication in Oxford University, 1995), S-8527 (KidneyInternational, 44 (6) (1993) 1352), pirinixique acid (Wy-14643) or 5,8,11,14-eicosane tetracid (ETYA).These different compounds in vitro or in vivo use with biology and/or the pharmacology use is compatible.
The sub-part of the activation of PPAR γ can be selected from natural or synthetic ligands.As native ligand, can mention lipid acid and eicosanoides (linolic acid for example, linolenic acid, 9-HODE, 5-HODE), as synthetic ligands, can enumerate thiazolidinedione, concrete as rosiglitazone (BRL49653), pioglitazone or troglitazone (for example referring to Krey G. and colleague thereof, " Mol.Endocrinol. " 11 (1997) 779-791 or Kliewer S. and Willson T. " (Curr.Opin.IN Gen.Dev. " 8 (1998) 576-581) or compound R G12525.
In addition, composition of the present invention can comprise activation of the PPAR of several cooperations, fiber hydrochlorate that particularly cooperates with retinene derivative or Carboxymethylcellulose salt analogue.
A further object of the invention is to use as previously defined composition or carrier, is expressed in vitro or the purpose nucleic acid in the cell in vitro.
To this, nucleic acid can be the coding purpose product (RNA, albumen, polypeptide, peptide etc.) any nucleic acid (DNA, RNA).The purpose product that can relate to agricultural food product, treatment, vaccine, marker etc.
The invention still further relates to above-mentioned composition or carrier and be used for being expressed in application aspect the product of cell purpose nucleic acid in vivo in preparation.
The invention still further relates to the method for regulating the express cell amplifying nucleic acid, this method comprises allows described cell contact with previously defined composition or carrier.
For at test tube or in vitro use, according to different schemes, cell can contact with composition of the present invention or carrier.Therefore, cells in culture can be with composition (a) and (b) of the present invention and (c) and optional (d), for example with comprise composition (a) and carrier (b) and direct cultivation in the presence of part (c).As available alternate scheme, cell at first can be with composition (a) and (b), and optional (d) cultivate (being integrated into particularly in the same carrier) together, then, secondly (after cultivating and randomly selecting to modify cell) can add composition (c).This a kind of scheme in back for example can be separated incubation period (or cell expansion phase) with the expression of nucleic acid phase.These tests can be at any equipment and are suitably carried out in the medium, preferably carry out under sterilising conditions in plate, box, bottle.Those skilled in the art are easy to determine the amount of cell, carrier and part according to information that provides among the embodiment and general knowledge thereof.
For in vivo using, cell (or organ, tissue etc.) is with simultaneously, separately or in certain hour mode at interval add composition (a) and (b) and (c), and optional (d) contacts.For this reason, usually by parenteral, adopt in intramuscular, intravenously, subcutaneous, intracutaneous, the tumour especially or three-dimensional location approach randomly adds composition (a) and (b) and (d) that choose wantonly with unique genetic constructs form.Can be by the application of being considered, target tissue and/or by the selection of the purpose product types direction of medication usage mode of transgenes encoding.For this medication, the present composition can comprise any reagent (cationic polymers, lipid etc.) that helps cell transfecting.In ad hoc fashion, adopt intramuscular method administration said composition, with " null dew " nucleic acid form, promptly in the presence of the transfection agents that does not have interpolation, use genetic constructs.
Similarly, adopting virus vector to add composition (a) and (b) and when (d) that choose wantonly, need not any additional transfection agents.
As described in the embodiment, part (c) can composition (a) and (b) and optional (d) before, add simultaneously or afterwards with it.
To this, for example can adopt oral, anus, intravenously, intraperitoneal or intramuscular administration part.
Those skilled in the art can adjust the dosage of use according to disclosed data in vivo in the document.Therefore, for example for insoluble form in water, be the 5-50 mg/kg as BRL49653 part typical doses, 30 mg/kg for example, such dosage can reach plasmid concentration at least near about 15 mcg/ml.For the water-soluble form (for example BRL49653 maleate) of the bigger part of its bioavailability, typical doses is lower, generally is lower than 5 mg/kg, for example the 0.01-1 mg/kg.Those skilled in the art obviously can adjust these dosage according to the construct that uses, part and the application of looking for and the effect of use.Usually, the result who lists among the embodiment shows that advantageously the present composition is lower than normally used dosage with part dosage can reach strong and adjustable expression in vivo.In addition, although can the repetitively administered part, the result who lists also shows, only expresses just very strong behind the single administration part.
Usually, according to the application of looking for, the carrier dosage of use can be 0.01-1000 microgram or more.
The present invention can be used at test tube, expresses the gene in dissimilar cells, tissue or the organ in vitro or in vivo.Especially, can relate to Mammals, preferably people's cell, tissue or organ.Myocyte's (or muscle) can be enumerated in illustrative ground, liver cell (or liver), heart cell (or heart, arterial wall or vessel wall), neurocyte (or brain, marrow etc.) or tumour cell (or tumour).
Preferably, construct of the present invention, composition and method are used for regulating the nucleic acid of expressing among myocyte's (or muscle) in vitro at test tube, or in vivo.The advantage that the result who lists among the embodiment has more particularly illustrated in vivo or the present invention presented in this class cell in test tube.
The invention still further relates to by contacting any cell of being modified with above-mentioned composition or carrier.
The invention still further relates to the application of above-mentioned composition, carrier or cell, wherein purpose nucleic acid is to be used at test tube, in vitro or in vivo the reporter gene (for example excretory luciferase or alkaline phosphatase) of (especially in myocyte or muscle) screening PPAR part.To this, the present invention has also described PPAR part authentication method, and this method comprises makes above-mentioned cell contact and prove purpose expression of nucleic acids (it is reporter gene preferably) with test molecule (or composition).For the activity of evaluation test compound, this expression observed expression when not having test compound or the reference part is arranged can also be compared.
The invention still further relates to as previously defined composition or carrier and making up transgenic animal, the particularly application of non-human mammal aspect, these transgenic animal are effectively for research in latent period or researchs such as bioavailability, mark.
Describe the present invention in more detail by following embodiment, these embodiment are considered to illustrative, rather than restrictive.
Description of drawings
Fig. 1: graphic interpretation explanation FTKpGL3 plasmid.
Fig. 2: graphic interpretation explanation Jx3S-TK-pGL3 plasmid.
Fig. 3: graphic interpretation explanation Jx3AS-TK-pGL3 plasmid.
Fig. 4: graphic interpretation explanation DR1x3S-TK-pGL3 plasmid.
Fig. 5: graphic interpretation explanation DR1x3AS-TK-pGL3 plasmid.
Fig. 6: the activity of the evoked promoter that the sarcoplast of mouse (C2C12) is estimated during transient transfection in test tube.1. 10 nanogram FTKpGL3 plasmids (a) of these cells, or Jx3S-TK-pGL3 plasmid (b), or Jx3AS-TK-pGL3 plasmid (c), or DR1x3S-TK-pGL3 plasmid (d), or DR1x3AS-TK-pGL3 plasmid (e); 2. the pSG5-hPPARg2 plasmid of increasing amount, and 3. 20 nanogram pRL-null plasmids carry out cotransfection.The activity of each evoked promoter is represented the uciferase activity by the standardized Photinus pyralis of the uciferase activity of Renilla reniformis.
Fig. 7: the activity of the evoked promoter that the sarcoplast of mouse (C2C12) is estimated during transient transfection in test tube.1. 10 nanogram FTKpGL3 plasmids (a) of these cells, or Jx3S-TK-pGL3 plasmid (b), or Jx3AS-TK-pGL3 plasmid (c), or DR1x3S-TK-pGL3 plasmid (d), or DR1x3AS-TK-pGL3 plasmid (e); 2. the pSG5-hPPARa of increasing amount (Koz) plasmid, and 3. 20 nanogram pRL-null plasmids carry out cotransfection.The activity of each evoked promoter is represented the uciferase activity by the standardized Photinus pyralis of the uciferase activity of Renilla reniformis.
Fig. 8: graphic interpretation explanation Jx5AS-CMV-pGL3 plasmid.
Fig. 9: the activity of the evoked promoter that the sarcoplast of mouse (C2C12) is estimated during transient transfection in test tube.1. 10 nanogram Jx5AS-TK-pGL3 plasmids (a) of these cells, or Jx5AS-CMV-pGL3 plasmid (b); 2. the pSG5-hPPARg2 plasmid of increasing amount, and 3. 20 nanogram pRL-null plasmids carry out cotransfection.The activity of each evoked promoter is represented the uciferase activity by the standardized Photinus pyralis of the uciferase activity of Renilla reniformis.
Figure 10: the activity of the evoked promoter that the sarcoplast of mouse (C2C12) is estimated during transient transfection in test tube.1. 10 nanogram JxnAS-TK-pGL3 plasmids of these cells; 2. 10 nanogram pSG5-hPPARg2 plasmids, and 3. 20 nanogram pRL-null plasmids carry out cotransfection.The activity of each evoked promoter is represented the uciferase activity by the standardized Photinus pyralis of the uciferase activity of Renilla reniformis.
Figure 11: the activity of the evoked promoter that the sarcoplast of mouse (C2C12) is estimated during transient transfection in test tube.1. 10 nanogram JxnAS-CMV-pGL3 plasmids of these cells; 2. 10 nanogram pSG5-hPPARg2 plasmids (a) or 50 nanogram pSG5-hPPARg2 plasmids (b), and 3. 20 nanogram pRL-null plasmids carry out cotransfection.The activity of each evoked promoter is represented the uciferase activity by the standardized Photinus pyralis of the uciferase activity of Renilla reniformis.
Figure 12: graphic interpretation explanation pSG5-hPPARg2g2 plasmid.
The comparison of Figure 13: hPPARg2 and hPPARg2g2 transcriptional regulatory gene.The sarcoplast of mouse (C2C12) is used 1. 10 nanogram Jx10AS-CMV-pGL3 plasmids; 2. the pSG5-hPPARg2 plasmid (a) of increasing amount or pSG5-hPPARg2g2 plasmid (b), and 3. 20 nanogram pRL-null plasmids carry out cotransfection.The activity of evoked promoter has been represented the uciferase activity with the standardized Photinuspyralis of uciferase activity of Renilla reniformis: the inducible factor of the BRL49653 that obtains with pSG5-hPPARg2 plasmid or pSG5-hPPARg2g2 plasmid.Divided by the activity in the presence of BRL49653, can calculate this inducible factor by the activity under existing with DMSO.
Figure 14: graphic interpretation explanation Jx10AS-CMV-EF-pGL3 plasmid.
Figure 15: the activity of the evoked promoter that the sarcoplast of mouse (C2C12) is estimated during transient transfection in test tube.1. 10 nanogram Jx10AS-CMV-pGL3 plasmids (a) of these cells, or Jx10AS-CMV-EF-pGL3 plasmid (b); 2. the pSG5-hPPARg2g2 plasmid of increasing amount, and 3. 20 nanogram pRL-null plasmids carry out cotransfection.The activity of each evoked promoter is represented the uciferase activity by the standardized Photinus pyralis of the uciferase activity of Renilla reniformis.
Figure 16: graphic interpretation explanation Jx5AS-TK-luc-hPPARg2 plasmid.
Figure 17: graphic interpretation explanation SV-g2-J10-C-pGL3 plasmid.
Figure 18: graphic interpretation explanation hPPARg2-CMV-Jx5AS-TK-pGL3 plasmid.
Figure 19: graphic interpretation explanation hPPARg2-CMV-Jx10AS-CMV-pGL3 plasmid.
Figure 20: the comparison of different inducible system versions in test tube.Each system version uses the sarcoplast (C2C12) of the abduction delivering box transfection mouse of identical mole number.To represent these results by the hCMV-IE promoter activity percentage ratio that uses the leading TK plasmid of pCMV-to obtain.By the activity of the activity when having DMSO to exist when having BRL49653 to exist, can calculate the BRL49653 inducible factor.1＝pSG5-hPPARg2+Jx5AS-TK-pGL3；2＝Jx5AS-TK-luc-hPPARg2；3＝pSG5-hPPARg2g2+Jx10AS-CMV-pGL3；4＝pSG5-hPPARg2+Jx10AS-CMV-pGL3；5＝SV-g2-J10-C-pGL3；6＝hPPARg2-CMV-Jx5AS-TK-pGL3；7＝hPPARg2-CMV-Jx10AS-CMV-pGL3；8＝hPPARg2-CMV-Jx15AS-CMV-pGL3；9＝hPPARg2-CMV-Jx20AS-CMV-pGL3。
Figure 21: BRL49653 and the comparison of RG12525 part in test tube.The sarcoplast of mouse (C2C12) is present in the hPPARg2-CMV-Jx10AS-CMV-pGL3 plasmid of (a) with 1. its expression cassette of 10 nanograms; 2. 10 nanogram pRL-null plasmids (b) carry out transfection.The activity of evoked promoter is represented the uciferase activity by the standardized Photinus pyralis of the uciferase activity of Renillareniformis.
Figure 22: the comparison of different inducible system versions in live body.Abduction delivering box at C57Bl/6 mouse (every group of 6 mouse) cranium side shin bone two side injections identical mole number for each system version.On every muscle, apply electrotransfer.The animal of handling adopts gavage to accept 30 mg/kg BRL49653 every day.Behind injection DNA four days, after animal via is slaughtered, take off muscle and measure uciferase activity.The leading TK of 1=pCMV-; 2=pSG5-hPPARg2+Jx10AS-CMV-pGL3; 3=pSG5-hPPARg2g2+Jx10AS-CMV-pGL3; 4=hPPARg2-CMV-Jx10AS-CMV-pGL3.
Figure 23: the comparison of different B RL49653 induction scheme in vivo.Comprise the DNA that its expression cassette of 1 microgram is present in the hPPARg2-CMV-Jx10AS-CMV-pGL3 plasmid in (a) in C57Bl/6 mouse (every group of 6 mouse) cranium side tibiale two side injections 10 micrograms.The activity that obtains with different induction schemes collects in the part (b).
Figure 24: graphic interpretation explanation pRDA02 plasmid.
Figure 25: induce kinetics with what inducible system obtained in vivo.(A) comprise the DNA mixture of 3 milligrams of pRDA02 plasmids and 3 milligrams of pSG5-hPPARg2 plasmids at ten C57Bl/6 mouse cranium side tibiale two side injections.At this moment on every muscle, apply electrotransfer.Behind injection DNA the 4th day, the 39th day then, adopt gavage with 30 mg/kg BRL49653 processing animal.Extract heparinemia at different time, use Phospha-Light
TM(Foster City CA), measures the enzymic activity of excretory alkaline phosphatase in the plasmid to box for Tropix, PE Biosystem.(B) comprise the DNA mixture of 3 milligrams of pRDA02 plasmids and 3 milligrams of pSG5-hPPARg2 plasmids at C57Bl/6 mouse (two groups each ten) cranium side tibiale two side injections.At this moment on every muscle, apply electrotransfer.Behind injection DNA four days, adopt gavage, animal or acceptable dose 30 mg/kg BRL49653 only, perhaps accept dose (30 mg/kg) every day in 5 days.At different time taking heparin blood, use Phospha-Light
TMBox (Tropix), the enzymic activity of hSeAP in the measurement blood plasma.The result who lists (inducible factor) is corresponding to hSeAP activity of measuring in meaningful that day and the active ratio that obtained in the 4th day.
Figure 26: more different in vivo PPARg parts and research is a kind of dose effect of part wherein.The DNA mixture that comprises 5 milligrams of pRDA02 plasmids and 5 milligrams of pSG5-hPPARg2 plasmids at C57Bl/6 mouse (five every group) cranium side shin bone two side injections.At this moment on every muscle, apply electrotransfer.Behind injection DNA six days (A) or ten days (B), adopt gavage, animal or with different PPARg part (A; BRL49653, Actos
TM(Takeda Phamaceuticals) and Avandia
TM(SmithKline Beecham)), perhaps use various dose BRL49653 (B) to handle.Extract heparinemia at different time, use Phospha-Light
TMBox (Tropix), the enzymic activity of hSeAP in the measurement blood plasma.The result who lists (inducible factor) is corresponding to the active active ratio that obtains with the 6th day (A) or the tenth day (B) of the hSeAP that measures in meaningful that day.
Figure 27: graphic interpretation explanation Jx10AS-CMV-VEGF
A165 plasmids.
Materials and methods
Normally used method in the molecular biology, for example plasmid DNA prepares extraction method, the cesium chloride gradient centrifugation of plasmid DNA, agarose gel electrophoresis method, dna fragmentation electroelution method of purification, the plasmid DNA precipitator method that in salt medium, adopt ethanol or Virahol to finish, conversion method in intestinal bacteria, all be that those skilled in the art know content and made to describe in a large number (Sambrook and colleague thereof, " molecular cloning, laboratory manual " (Molecular Cloning in the literature, a Laboratory Manual), Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.1989).
Be used to clone the pGL3-Basic plasmid in different promoters district and the plasmid (Promega Corportion) that the pRL-null plasmid all is commercial source, pSG5 (Stratagene), pBluescript IISK+ (Stratagene) and pSL301 (Invitrogen Corportion) plasmid also is the plasmid of commercial source.Structure pSG5-hPPARg2 expression plasmid (Fajas and colleague thereof were described in the front, " journal of biological chemistry " 272,18779-18789) and pSG5-hPPARa (Koz) (Gervois P. and colleague thereof, " Mol.Endocrinol. " 13 (1999) 400-409) (1997).
The leading TK plasmid of structure pCMV-was also described among french patent application FR98/120000 25/09/98 and the U.S. Patent application US SN 60/123298 (provisional application).
It should be noted that this plasmid makes up in the following manner.The pCGN expression vector (" cell " that Tanaka and colleague thereof described, 60 (1990) 375-386) comprise the CMV promotor (522/+72), in the sequence upstream of coding hemagglutinin antigenic determinant, (+51/+101) " caulody " fusion of the tk gene of this promotor and HSV.PGCN plasmid (10 nanogram) is used as the matrix of ACP amplification.Once the primer of Shi Yonging was as follows:
-6718 primers (5 ' CCCGTTACATAACTTACGGTAAATGGCCCG3 ') (SEQ ID NO:26), this primer and-522 CMV promotor (at 8 Nucleotide in the downstream, EcoRI site of pGCN) hydridization.
-6719 primers (5 ' GGGACGCGCTTCACAAGGCGCTGGCCGAA3 ') (SEQ ID NO:27), this primer generation hydridization is till 101 of tk " leader ".Explain the Ncol site that the first Nucleotide G of employing boldface letter mark is used to repair pGL3-Basic as following.
After the ACP fragment that so obtains was purified, the polynucleotide kinase with the T4 phage carried out phosphorylation (New Englands Biolabs) again.Simultaneously, pGL3-Basic carrier (Promega) is used the Ncol linearizing, and purifying uses Klenow archaeal dna polymerase (Boehringer Manheim) to handle, so that fill and lead up the Ncol site then.This carrier uses alkaline phosphatase (Boehringer Manheim) to carry out dephosphorylation then, is used further to insert phosphorylation ACP fragment.So, have only 5 ' (6718 primers partly when the downstream, HindIII site that be used in pGL3-Basic,-522 of CMV) make CMV-leader tk fragment orientation, and its 3 ' end (6719 primers, leader tk) with the Ncol site (first ATG of luciferase) of pGL3-Basic when being connected, the guanosine of 6719 primers (G) can be repaired the Ncol site.The leading TK of plasmid called after pCMV-that obtains.
According to the producer's recommendation, by using the DNA thermo cycler
TM(Perkin Elmer Cetus) can carry out the dna fragmentation enzymatic amplification by ACP (by the polymeric enzymatic amplification chain) technology.
According to the producer's recommendation, use electroporation apparatus in Bacillus coli cells, to carry out the electroporation of plasmid DNA.
Adopt the method (" Proc.Natl.Acad. Sci.U.S.A. " 74, (1977) 5463-5467) of Sanger and colleague's development thereof, recommend, the test kit that provides by the applying biological system is provided, confirm nucleotide sequence according to the producer.
At the DMEM that replenishes 10% foetal calf serum (SVF)
TMCultivate the sarcoplast of C2C12 mouse in the medium (Life Technologies Inc.).In 37 ℃ of baking ovens, at humid atmosphere and 5%CO
2Divide to depress and cultivate.
Carry out transfection in 24 orifice plates, each transfection is carried out three times.Twenty four hours before transfection, each hole is seeded in DMEM
TMIn the medium 3 * 10
4Individual cell.For each hole, 500 nanogram plasmid DNA (purpose plasmid and pBluescript II SK+, to add to 500 nanograms) and RPR120535 B positively charged ion lipid (WO97/18185), according to every micrograms of DNA 6 nmole lipids, at the DMEM that comprises 150 mmole NaCl and 50 mmole supercarbonates
TMMix in the medium (last 20 microlitres).After at room temperature 20 minutes, there be not under the situation of SVF 20 microlitre DNA/ lipid mixtures and these cells contacting 2 hours.At this moment developing medium adds SVF and ULTROSER
TM(BioSepra Inc.) is so that final concn reaches 10% or 2% respectively.With SVF or ULTROSER
TMSimultaneously, the PPAR part that will be dissolved among the DMSO adds in the developing medium.After transfection 48 hours, take out developing medium, clean cell twice with PBS (Life Technologies Inc.).At this moment according to supplier's recommendation, use Dual-Luciferase Reporter AssaySystem
TM(Promega), measure the uciferase activity of Renilla pyralis and the uciferase activity of Renillarenifomis.
Carry out the transgenosis test in vivo with the female mouse of 6 week C57BI/6 in age.These animals are anaesthetized by the intraperitoneal mode with the mixture of 250 microlitre ketamines (Rh ne M é rieux is 10 mg/ml at last)/xylazine (Bayer Pharma is 0.3 mg/ml at last).At this moment, the injection total amount is 10 micrograms of DNA in each cranium side tibialis.Then, each pawl is applied in electric field (frequency 1Hz; 250 volts of/centimetre following 4 pulses, each pulse is 20 milliseconds).In the whole test phase, adopt gavage in every morning, allow the solution of these animals received 30 mg/kg BRL49653 (SmithKline Beecham) in 1% (weight/volume) carboxy cellulose, perhaps just accept 1% carboxy cellulose.Behind metastatic gene four days, slaughter these animals, at Lysing Matrix
TM(BIO 101, PLB Inc.) for pipe
TMTake off muscle in the cytolysis damping fluid (Promega Corporation).Use FastPrep
TM(BIO 101, Inc.) grind muscle 25 seconds with 6.5 meter per seconds, can extract luciferase like this.Recommend according to supplier, at this moment use Luciferase Assay System
TMBox (Promega Corporation) is measured the uciferase activity of Photinuspyralis.
Embodiment
Embodiment 1: make up evoked promoter and make up the expression plasmid that comprises these promotors with PPAR
1.1 FTKpGL3 plasmid
Use pBLCAT2 plasmid (Luckow B. and Schutz G., " nucleic acids research " (Nucleic AcidsRes.), 15 (1987) 5490) as matrix and utilize 5 ' CGA CTC TAG AAG ATC TTG CCC CGCCCA GCG 3 ' (SEQ ID NO:28) and 5 ' TCG CCA AGC TTC TCG TGA TCT GCG GCA 3 ' (SEQID NO:2) oligonucleotide as primer by ACP amplification dna fragmentation corresponding to a part of herpes simplex types 1 virus TK gene promoter, compare with transcription initiation site, this fragment bit-105 is between+56.This fragment is with BaIII and HindIII digestion, then cloning in the pGL3-Basic plasmid with BaIII and HindIII digestion in advance, so that obtain the FTKpGL3 plasmid.The schematic view illustrating of this plasmid is in Fig. 1.
1.2 JxnS-TK-pGL3 plasmid
Use J3TK-pGL3 plasmid (Vu-Dac N. and colleague thereof, " J.Clin.Invest. " 96 (1995) 741-750) is as matrix and utilize 3RDA37 (5 ' ACG TGT CGA CAC TAG TGG CTAGAG GAT CTC TAC CAG G3 '; SEQ ID NO:3 ') and 4RDA48 (5 ' CGA TGG TAC CCT CGAGCA ATG TGC TAG CGA GAT CCT TCA ACC TTT ACC 3 '; SEQ ID NO:4) oligonucleotide makes the dna fragmentation amplification in the promotor J site that comprises one or more (n) people ApoA-II gene as primer by ACP.This fragment digests with Xhol and Spel, then in the FTKpGL3 plasmid that digests with Xhol and Nhel in advance, clone along TK (S) minimal promoter transcriptional orientation, so that obtain Jx1S-TK-pGL3, Jx2S-TK-pGL3 and Jx3S-TK-pGL3 plasmid according to the J number of sites that exists.The synoptic diagram of Jx3S-TK-pGL3 plasmid as shown in Figure 2.
That J3TKpGL3 plasmid and 3RDA37 and 4RDA48 oligonucleotide are increased by ACP as primer by using, as to use Xhol and Spel digestion dna fragmentation, also cloning in the Jx3S-TK-pGL3 plasmid with Xhol and Nhel digestion in advance, so that obtain Jx4S-TK-pGL3, Jx5S-TK-pGL3 and Jx6S-TK-pGL3 plasmid according to the J number of sites that exists.
1.3 JxnAS-TK-pGL3 plasmid
It is different aspect the J of A JxnAS-TK-pGL3 plasmid in being present in evoked promoter is site-directed with the JxnS-TK-pGL3 plasmid.By using the J3TKpGL3 plasmid as matrix, amplification comprises the dna fragmentation in the promotor J site of one or more people ApoA-II genes by ACP as primer for 3RDA37 and 4RDA48 oligonucleotide.This fragment digests with Sall and Nhel, then in the FTXpGL3 plasmid that digests in advance with Xhol and Nhel, transcribe reverse direction and clone along TK (AS) minimal promoter, so that obtain Jx1AS-TK-pGL3, Jx2AS-TK-pGL3 and Jx3AS-TK-pGL3 plasmid according to the J number of sites that exists.The synoptic diagram of Jx3AS-TK-pGL3 plasmid as shown in Figure 3.
That J3TKpGL3 plasmid and 3RDA37 and 4RDA48 oligonucleotide are increased by ACP as primer by using, as to use Kpnl and Spel digestion dna fragmentation, also in the Jx3AS-TK-pGL3 plasmid that digests with Kpnl and Nhel in advance, clone along opposite direction (AS), so that obtain Jx4AS-TK-pGL3 and Jx5AS-TK-pGL3 plasmid according to the J number of sites that exists.
1.4 DR1xnS-TK-pGL3 plasmid
These plasmids comprise be known as total DR1 consensus sequence (AGGTCA A AGGTCA, SEQ ID NO:5) as composition (PPRE) to the PPAR reaction.By using 1RDA69 (5 ' ACG TGT CGA CAC TAGTCA AAA CTA GGT CAA AGG TCA CGG AAA ACT AGG TCA AAG GTC ACG GAA AACTAG3 '; SEQ ID NO:6) and 2RDA64 (5 ' CGA TGG TAC CCT CGA GCA ATG TGC TAG CCGTGA CCT TTG ACC TAG TTT TCC GTG ACC TTT GAC C 3 '; SEQ ID NO:7) oligonucleotide increases by ACP as primer and comprises the dna fragmentation in one or more total DR1 site.This fragment in the FTKpGL3 plasmid that digests with Xhol and Nhel in advance, is cloned along differently-oriented directivity, so that obtain DR1x2S-TK-pGL3 and DR1x3S-TK-pGL3 plasmid according to the total DR1 number of sites that exists then with Xhol and Spel digestion.The synoptic diagram of DR1x3S-TK-pGL3 plasmid as shown in Figure 4.
When using 1RDA69 and 2RDA64 oligonucleotide as primer by the dna fragmentation with Xhol and Spel digestion of ACP amplification, also in the DR1x3S-TK-pGL3 plasmid that digests in advance with Xhol and Nhel, clone, so that obtain DR1x5S-TK-pGL3, DR1x6S-TK-pGL3 and DR1x7S-TK-pGL3 plasmid according to the total DR1 number of sites that exists.
1.5 DR1xnAS-TK-pGL3 plasmid
Different aspect DR1xnAS-TK-pGL3 plasmid and the total DR1 site of DR1xnS-TK-pGL3 plasmid in being present in evoked promoter directed.Comprise the dna fragmentation of one or more total DR1 sequences by using 1RDA69 and 2RDA64 oligonucleotide to increase by ACP as primer.This fragment is with Sall and Nhel digestion, then in the FTKpGL3 plasmid that digests with Xhol and Nhel in advance, along cloning in the other direction, so that obtain DR1x2AS-TK-pGL3 and DR1x3AS-TK-pGL3 plasmid according to the total DR1 number of sites that exists.The synoptic diagram of DR1x3AS-TK-pGL3 plasmid as shown in Figure 5.
By use 1RDA69 and 2RDA64 oligonucleotide as primer by the Kpnl of ACP amplification and the dna fragmentation of Spel digestion, also cloning in the DR1x3AS-TK-pGL3 plasmid with Kpnl and Nhel digestion in advance, so that obtain DR1x5AS-TK-pGL3 and DR1x6AS-TK-pGL3 plasmid according to the total DR1 number of sites that exists.
Embodiment 2:PPAR is to the wholesomeness of differential responses composition
2.1. use the system of hPPARg2
In the sarcoplast transient transfection of mouse (Fig. 6), once estimated the activity of evoked promoter when using hPPARg2 as the transcriptional regulatory gene.These results show, according to the reacted constituent (PPRE) that uses, use the inducing action activity last with activating the back of the part of hPPARg2 (BRL49653) all to change thereupon.Obtain optimum when using the J site as PPRE.In addition, the PPRE direction is also very important.Under the situation in J site, AS direction more favourable (part c).
2.2. use the system of hPPARa
Use hPPARa to come together in Fig. 7 as the resulting result of transcriptional regulatory gene.Opposite with hPPARg2, for hPPARg2, total DR1 is only best PPRE (part d and e).
Therefore, these results show functionality of (1) plasmid of the present invention and the PPAR that (2) basis is selected from inducible system, importantly select the only PPRE of transcriptional regulatory gene.Because part exists, this selection may influence inducible factor, but also the activity level that the back is reached is induced in influence.Self-evident, other PPRE also can be used for system of the present invention.
Embodiment 3: make up evoked promoter with PPAR, they comprise the minimal promoter except that the HSV1-TK promotor, for example as the hCMV-IE minimal promoter
3.1. make up the plasmid that comprises the hCMV-IE minimal promoter
By using pCMV β plasmid (Clontech) as matrix and utilize 5RDA32 (5 ' ACG TAGATC TCG GTA GGC GTG TAC GGT GGG AG 3 '; SEQ ID NO:8) and 6RDA29 (5 ' ACG TAAGCT TCT ATG GAG GTC AAA ACA GC 3 '; SEQ ID NO:9) oligonucleotide is as primer, adopts the ACP amplification to comprise the dna fragmentation of hCMV-IE minimal promoter (with respect to transcription initiation site from-54 to+48).This fragment is cloned in the FTKpGL3 plasmid that digests in advance with HindIII and BqIII, so that obtain the FCMVpGL3 plasmid then with HindIII and BqIII digestion.
With BgIII and Nhel digestion Jx5AS-TK-pGL3 plasmid, so that separate the 179pb BgIII-Nhel fragment that comprises 5 J site replica.This fragment is inserted into the FCMVpGL3 plasmid that digests in advance with BgIII and Nhel so that obtain the Jx5AS-CMV-pGL3 plasmid.Jx5AS-CMV-pGL3 plasmid synoptic diagram as shown in Figure 8.
With Sphl and Nhel digestion Jx5AS-CMV-pGL3 plasmid, isolate and comprise 5 J site replica, the 982pb Sphl-Nhel fragment of the gene 5 ' part of the plain enzyme of hCMV-IE minimal promoter and coding fluorescence.This fragment is inserted in the Jx5AS-CMV-pGL3 plasmid that digests in advance with Sphl and Spel so that obtain the Jx10AS-CMV-pGL3 plasmid.According to same conception, can obtain Jx15AS-CMV-pGL3 and Jx20AS-CMV-pGL3 plasmid
3.2. comprise the plasmid activity of hCMV-IE minimal promoter
When transient transfection, the minimal promoter that can be used in the inducible system was carried out relatively.The result who comes together among Fig. 9 shows that according to minimal promoter, the last activity after inducing can increase or reduce one times.These results show especially as if under test conditions, the CMV promotor provides higher activity.Certainly, other minimal promoter as not comprising the promotor of TATA box, also can use.
Embodiment 4: the importance that is reacted into mark that exists in evoked promoter
When transient transfection, the optimization that is present in the PPRE number in the evoked promoter is studied.The result who lists in Figure 10 shows that PPRE replica quantity is big more, and the inducible factor of part and induced activity are just high more.On the contrary, if this number is too big, inducible factor and induced activity decrease simultaneously, no matter how the hPPAPRg2 that exists in test amount is not always the case (Figure 11).Best PPRE number is 10-15 seemingly.
Embodiment 5: the part with PPAR makes up strong inductive transcriptional regulatory gene
5.1. make up the transcriptional regulatory gene that comprises two part sequence replica.Make up the pSG5-hPPARg2g2 plasmid
By using the pSG5-hPPARg2 plasmid as matrix and utilize 20RDA21 (5 ' GGT TTG CTGAAT GTG AAG CCC 3 '; SEQ ID NO:10) and 21RDA42 (5 ' AGT CTC TAG AGC TAC GCGTAC AAG TCC TTG TAG ATC TCC TGC3 '; SEQ ID NO:11) oligonucleotide adopts the ACP amplification to be marked as the dna fragmentation of A as primer, and this fragment comprises the complementary DNA district of the hPPARg2 of the C-end, F district of encoding.By using the pSG5-hPPARg2 plasmid as matrix and utilize 22RDA32 (5 ' AGTCAC GCG TGG GCG ATC TTG ACA GGA AAG AC 3 '; SEQ ID NO:12) and 23RDA21 (5 ' GCC TTT GAG TGA GCT GAT ACC 3 '; SEQ ID NO:13) oligonucleotide adopts the ACP amplification to be marked as the dna fragmentation of B as primer, and this fragment comprises the hPPARg2 complementary DNA district in coding E and F district., clone in the pSG5-hPPARg2 plasmid that digests in advance with Sacl and Xbal with B fragment with the A fragment of Sacl and Mlul digestion, obtain the pSG5-hPPARg2g2 plasmid with Mlul and Xbal digestion.Its synoptic diagram this plasmid as shown in Figure 8 comprises the complementary DNA of encoding transcription regulatory gene (being designated as hPPARg2g2), and this regulatory gene comprises two E and F district replica, i.e. two part sequences.
List the sufficient sequence (SEQ ID NO:24) of PPAR γ 2 γ 2 below:
MGETLGDSPIDPESDSFTDTLSANISQEMTMVDTEMPFWPTNFGISSVDLSVMEDHSHSFDI
KPFTTVDFSSISTPHYEDIPFTRTDPVVADYKYDLKLQEYQSAIKVEPASPPYYSEKTQLYN
KPHEEPSNSLMAIECRVCGDKASGFHYGVHACEGCKGFFRRTIRLKLIYDRCDLNCRIHKKS
RNKCQYCRFQKCLAVGMSHNAIRFGRMPQAEKEKLLAEISSDIDQLNPESADLRALAKHLYD
SYIKSFPLTKAKARAILTGKTTDKSPFVIYDMNSLMMGEDKIKFKHITPLQEQSKEVAIRIF
QGCQFRSVEAVQEITEYAKSIPGFVNLDLNDQVTLLKYGVHEIIYTMLASLMNKDGVLISEG
QGFMTREFLKSLRKPFGDFMEPKFEFAVKFNALELDDSDLAIFIAVIILSGDRPGLLNVKPI
EDIQDNLLQALELQLKLNHPESSQLFAKLLQKMTDLRQIVTEHVQLLQVIKKTETDMSLHPL
LQEIYKDLYAWAILTGKTTDKSPFVIYDMNSLMMGEDKIKFKHITPLQEQSKEVAIRIFQGC
QFRSVEAVQEITEYAKSIPGFVNLDLNDQVTLLKYGVHEIIYTMLASLMNKDGVLISEGQGF
MTREFLKSLRKPFGDFMEPKFEFAVKFNALELDDSDLAIFIAVIILSGDRPGLLNVKPIEDI
QDNLLQALELQLKLNHPESSQLFAKLLQKMTDLRQIVTEHVQLLQVIKKTETDMSLHPLLQE
IYKDLY
The sequence of C-end parts that comprises PPAR γ 2 γ 2 in E and F district is following SEQ ID NO:25 sequence:
MMGEDKIKFKHITPLQEQSKEVAIRIFQGCQFRSVEAVQEITEYAKSIPGFVNLDLNDQVTL
LKYGVHEIIYTMLASLMNKDGVLISEGQGFMTREFLKSLRKPFGDFMEPKFEFAVKFNALEL
DDSDLAIFIAVIILSGDRPGLLNVKPIEDIQDNLLQALELQLKLNHPESSQLFAKLLQKMTD
LRQIVTEHVQLLQVIKKTETDMSLHPLLQEIYKDLYAWAILTGKTTDKSPFVIYDMNSLMMG
EDKIKFKHITPLQEQSKEVAIRIFQGCQFRSVEAVQEITEYAKSIPGFVNLDLNDQVTLLKY
GVHEIIYTMLASLMNKDGVLISEGQGFMTREFLKSLRKPFGDFMEPKFEFAVKFNALELDDS
DLAIFIAVIILSGDRPGLLNVKPIEDIQDNLLQALELQLKLNHPESSQLFAKLLQKMTDLRQ
IVTEHVQLLQVIKKTETDMSLHPLLQEIYKDLY
5.2.pSG5-hPPARg2g2 the activity of plasmid
The result who lists among Figure 13 shows, if when using hPPARg2g2 as the transcriptional regulatory gene, and induced activity lower (Figure 13 a and b), when then using this regulatory gene, (Figure 13 c) is higher significantly for the inducible factor of part.Difference between two kinds of transcriptional regulatory genes can be used following facts explain: for hPPARg2g2, the ground noise of system is not low when having part to exist, although there is a certain amount of regulatory gene, but still keeps low-level.On the other hand, the hPPARg2g2 amount increases big more, and induced activity is also strong more, uses and it seems that the system of saturated hPPARg2 is not this situation just.
Therefore, the existence of second part sequence (hPPARg2g2) is given the transcriptional regulatory gene with bigger inducibility by part.
Embodiment 6: the last activity that improves evoked promoter
6.1. make up the abduction delivering box that comprises the hEF1a intron.Make up the Jx10AS-CMV-EF-pGL3 plasmid
By using 25RDA35 (5 ' AGT CAC TAG TAA GCT TTT TGC CGC CAG AAC ACA GG3 '; SEQ ID NO:14) and 26RDA36 (5 ' AGT CAC TAG TCC ATG GCT GCC CAG TGC CTCACG ACC 3 '; SEQ ID NO:15) oligonucleotide comprises the dna fragmentation of first intron of gene of the hEF1a that encodes (with respect to transcription initiation site from+16 to+984 by ACP amplification as primer; Enter the numbering of Genbank: E02627).This fragment is cloned in the Jx10AS-CMV-pGL3 plasmid that digests in advance with HindIII and NcoI then with HindIII and NcoI digestion, obtains the Jx10AS-CMV-EF-pGL3 plasmid.Jx10AS-CMV-EF-pGL3 plasmid synoptic diagram as shown in figure 14.
6.2.Jx10AS-CMV-EF-pGL3 the activity of plasmid
In order to improve the last activity of system, the clone is arranged in the enhancer sequence of first intron of hEF1a gene near evoked promoter.The result who lists among Figure 15 shows that the existence that strengthens the subarea has improved the induced activity of system, and it is still like this no matter how many transcriptional regulatory gene usage quantitys is.
Embodiment 7: make up the plasmid that comprises transcriptional regulatory expression casette and abduction delivering box simultaneously
7.1.Jx5AS-TK-luc-hPPARg2 plasmid
With Mlul and Scal digestion pSG5-hPPARa (Koz) plasmid, so that separate the 1229pb Mlul-Scal fragment in the 3 ' district that comprises the hPPARa complementary DNA.This fragment is inserted in the pSL-301 plasmid that digests in advance with Mlul and Smal, obtains pSL-3 ' hPPARa plasmid.
With Sall and Mlul digestion pSG5-hPPARa (Koz) plasmid, so that separate the 1406pb Mlul-Scal fragment in the 5 ' district that comprises SV40 virus early promoter and hPPARa complementary DNA.This fragment is inserted pSL-3 ' the hPPARa plasmid that digests in advance with Xhol and Mlul, obtains the pSL-hPPARa plasmid.
With Spel and Sall digestion pSL-hPPARa plasmid, so that separate the 2664pb Spel-Sall fragment that comprises SV40 virus early promoter and hPPARa complementary DNA.This fragment is inserted in the pBluescript II SK+ plasmid that digests in advance with Spel and Sall, obtains the pBS-hPPARa plasmid.
With Avrll and Sacl digestion pSG5-hPPARg2 plasmid, so that separate the 2070pb Avrll-Sacl fragment (being labeled as C) in the 5 ' district that comprises the hPPARg2 complementary DNA.By using the pSG5-hPPARg2 plasmid as matrix and utilize 10RDA21 (5 ' CAG GTT TGC TGA ATG TGA AGC 3 '; SEQ ID NO:16) and 11RDA40 (5 ' TGA CGT GTC GAC CTA GTA CAA GTC CTT GTA GAT CTC CTGC3 '; SEQ ID NO:17) oligonucleotide is as primer; Amplification comprises the dna fragmentation (being labeled as D) in 3 ' district of hPPARg2 complementary DNA by ACP.In the pBS-hPPARa plasmid that digests in advance with Avrll and Sall, make and adopt the fragment C and the fragment D of Sacl and Sall digestion to clone together, obtain the pBS-hPPARg2 plasmid.
Use Kpnl and Sall digestion Jx5AS-TK-pGL3 plasmid, so that be separated in the 2324pb Kpnl-Sall fragment that evoked promoter control comprises the luc+ gene down.This fragment is inserted in the pBS-hPPARg2 plasmid that digests in advance with Kpnl and Sall, obtains the Jx5AS-TK-luc-hPPARg2 plasmid.Jx5AS-TK-luc-hPPARg2 plasmid synoptic diagram as shown in figure 16.
7.2.SV-g2-J10-C-pGL3 plasmid
With Notl and Sall digestion pBS-hPPARg2 plasmid, so that be separated in the 2622pb Notl-Sall fragment (being labeled as E) that the control of SV40 early promoter comprises the hPPARg2 complementary DNA down.By using the FTK-pGL3 plasmid as matrix and utilize 18RDA31 (5 ' AGT CGT CGA CGC TTC GAG CAG ACATGA TAA G 3 '; SEQ ID NO:18) and 19RDA35 (5 ' AGT CGC TAG CGA CGG ATC CTTATC GAT TTT ACC AC 3 '; SEQ ID NO:19) oligonucleotide adopts the ACP amplification to comprise the dna fragmentation (being labeled as F) in the polyadenylation site of SV40 virus as primer.In the Jx10AS-CMV-pGL3 plasmid that digests in advance with Notl and Nhel, make and adopt the fragment E and the fragment F of Sall and Nhel digestion to clone together, obtain the SV-g2-J10-C-pGL3 plasmid.SV-g2-J10-C-pGL3 plasmid synoptic diagram as shown in figure 17.
7.3.hPPARg2-CMV-Jx5AS-TK-pGL3 plasmid
By using the Jx5AS-TK-luc-hPPARg2 plasmid as matrix and utilize 12RDA50 (5 ' GTCAGC TAG CCT ACT CGA GCC ACC ATG GGT GAA ACT CTG GGA GAT TCT CC 3 '; SEQID NO:20) and 13RDA42 (5 ' TAC GGG GTA CCC AGA CAT GAT AAG ATA CAT TGA TGAGTT TGG 3 '; SEQ ID NO:21) oligonucleotide adopts the ACP amplification to comprise the dna fragmentation (being labeled as G) of hPPARg2 complementary DNA as primer.By using pCMV β plasmid as matrix and utilize 14RDA33 (5 ' GTC AGC TAG CCG GTA GGC GTG TAC GGT GGG AGG 3 '; SEQ ID NO:22) and 15RDA33 (5 ' TAC GCT CGA GCT TCT ATG GAG GTC AAA ACA GCG 3 '; SEQID NO:23) oligonucleotide adopts the ACP amplification to comprise the dna fragmentation (being labeled as H) in hCMV-IE minimal promoter (with respect to the transcripting starting site, from-54 to+48) as primer.In the Jx5AS-TK-pGL3 plasmid that digests in advance with Kpnl and Nhel, adopt the fragment G of Kpn and Xhol digestion and the fragment H of employing Xhol and Nhel digestion to clone together, obtain the hPPARg2-CMV-Jx5AS-TK-pGL3 plasmid.HPPARg2-CMV-Jx5AS-TK-pGL3 plasmid synoptic diagram as shown in figure 18.
7.4.hPPARg2-CMV-JxnAS-CMV-pGL3 plasmid
With Nhel and Sphl digestion Jx5AS-CMV-pGL3 plasmid, so that be separated in the 982pb Nhel-Sphl fragment that evoked promoter control comprises luc+ gene 5 ' district down.This fragment is inserted in the hPPARg2-CMV-Jx5AS-TK-pGL3 plasmid that digests in advance with Spel and Sphl, obtains the hPPARg2-CMV-Jx10AS-CMV-pGL3 plasmid.HPPARg2-CMV-Jx10AS-CMV-pGL3 plasmid synoptic diagram as shown in figure 19.
With Nhel and Sphl digestion Jx5AS-CMV-pGL3 plasmid, so that be separated in the 982pb Nhel-Sphl fragment that evoked promoter control comprises luc+ gene 5 ' district down.This fragment is inserted in the hPPARg2-CMV-Jx10AS-CMV-pGL3 plasmid that digests in advance with Spel and Sphl, obtains the hPPARg2-CMV-Jx15AS-CMV-pGL3 plasmid.
With Nhel and Sphl digestion Jx10AS-CMV-pGL3 plasmid, so that be separated in the 1151pb Nhel-Sphl fragment that evoked promoter control comprises luc+ gene 5 ' district down.This fragment is inserted in the hPPARg2-CMV-Jx10AS-CMV-pGL3 plasmid that digests in advance with Spel and Sphl, obtains the hPPARg2-CMV-Jx20AS-CMV-pGL3 plasmid.
Embodiment 8: the comparison of different in vitro inducible system versions
Figure 20 has compiled the result who obtains with multiple inducible system version in vitro.These results show, with (the Figure 20 of system that only has a plasmid, curve 2 and 5-9) the same, it is functional using system's (Figure 20, curve 1,3 and 4) of two plasmids: the existence that is to say PPARg (being BRL49653 at this) part has obviously strengthened the genetic expression that places under the evoked promoter control.Should also be noted that the part inducible factor is higher than 30 for some system (Figure 20, curve 3 and 7-9), and for system shown in Figure 20 curve 4, the activity after inducing equals the activity of strong promoter, as the hCMV-IE promoter activity.
Embodiment 9: different PPAR parts can the activation-inducing system
9.1. use the system of hPPARg2
Result among Figure 21 shows the hPPARg part except that BRL49653, is RG12525 (the RPR part of hPPARg) at this, can be used for the activation-inducing system.Under 100 micro-molar concentrations, handle even obtain comparing stronger inducing action with the inducing action that adopts BRL49653 to obtain with RG12525.Other part of all of hPPARg therefore can be as the inductor of system.
9.2. use the system of hPPARa
The same with the system that uses hPPARg, use hPPARa can to activate with any other part of for example Carboxymethylcellulose salt or WY-14643 or hPPARa as the system of transcriptional regulatory gene.
Embodiment 10: in vivo can the activation-inducing system in muscle
Figure 22 has compiled the inducible system resulting result in live body muscle who uses different editions.These results show, for the version (Figure 22, curve 2-4) of three kinds of tests, adopt gavage to handle the activity that can obviously improve evoked promoter in muscle with the hPPARg part.Inducible factor is, Figure 22 curve 2 versions are * 14, and Figure 22 curve 3 versions are * 8, and Figure 22 curve 4 versions are * 24.In addition, for one of them version (Figure 22 curve 2), the activity that is reached in the animal body of being handled by BRL49653 is in the same order of magnitude with the activity of strong promoter such as the hCMV-IE promotor.
The result who lists in Figure 23 shows that also only the applied once part just can be induced this system, and this using can be carried out before or after transgenosis.This test shows that also the dosage than the little twice of common dose just can reach same inducible factor.
Using the system of PPAR nuclear receptor as the transcriptional regulatory gene, in vivo is functional therefore, can induce this system by oral PPAR part.
Embodiment 11: the plasmid of the gene that structure can the abduction delivering secretory product
11.1. make up the pRDA02 plasmid
With HindIII and Mlul digestion Jx10AS-CMV-pGL3 plasmid, so that separate the 459pbHindIII-Mlul fragment.This fragment is inserted in the pXL3010 plasmid that digests in advance with HindIII and Mlul (Bettan M. and colleague thereof, " Anal.Biochem. ", 271 (1999) 187-189), obtains the pRDA02 plasmid.This plasmid comprises the complementary DNA of the gene of coding placental alkaline phosphatase secreted form (hSeAP), and its Phosphoric acid esterase is expressed by utilizing PPAR to be in the control of inductive promotor down as the system of transcriptional regulatory gene.PRDA02 plasmid schematic view illustrating is in Figure 24.
Embodiment 12: inducible system can be regulated the plasmid concentration in the secretory protein in vivo
The result who lists among Figure 25 shows, when using inducible system, can regulate at any time by the plasmid concentration in the muscle excretory albumen, and this point can realize by an oral PPAR part only.After using part two days, increase by 17 times (Figure 25 A) of hSeAP plasmid concentration, return to its basal level after the week.Between the 21st day to the 39th day, observe hSeAP immune response, and show as this albumen plasmid concentration reduction the people source.Although there is this immune response, still may carry out second induction duration (Figure 25 A).
Indicated as Figure 25 B, repeatedly take part every day, in the time identical with the treatment time, inducible system also can make the hSeAP plasmid concentration keep high level.
Embodiment 13: different PPAR parts can activate inducible system in vivo, and with the mode activation-inducing system of dependent dose
BRL49653 (the Avandia that presents the sale form that is used for the treatment of type ii diabetes
TM, SmithKlineBeecham) with the pioglitazone (Actos that is the sale form that is used for this same treatment
TM: TakedaPharmaceuticals) also can activation-inducing system (Figure 26 A).Figure 26 B shows that also inducible factor is directly related with the part dosage of use.
Use the PPAR nuclear receptor therefore can very accurately control the plasmid content of secretory protein as the system of transcriptional regulatory gene.In addition, when using different PPAR parts, can reach this adjusting.
Embodiment 14: making up its product of abduction delivering to be the plasmid of the gene of angiogenesis factor
14.1. make up Jx10AS-CMV-VEGF
A165 plasmids
By the total RNA of people's placenta by reverse transcription and PCR human cloning VEGF165 read area (Clontech) (people such as Houck, " Mol.Endocrinol. " 12 (1991) 1806-1814), insert the pBluescript plasmid (Stratagene) of the tardigrades polyA of the CMV E/P promotor comprise-522 to+72 and SV40 then, so that obtain the pXL3218 plasmid.This plasmid is then with HindIII and BsrGI digestion, so that separate 482pb HindIII-BsrGI Segment A.Also available BsrGI of pXL3218 plasmid and BamHI digestion are so that separate 390pb BsrGI-BamHI fragment B.A and B fragment are inserted in the Jx10AS-CMV-pGL3 plasmid of using HindIII and BamHI digestion in advance, so that obtain Jx10AS-CMV-VFGF
A165 plasmids.This plasmid comprises coding VEGF
A165 gene complementation DNA is by using the system of PPAR as the transcriptional regulatory gene, VEGF expression under evoked promoter control
A165.Jx10AS-CMV-VEGF
AThe plasmid synoptic diagram as shown in figure 27.
This plasmid for example can be used for controlling at any time with therapeutic purpose the angiogenic activity of VEGF.
Sequence table＜110〉Aventis Pharma S.A＜120〉use PPAR nuclear receptor and part thereof
＜130〉＜140〉＜141〉＜160〉28＜170〉2.1＜210〉1＜211〉19＜212〉ADN＜213〉＜400〉1tcaaccttta ccctggtag 19＜210〉2＜211〉27＜212〉ADN＜213〉＜400〉2tcgccaagct tctcgtgatc tgcggca 27＜210〉3＜211〉37＜212〉ADN＜213〉＜400〉3acgtgtcgac actagtggct agaggatctc taccagg 37＜210〉4＜211〉48＜212〉ADN＜213〉＜400〉4cgatggtacc ctcgagcaat gtgctagcga gatccttcaa cctttacc 48＜210〉5＜211〉13＜212〉ADN＜213〉＜400〉5aggtcaaagg tca 13＜210〉6＜211〉69＜212〉ADN＜213〉＜400〉6acgtgtcgac actagtcaaa actaggtcaa aggtcacgga aaactaggtc aaaggtcacg 60gaaaactag 69＜210〉7＜211〉64＜212〉ADN＜213〉＜400〉7cgatggtacc ctcgagcaat gtgctagccg tgacctttga cctagttttc cgtgaccttt 60gacc 64＜210〉8＜211〉32＜212〉ADN＜213〉＜400〉8acgtagatct cggtaggcgt gtacggtggg ag 32＜210〉9＜211〉29＜212〉ADN＜213〉＜400〉9acgtaagctt ctatggaggt caaaacagc 29＜210〉10＜211〉21＜212〉ADN＜213〉＜400〉10ggtttgctga atgtgaagcc c 21＜210〉11＜211〉42＜212〉ADN＜213〉＜400〉11agtctctaga gctacgcgta caagtccttg tagatctcct gc 42＜210〉12＜211〉32＜212〉ADN＜213〉＜400〉12agtcacgcgt gggcgatctt gacaggaaag ac 32＜210〉13＜211〉21＜212〉ADN＜213〉＜400〉13gcctttgagt gagctgatac c 21＜210〉14＜211〉35＜212〉ADN＜213〉＜400〉14agtcactagt aagctttttg ccgccagaac acagg 35＜210〉15＜211〉36＜212〉ADN＜213〉＜400〉15agtcactagt ccatggctgc ccagtgcctc acgacc 36＜210〉16＜211〉21＜212〉ADN＜213〉＜400〉16caggtttgct gaatgtgaag c 21＜210〉17＜211〉40＜212〉ADN＜213〉＜400〉17tgacgtgtcg acctagtaca agtccttgta gatctcctgc 40＜210〉18＜211〉31＜212〉ADN＜213〉＜400〉18agtcgtcgac gcttcgagca gacatgataa g 31＜210〉19＜211〉35＜212〉ADN＜213〉＜400〉19agtcgctagc gacggatcct tatcgatttt accac 35＜210〉20＜211〉50＜212〉ADN＜213〉＜400〉20gtcagctagc ctactcgagc caccatgggt gaaactctgg gagattctcc 50＜210〉21＜211〉42＜212〉ADN＜213〉＜400〉21tacggggtac ccagacatga taagatacat tgatgagttt gg 42＜210〉22＜211〉33＜212〉ADN＜213〉＜400〉22gtcagctagc cggtaggcgt gtacggtggg agg 33＜210〉23＜211〉33＜212〉ADN＜213〉＜400〉23tacgctcgag cttctatgga ggtcaaaaca gcg 33＜210〉24＜211〉750＜212〉PRT＜213〉＜400〉24Met Gly Glu Thr Leu Gly Asp Ser Pro Ile Asp Pro Glu Ser Asp Ser 1 5 10 15Phe Thr AsD Thr Leu Ser Ala Asn Ile Ser Gln Glu Met Thr Met Val
20 25 30Asp?Thr?Glu?Met?Pro?Phe?Trp?Pro?Thr?Asn?Phe?Gly?Ile?Ser?Ser?Val
35 40 45Asp?Leu?Ser?Val?Met?Glu?Asp?His?Ser?His?Ser?Phe?Asp?Ile?Lys?Pro
50 55 60Phe?Thr?Thr?Val?Asp?Phe?Ser?Ser?Ile?Ser?Thr?Pro?His?Tyr?Glu?Asp?65 70 75 80Ile?Pro?Phe?Thr?Arg?Thr?Asp?Pro?Val?Val?Ala?Asp?Tyr?Lys?Tyr?Asp
85 90 95Leu?Lys?Leu?Gln?Glu?Tyr?Gln?Ser?Ala?Ile?Lys?Val?Glu?Pro?Ala?Ser
100 105 110Pro?Pro?Tyr?Tyr?Ser?Glu?Lys?Thr?Gln?Leu?Tyr?Asn?Lys?Pro?His?Glu
115 120 125Glu?Pro?Ser?Asn?Ser?Leu?Met?Ala?Ile?Glu?Cys?Arg?Val?Cys?Gly?Asp
130 135 140Lys?Ala?Ser?G1y?Phe?His?Tyr?G1y?Val?His?Ala?Cys?Glu?Gly?Cys?Lys145 150 155 160Gly?Phe?Phe?Arg?Arg?Thr?Ile?Arg?Leu?Lys?Leu?Ile?Tyr?Asp?Arg?Cys
165 170 175Asp?Leu?Asn?Cys?Arg?Ile?His?Lys?Lys?Ser?Arg?Asn?Lys?Cys?Gln?Tyr
180 185 190Cys?Arg?Phe?Gln?Lys?Cys?Leu?Ala?Val?Gly?Met?Ser?His?Asn?Ala?Ile
195 200 205Arg?Phe?Gly?Arg?Met?Pro?Gln?Ala?Glu?Lys?Glu?Lys?Leu?Leu?Ala?Glu
210 215 220Ile?Ser?Ser?Asp?Ile?Asp?Gln?Leu?Asn?Pro?Glu?Ser?Ala?Asp?Leu?Arg225 230 235 240Ala?Leu?Ala?Lys?His?Leu?Tyr?Asp?Ser?Tyr?Ile?Lys?Ser?Phe?Pro?Leu
245 250 255Thr?Lys?Ala?Lys?Ala?Arg?Ala?Ile?Leu?Thr?Gly?Lys?Thr?Thr?Asp?Lys
260 265 270Ser?Pro?Phe?Val?Ile?Tyr?Asp?Met?Asn?Ser?Leu?Met?Met?Gly?Glu?Asp
275 280 285Lys?Ile?Lys?Phe?Lys?His?Ile?Thr?Pro?Leu?Gln?Glu?Gln?Ser?Lys?Glu
290 295 300Val?Ala?Ile?Arg?Ile?Phe?Gln?Gly?cys?Gln?Phe?Arg?Ser?Val?Glu?Ala305 310 315 320Val?Gln?Glu?Ile?Thr?Glu?Tyr?Ala?Lys?Ser?Ile?Pro?Gly?Phe?Val?Asn
325 330 335Leu?Asp?Leu?Asn?Asp?Gln?Val?Thr?Leu?Leu?Lys?Tyr?Gly?Val?His?Glu
340 345 350Ile?Ile?Tyr?Thr?Met?Leu?Ala?Ser?Leu?Met?Asn?Lys?Asp?Gly?Val?Leu
355 360 365Ile?Ser?Glu?Gly?Gln?Gly?Phe?Met?Thr?Arg?Glu?Phe?Leu?Lys?Ser?Leu
370 375 380Arg?Lys?Pro?Phe?Gly?Asp?Phe?Met?Glu?Pro?Lys?Phe?Glu?Phe?Ala?Val385 390 395 400Lys?Phe?Asn?Ala?Leu?Glu?Leu?Asp?Asp?Ser?Asp?Leu?Ala?Ile?Phe?Ile
405 410 415Ala?Val?Ile?Ile?Leu?Ser?Gly?Asp?Arg?Pro?Gly?Leu?Leu?Asn?Val?Lys
420 425 430Pro?Ile?Glu?Asp?Ile?Gln?Asp?Asn?Leu?Leu?Gln?Ala?Leu?Glu?Leu?Gln
435 440 445Leu?Lys?Leu?Asn?His?Pro?Glu?Ser?Ser?Gln?Leu?Phe?Ala?Lys?Leu?Leu
450 455 460Gln?Lys?Met?Thr?Asp?Leu?Arg?Gln?Ile?Val?Thr?Glu?His?Val?Gln?Leu465 470 475 480Leu?Gln?Val?Ile?Lys?Lys?Thr?Glu?Thr?Asp?Met?Ser?Leu?His?Pro?Leu
485 490 495Leu?Gln?Glu?Ile?Tyr?Lys?Asp?Leu?Tyr?Ala?Trp?Ala?Ile?Leu?Thr?Gly
500 505 510Lys?Thr?Thr?Asp?Lys?Ser?Pro?Phe?Val?Ile?Tyr?Asp?Met?Asn?Ser?Leu
515 520 525Met?Met?Gly?Glu?Asp?Lys?Ile?Lys?Phe?Lys?His?Ile?Thr?Pro?Leu?Gln
530 535 540Glu?Gln?Ser?Lys?Glu?Val?Ala?Ile?Arg?Ile?Phe?Gln?Gly?Cys?Gln?Phe545 550 555 560Arg?Ser?Val?Glu?Ala?Val?Gln?Glu?Ile?Thr?Glu?Tyr?Ala?Lys?Ser?Ile
565 570 575Pro?Gly?Phe?Val?Asn?Leu?Asp?Leu?Asn?Asp?Gln?Val?Thr?Leu?Leu?Lys
580 585 590Tyr?Gly?Val?His?Glu?Ile?Ile?Tyr?Thr?Met?Leu?Ala?Ser?Leu?Met?Asn
595 600 605Lys?Asp?Gly?Val?Leu?Ile?Ser?Glu?Gly?Gln?Gly?Phe?Met?Thr?Arg?Glu
610 615 620Phe?Leu?Lys?Ser?Leu?Arg?Lys?Pro?Phe?Gly?Asp?Phe?Met?Glu?Pro?Lys625 630 635 640Phe?Glu?Phe?Ala?Val?Lys?Phe?Asn?Ala?Leu?Glu?Leu?Asp?Asp?Ser?Asp
645 650 655Leu?Ala?Ile?Phe?Ile?Ala?Val?Ile?Ile?Leu?Ser?Gly?Asp?Arg?Pro?Gly
660 665 670Leu?Leu?Asn?Val?Lys?Pro?Ile?Glu?Asp?Ile?Gln?Asp?Asn?Leu?Leu?Gln
675 680 685Ala?Leu?Glu?Leu?Gln?Leu?Lys?Leu?Asn?His?Pro?Glu?Ser?Ser?Gln?Leu
690 695 700Phe?Ala?Lys?Leu?Leu?Gln?Lys?Met?Thr?Asp?Leu?Arg?Gln?Ile?Val?Thr705 710 715 720Glu?His?Val?Gln?Leu?Leu?Gln?Val?Ile?Lys?Lys?Thr?Glu?Thr?Asp?Met
725 730 735Ser?Leu?His?Pro?Leu?Leu?Gln?Glu?Ile?Tyr?Lys?Asp?Leu?Tyr
740 745 750＜210〉25＜211〉467＜212〉PRT＜213〉homo sapiens＜400〉25Met Met Gly Glu Asp Lys Ile Lys Phe Lys His Ile Thr Pro Leu Gln, 15 10 15Glu Gln Ser Lys Glu Val Ala Ile Arg Ile Phe Gln Gly Cys Gln Phe
20 25 30Arg?Ser?Val?Glu?Ala?Val?Gln?Glu?Ile?Thr?Glu?Tyr?Ala?Lys?Ser?Ile
35 40 45Pro?Gly?Phe?Val?Asn?Leu?Asp?Leu?Asn?Asp?Gln?Val?Thr?Leu?Leu?Lys
50 55 60Tyr?Gly?Val?His?Glu?Ile?Ile?Tyr?Thr?Met?Leu?Ala?Ser?Leu?Met?Asn?65 70 75 80Lys?Asp?Gly?Val?Leu?Ile?Ser?Glu?Gly?Gln?Gly?Phe?Met?Thr?Arg?Glu
85 90 95Phe?Leu?Lys?Ser?Leu?Arg?Lys?Pro?Phe?Gly?Asp?Phe?Met?Glu?Pro?Lys
100 105 110Phe?Glu?Phe?Ala?Val?Lys?Phe?Asn?Ala?Leu?Glu?Leu?Asp?Asp?Ser?Asp
115 120 125Leu?Ala?Ile?Phe?Ile?Ala?Val?Ile?Ile?Leu?Ser?Gly?Asp?Arg?Pro?Gly
130 135 140Leu?Leu?Asn?Val?Lys?Pro?Ile?Glu?Asp?Ile?Gln?Asp?Asn?Leu?Leu?Gln145 150 155 160Ala?Leu?Glu?Leu?Gln?Leu?Lys?Leu?Asn?His?Pro?Glu?Ser?Ser?Gln?Leu
165 170 175Phe?Ala?Lys?Leu?Leu?Gln?Lys?Met?Thr?Asp?Leu?Arg?Gln?Ile?Val?Thr
180 185 190Glu?His?Val?Gln?Leu?Leu?Gln?Val?Ile?Lys?Lys?Thr?Glu?Thr?Asp?Met
195 200 205Ser?Leu?His?Pro?Leu?Leu?Gln?Glu?Ile?Tyr?Lys?Asp?Leu?Tyr?Ala?Trp
210 215 220Ala?Ile?Leu?Thr?Gly?Lys?Thr?Thr?Asp?Lys?Ser?Pro?Phe?Val?Ile?Tyr225 230 235 240Asp?Met?Asn?Ser?Leu?Met?Met?Gly?Glu?Asp?Lys?Ile?Lys?Phe?Lys?His
245 250 255Ile?Thr?Pro?Leu?Gln?Glu?Gln?Ser?Lys?Glu?Val?Ala?Ile?Arg?Ile?Phe
260 265 270Gln?Gly?Cys?Gln?Phe?Arg?Ser?Val?Glu?Ala?Val?Gln?Glu?Ile?Thr?Glu
275 280 285Tyr?Ala?Lys?Ser?Ile?Pro?Gly?Phe?Val?Asn?Leu?Asp?Leu?Asn?Asp?Gln
290 295 300Val?Thr?Leu?Leu?Lys?Tyr?Gly?Val?His?Glu?Ile?Ile?Tyr?Thr?Met?Leu305 310 315 320Ala?Ser?Leu?Met?Asn?Lys?Asp?Gly?Val?Leu?Ile?Ser?Glu?Gly?Gln?Gly
325 330 335Phe?Met?Thr?Arg?Glu?Phe?Leu?Lys?Ser?Leu?Arg?Lys?Pro?Phe?Gly?Asp
340 345 350Phe?Met?Glu?Pro?Lys?Phe?Glu?Phe?Ala?Val?Lys?Phe?Asn?Ala?Leu?Glu
355 360 365Leu?Asp?Asp?Ser?Asp?Leu?Ala?Ile?Phe?Ile?Ala?Val?Ile?Ile?Leu?Ser
370 375 380Gly?Asp?Arg?Pro?Gly?Leu?Leu?Asn?Val?Lys?Pro?Ile?Glu?Asp?Ile?Gln385 390 395 400Asp?Asn?Leu?Leu?Gln?Ala?Leu?Glu?Leu?Gln?Leu?Lys?Leu?Asn?His?Pro
405 410 415Glu?Ser?Ser?Gln?Leu?Phe?Ala?Lys?Leu?Leu?Gln?Lys?Met?Thr?Asp?Leu
420 425 430Arg?Gln?Ile?Val?Thr?Glu?His?Val?Gln?Leu?Leu?Gln?Val?Ile?Lys?Lys
435 440 445Thr?Glu?Thr?Asp?Met?Ser?Leu?His?Pro?Leu?Leu?Gln?Glu?Ile?Tyr?Lys
450 455 460Asp Leu Tyr465＜210〉26＜211〉30＜212〉ADN＜213〉homo sapiens＜400〉26cccgttacat aacttacggt aaatggcccg 30＜210〉27＜211〉30＜212〉ADN＜213〉homo sapiens＜400〉27gggacgcgct tctacaaggc gctggccgaa 30＜210〉28＜211〉30＜212〉ADN＜213〉homo sapiens＜400〉28cgactctaga agatcttgcc ccgcccagcg 30
Claims (30)
1, composition wherein comprises:
(a) first composition, it comprises the purpose nucleic acid that is under the evoked promoter control, and this evoked promoter comprises composition and the minimum transcripting promoter to the PPAR reaction,
(b) second composition, it is included in the evoked promoter control nucleic acid of coding PPAR down, so that they side by side, use respectively or with certain hour at interval.
2,, it is characterized in that it also comprises according to the composition of claim 1:
(c) PPAR part
So that side by side, use at interval respectively or with certain hour.
3,, it is characterized in that different genetic constructs has described composition (a) and (b) according to the composition of claim 1 or 2.
4,, it is characterized in that described composition (a) and (b) be integrated in the same genetic constructs according to the composition of claim 1 or 2.
5,, it is characterized in that described genetic constructs is plasmid or virus vector according to the composition of claim 3 or 4.
6,, it is characterized in that the composition to the PPAR reaction comprises the site of one or more PPAR chains according to each composition among the claim 1-5.
7,, it is characterized in that described composition to the PPAR reaction comprises one or more sites of the functional variant of SEQID NO:1 sequence or this sequence according to the composition of claim 6.
8,, it is characterized in that described composition to the PPAR reaction comprises one or more sites of the functional variant of SEQID NO:5 sequence or this sequence according to the composition of claim 6.
9, each composition among the claim 6-8 is characterized in that described reacted constituent comprises nearly 30 chain sites, preferably 3-20 chain site, more preferably 5-15 chain site.
10, each composition among the claim 1-9 is characterized in that described minimal promoter is one or more cytogene or the virogene promotors with nonessential region of transcriptional activity of disappearance.
11, each composition among the claim 1-10 is characterized in that described evoked promoter also comprises amplification region.
12, each composition among the claim 1-11 is characterized in that described minimal promoter is with identical to the direction of PPAR reacted constituent.
13, each composition among the claim 1-11 is characterized in that minimal promoter and opposite to the direction of PPAR reacted constituent.
14, each composition among the claim 1-13, nucleic acid codified PPAR α or the PPAR γ of the PPAR that it is characterized in that encoding.
15, each composition among the claim 1-14, the nucleic acid codified of the PPAR that it is characterized in that encoding comprises the modification PPAR in a plurality of part chains site.
16, each composition among the claim 1-15 is characterized in that it also comprises composition (d), and this composition is included in the transcripting promoter control nucleic acid of coding RXR down.
17, the carrier that comprises described composition of claim (a) and composition (b).
18,, it is characterized in that the direction of described composition (a) and composition (b) is opposite according to the carrier of claim 17.
19, according to the carrier of claim 17 or 18, it is characterized in that the evoked promoter of described composition (a) and the transcripting promoter of composition (b) are integrated in the carrier, can regulate bidirectional promoter so that generate.
20, according to the carrier of claim 19, it is characterized in that in 5 '-3 ' direction, it comprises first nucleic acid of the PPAR that encodes, first minimum transcripting promoter of described first expression of nucleic acid of control, one or more are to the PPAR reacted constituent, second minimum transcripting promoter, and second nucleic acid of the purpose product of under described second minimum transcripting promoter control, encoding.
21, according to each carrier among the claim 17-20, it is characterized in that it also comprises 16 composition (d).
22, among the claim 1-16 among each composition or the claim 17-21 each carrier express the application of purpose nucleic acid in vitro or in the invisible spectro cell.
23, among the claim 1-16 among each composition or the claim 17-21 each carrier be used for intravital cell in preparation and express application in the product of purpose nucleic acid.
24, in vitro or or invisible spectro cell in regulate express nucleic acid method, this method comprise among described cell and the claim 1-16 each composition or claim 17-21 in each carrier contact.
25,, it is characterized in that this method relates to mammalian cell, preferably people's cell according to the method for claim 24.
26,, it is characterized in that this method relates to muscle cell according to the method for claim 25.
27, by with claim 1-16 in each composition or claim 17-21 in each carrier contact the cell of modifying.
28, the PPAR of Xiu Shiing, it comprises a plurality of part chains site.
29, the nucleic acid of the PPAR of coding claim 28.
30, the authentication method of PPAR part, this method comprise that the cell that makes claim 27 contacts with the test molecule, proves the purpose expression of nucleic acids again.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR99/07957 | 1999-06-22 | ||
| FR9907957A FR2795425B1 (en) | 1999-06-22 | 1999-06-22 | PHARMACOLOGICAL REGULATION OF EXPRESSION SYSTEM USING NUCLEAR RECEPTORS BY AND THEIR LIGANDS |
| US14972199P | 1999-08-20 | 1999-08-20 | |
| US60/149,721 | 1999-08-20 |
Publications (1)
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|---|---|
| CN1370240A true CN1370240A (en) | 2002-09-18 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN00811928A Pending CN1370240A (en) | 1999-06-22 | 2000-06-22 | Regulation system of expression using nuclear PPAR receptors |
Country Status (14)
| Country | Link |
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| EP (1) | EP1190084A1 (en) |
| JP (1) | JP2003503034A (en) |
| KR (1) | KR20020028909A (en) |
| CN (1) | CN1370240A (en) |
| AU (1) | AU6164500A (en) |
| BR (1) | BR0011897A (en) |
| CA (1) | CA2375869A1 (en) |
| CZ (1) | CZ20014609A3 (en) |
| HU (1) | HUP0203584A2 (en) |
| IL (1) | IL147104A0 (en) |
| MX (1) | MXPA01013227A (en) |
| NO (1) | NO20016347L (en) |
| NZ (1) | NZ516678A (en) |
| WO (1) | WO2000078986A1 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2403189A1 (en) * | 2000-03-14 | 2001-09-20 | Charbel Massaad | Inflammation-inducible hybrid promoters, vectors containing same and uses thereof |
| EP1288303A1 (en) * | 2001-08-23 | 2003-03-05 | Aventis Pharma S.A. | Inducible expression systems employing PPAR transcriptional activators |
| WO2003012113A2 (en) * | 2001-08-02 | 2003-02-13 | Gencell S.A. | Inducible expression systems employing ppar transcriptional activators |
| WO2014117945A2 (en) * | 2013-02-04 | 2014-08-07 | Eth Zurich | Designer circuit controlling diet-induced obesity |
| CN107779454A (en) * | 2016-08-31 | 2018-03-09 | 上海米络凯生物科技有限公司 | The structure of medicaments sifting model based on PPAR γ signal paths and application |
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| JPH10502256A (en) * | 1994-07-01 | 1998-03-03 | ザ ソールク インスチチュート フォア バイオロジカル スタディズ | Mammalian peroxisome proliferator-activated receptor and uses thereof |
| FR2755699B1 (en) * | 1996-11-08 | 1998-12-18 | Rhone Poulenc Rorer Sa | NEW CONSTRUCTIONS AND VECTORS FOR TARGETED AND INDUCTIBLE GENE EXPRESSION |
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2000
- 2000-06-22 AU AU61645/00A patent/AU6164500A/en not_active Abandoned
- 2000-06-22 CZ CZ20014609A patent/CZ20014609A3/en unknown
- 2000-06-22 CA CA002375869A patent/CA2375869A1/en not_active Abandoned
- 2000-06-22 IL IL14710400A patent/IL147104A0/en unknown
- 2000-06-22 CN CN00811928A patent/CN1370240A/en active Pending
- 2000-06-22 HU HU0203584A patent/HUP0203584A2/en unknown
- 2000-06-22 NZ NZ516678A patent/NZ516678A/en unknown
- 2000-06-22 JP JP2001505726A patent/JP2003503034A/en not_active Withdrawn
- 2000-06-22 MX MXPA01013227A patent/MXPA01013227A/en unknown
- 2000-06-22 KR KR1020017016447A patent/KR20020028909A/en not_active Application Discontinuation
- 2000-06-22 WO PCT/FR2000/001744 patent/WO2000078986A1/en not_active Application Discontinuation
- 2000-06-22 BR BR0011897-4A patent/BR0011897A/en not_active IP Right Cessation
- 2000-06-22 EP EP00948058A patent/EP1190084A1/en not_active Withdrawn
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Also Published As
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| IL147104A0 (en) | 2002-08-14 |
| BR0011897A (en) | 2002-03-26 |
| NZ516678A (en) | 2004-04-30 |
| CZ20014609A3 (en) | 2002-05-15 |
| WO2000078986B1 (en) | 2001-04-19 |
| HUP0203584A2 (en) | 2003-02-28 |
| EP1190084A1 (en) | 2002-03-27 |
| NO20016347L (en) | 2002-02-19 |
| MXPA01013227A (en) | 2002-06-04 |
| CA2375869A1 (en) | 2000-12-28 |
| KR20020028909A (en) | 2002-04-17 |
| JP2003503034A (en) | 2003-01-28 |
| NO20016347D0 (en) | 2001-12-21 |
| AU6164500A (en) | 2001-01-09 |
| WO2000078986A1 (en) | 2000-12-28 |
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