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WO2002033104A2 - Systeme de regulation des genes a base d'antibiotique - Google Patents

Systeme de regulation des genes a base d'antibiotique Download PDF

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WO2002033104A2
WO2002033104A2 PCT/IB2001/001963 IB0101963W WO0233104A2 WO 2002033104 A2 WO2002033104 A2 WO 2002033104A2 IB 0101963 W IB0101963 W IB 0101963W WO 0233104 A2 WO0233104 A2 WO 0233104A2
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protein
domain
ptr
sequence
polypeptide
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PCT/IB2001/001963
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WO2002033104A3 (fr
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Martin Fussenegger
James E. Bailey
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Cistronics Cell Technology Gmbh
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Publication of WO2002033104A3 publication Critical patent/WO2002033104A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8237—Externally regulated expression systems
    • C12N15/8238—Externally regulated expression systems chemically inducible, e.g. tetracycline

Definitions

  • the invention relates to a novel system for gene regulation in eukaryotic cells, and methods of using the same for protein production.
  • the invention provides a new system for antibiotic-regulated gene expression in eukaryotic cells, including plant and mammalian cells, based on sequences from Actinomycetes antibiotic resistance promoters, polypeptides that bind to the same in an antibiotic responsive manner, and nucleotides encoding such polypeptides.
  • FIG 10 A Graphical representation of the PLPpOFF expression vector pMF276.
  • pMF276 harbors the PIT expression unit which is driven by the constitutive promoter of the cauliflower mosaic virus gene 35S (P CaMV3 ss) m & terminated by the polyadenylation site derived from the octopine synthase gene (pA 0CS ).
  • PIT is a fusion protein of the Streptomyces coelicolor Pip protein and the NP16 transactivation domain of the Herpes simplex virus.
  • Figure 10B Graphical representation of the PIPpOFF expression vectors pMF279/281.
  • a nucleic acid which is hybridizable to a PIP nucleic acid under conditions of moderate stringency is provided.
  • procedures using such conditions of moderate stringency are as follows: Filters containing
  • a nucleic acid which is hybridizable to a PIP nucleic acid under conditions of high stringency is provided.
  • procedures using such conditions of high stringency are as follows: Prehybridization of filters containing DNA is carried out for 8 h to overnight at 65 °C in buffer composed of 6X SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PNP, 0.02% Ficoll, 0.02% BSA, and
  • New PIP proteins can also be isolated by binding to P ptr sequences.
  • the polynucleotide with the P ptr sequence is immobilized on a matrix and ideally packed in a column.
  • Bacterial or eukaryotic extracts are applied to the column under conditions which allow PIP homologs to bind to the immobilized target sequence.
  • the PIP homolog is eluted by suitable conditions (e.g., addition of antibiotic) and the sequence of the purified protein determined and the corresponding gene cloned.
  • PIP proteins of the invention include polypeptides that bind to P ptr sequences, as well as fusion proteins containing a first polypeptide that binds to P ptr sequences operatively linked to a second polypeptide which activates or represses transcription in eukaryotic cells.
  • operatively linked means that the two proteins are covalently or non-covalently bound to one another in such a manner that they retain their functional activities of binding to
  • repression of transcription is meant that the rate of transcriptional initiation is decreased from the nucleotide sequence to be transcribed that is operatively linked to a P ptr sequence when the fusion protein that represses transcription is bound to the P ptr sequence, as opposed to when it it not bound.
  • Acidic transcription activation domains proline-rich transcription activation domains, serine/threonine-rich transcription activation domains and glutamine-rich transcription ⁇ activation domains can all be used in the compositions and methods of the invention.
  • NP16 polypeptides and amino acid residues 753-881 of GAL4 are acidic activating domains.
  • Another polypeptide that activates transcription is the p65 domain of ⁇ F- ⁇ B (Schmitz and Baeuerle, 1991, EMBO J. 10:3805-3817).
  • proline-rich activation domains 5 include amino acid residues 399-499 of CTF/NFl and amino acid residues 31-76 of AP2.
  • a PIP of the invention can be fused to a polypeptide domain (e.g., a dimerization domain) capable of mediating a protein-protein interaction with a transcriptional activator protein, such as an endogenous activator present in a host cell.
  • a polypeptide domain e.g., a dimerization domain
  • transcriptional activator protein such as an endogenous activator present in a host cell.
  • Non-covalent interactions between DNA binding domains and transactivation domains are known in the art
  • the fusion protein that binds P ptr can be used to repress transcription.
  • the first polypeptide is operatively linked, as described above, to
  • Proteins and polypeptide domains within proteins which can function to repress transcription in eukaryotic cells have been described in the art (for reviews see, e.g., Renkawitz, R., 1990,
  • a polypeptide that represses transcription in eukaryotic cells is intended to include polypeptides which act either directly or indirectly to repress transcription.
  • "repression" of transcription is intended to mean a diminution in the level or amount of transcription of a target gene compared to the level or amount of transcription prior to regulation by the transcriptional inhibitor protein. Transcriptional inhibition may be partial or complete.
  • a transcriptional "repressor” or “silencer” domain as described herein is a polypeptide domain that retains its ability to repress transcription when the domain is transferred to a heterologous protein. Proteins which have been demonstrated to have repressor domains that can function when transferred to a heterologous protein include the v-erbA oncogene product
  • proteins which have transcri •pti •onal repressor activity in eukaryotic cells include the Drosophila homeodomain protein even-skipped (eve) (Han and Manley,
  • SF1 see Targa, et al, 1992, Biochem. Biophys. Res. Comm.. 188:416-423
  • Drosophila hunchback protein see Zhang, et al, 1992, Proc. Natl. Acad. Sci. USA 89:7511-7515
  • Drosophila knirps protein see Gerwin, et al, 1994, Mol. Cell. Biol.
  • Non-limiting examples of polypeptide domains that can be used as silencing domains include: amino acid residues 120-410 of the thyroid hormone receptor alpha (THRalpha.), amino acid residues 143-403 of the retinoic acid receptor alpha (RARalpha.), amino acid 5 residues 186-232 of knirps, the N-terminal region of WT 1 (see Anant, supra), the N-terminal region of Oct-2.1 (see Lillycrop, supra), a 65 amino acid domain of E4BP4 (see Cowell and Hurst, supra) and the N-terminal zinc finger domain of ZF5 (see Numoto, supra). Moreover, shorter or longer polypeptide fragments encompassing these regions that still retain full or partial repression activity are also contemplated.
  • novel transcriptional repressor domains which can be identified by standard techniques (e.g., reporter gene constructs), are within the scope of the invention.
  • Construction of the nucleic acids of the invention can be accomplished by those of the invention
  • the host cells for use in the methods and compositions of the invention can also be any eukaryotic cell such as mammalian cells, fungal cells, plant cells and microbial parasites.
  • Suitable host cells include, for example, mammalian cells such as CHO-Kl, BHK-21, HeLa , COS-7, HEK 293, HT1080, PC12, MDCK, C2C12, Jurkat,
  • NIH3T3, K-562, TF-1, P19 or plant cells such as those derived from barley, wheat, rice, soybean, potatoe, tobacco (e.g. Nicotiana tabacum SRI) and arabidopsis.
  • Suitable hosts also include plant-derived hairy roots such as those derived from Artemisia, Atropa, Beta, Brugmansia and others such as those described in Shanks and Morgan, 1999, Curr. Opin.
  • Suitable cell lines of mammalian and plant origin are well known to those of skill in the art and include, for example, those described in ATCC Cell Lines and Hybridomas 8 th Edition, 1994, American Type Culture Collection, Rockville, MD.
  • Methods of genetically engineering a host cell to contain the nucleic acids of the 5 invention are well known to those of skill in the art and include transformation, transfection, and electroporation.
  • the nucleic acids can be carried extracliromasomally or on the chromosome. Integration can be random, homologous, or site-specific recombination.
  • mice 20 rabbits, transgenic cattle, transgenic goats, and other transgenic animal species, particularly mammalian species, known in the art. Additionally, bovine, ovine, and porcine species, other members of the rodent family, e.g. rat, as well as rabbit and guinea pig, and non-human primates, such as chimpanzee, may be used to practice the present invention. Particularly 2 preferred animals are rats, rabbits, guinea pigs, and most preferably mice.
  • the "transgenic non-human animals" of the invention are produced by introducing transgenes into the germline of the non-human animal.
  • Embryonal target cells at various developmental stages can be used to introduce transgenes.
  • One common method is to incubate embryos in vitro for about 1-7 days, depending on the species, and then reimplant them into a surrogate host.
  • the progeny of transgenically manipulated embryos can be tested for the presence of 5 the construct by any means known to those of skill in the art including, for example, Southern blot analysis or PCR analysis of a segment of tissue. If one or more copies of the exogenous cloned construct is stably integrated into the genome of such transgenic embryos, it is possible to establish permanent transgenic mammallian lines carrying the transgenically added construct.
  • the exogenous genetic material is preferentially inserted into the nucleic genetic material by micromjection. Micromjection of cells and cellular structures is known and is used in the art. Reimplantation is accomplished using standard methods. Usually, the surrogate host 5 is anesthetized, and the embryos are inserted into the oviduct. The number of embryos implanted into a particular host will vary by species, but will usually be comparable to the number of off spring the species naturally produces.
  • Transgenic offspring of the surrogate host may be screened for the presence and/or expression of the transgene by any suitable method. Screening is often accomplished by Southern blot or Northern blot analysis, using a probe that is complementary to at least a portion of the transgene. Western blot analysis using an antibody against the protein encoded by the transgene may be employed as an alternative or additional method for screening for the presence of the transgene product.
  • DNA is prepared from tail tissue and analyzed 25 by Southern analysis or PCR for the transgene.
  • the tissues or cells believed to express the transgene at the highest levels are tested for the presence and expression of the transgene using Southern analysis or PCR, although any tissues or cell types may be used for this analysis.
  • suitable biochemical assays such as enzyme and/or immunological assays, histological stains for particular marker or enzyme activities, flow cytometric analysis, and the like.
  • Analysis of the blood may also be useful to detect the presence of the transgene product in the blood, as well as to evaluate the effect of the transgene on the levels of various
  • Progeny of the transgenic animals may be obtained by mating the transgenic animal with a suitable partner, or by in vitro fertilization of eggs and/or sperm obtained from the transgenic animal. Where mating with a partner is to be performed, the partner may or may not be transgenic and/or a knockout; where it is transgenic, it may contain the same or a
  • the partner may be a parental line.
  • the fertilized embryo may be implanted into a surrogate host or incubated in vitro, or both.
  • the progeny may be evaluated for the presence of the transgene using methods described above, or other appropriate methods.
  • Suitable mammalian expression vectors include pSG5, pCMV-Script, pcDNA3.1, pcDNA4 series, pEFl, pBK-CMV, pBK-RSV, pSBC-1, pSBC-2.
  • Suitable viral vectors include adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors and other viral vectors known to those of skill in the art.
  • Suitable retroviral and/or lentiviral vectors include, for example, pLAPSN, pLHCX, pLIB, pLNCX2, pLNHX, pLBCX, pLXRN, pLXSN, pMSCVneo, pSIR and lentiviral vectors such as those described by Naldini and Nerma, 1999, 47-60 (and references therein) in Friedman (ed.) The development of human gene therapy. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).
  • Suitable plant expression vectors include, for example, pBI, pRT99, pGPTV series, pGSN4, pBI 121, pBI221, pBHOl, pKINI105, pBI ⁇ 19, pPZP121, and the pCAMBIA family). Additional suitable vectors will be apparent to those of skill in the art and inlcude, for example, those described in ATCC Catalogue of Recombinant DNA Materials 3 rd Edition, 1993, American Type Culture Collection, Rockville, MD.
  • a P ptr -linked gene is defined herein as a promoter which directs the expression of a coding sequence, wherein the promoter and coding sequence are operatively linked to a P ptr sequence.
  • the promoter is a eukaryotic promoter.
  • operatively linked in this context is meant that the P ptr sequence is placed proximal to the promoter, either 5' to, or 3' to, or within the sequence of the promoter, such that when a fusion protein that modulates transcription is bound to the P ptr sequence, initiation of transcription at the promoter is affected.
  • the coding sequence operatively linked to the promoter can encode for any gene product for which regulated expression is desired, and can be exogenous to the host cell or endogenous.
  • endogenous coding sequence is meant coding sequence that is naturally present in the host cell and not introduced into the host cell via transformation techniques.
  • Exogenous coding sequence is not endogenous coding sequence. Coding sequences include not only sequences encoding proteins, but also other coding sequences, e.g., encoding antisense gene products, ribozymes, etc. Further, the coding sequences can be multicistronic (see, for example, U.S. Application No. 08/948,381, filed October 9, 1997).
  • Production of any gene product can be regulated using the compositions and methods of the present invention.
  • production of a marker gene product such as green fluorescent protein
  • production of a model secreted gene product such as SEAP
  • the invention finds particular use in the production of products of industrial or pharmaceutical interest such as industrial enzymes (e.g. proteases, cellulases, glycosidases, or ligninases), interferons (e.g.
  • tumor suppressor gene products to regulate proliferation of the host cells of the invention.
  • Regulated expression of tumor suppressor gene products are particularly useful for a variety of applications. For example, one may want the host cells to undergo a rapid proliferation phase followed by a production phase where cellular energies are devoted to protein production, or a rapid proliferation phase in vitro followed by regulated growth in vivo (see, for example, U.S. Application No. 08/948,381, filed October 9, 1997, the disclosure of which is incorporated by reference).
  • tumor suppressor gene products are intracellular proteins that block the cell cycle at a cell cycle checkpoint by interaction with cyclins, Cdks or cyclin-Cdk complexes, or by induction of proteins that do so.
  • tumor suppressor gene products inhibit the cyclin-dependent progression of the cell cycle.
  • Particularly preferred tumor suppressor gene products act on the Gl-S transition of the cell cycle.
  • the invention encompasses the use of any tumor suppressor gene product which performs this function, whether known or yet to be discovered.
  • tumor suppressor genes include p21, p27, p53 (and particularly, the p53175P mutant allele), p57, pl5, pl6, pl8, pl9, p73,
  • survival factors are intracellular proteins that prevent apoptosis such as bcl-2, bcl-x L , E1B-19K, mcl-1, crmA, abl, p35, bag-1, A20, LMP-1, Tax,
  • the tumor suppressor gene(s) is expressed concomitantly with a factor that stabilizes the tumor suppressor gene product in the cell.
  • stabilizing factors are members of the CAAT enhancer binding protein family.
  • p21 protein activity is stabilized when coexpressed with C/EBP ⁇ .
  • C/EBP ⁇ specifically induces transcription of the endogenous p21 gene.
  • C/EBP ⁇ functions as both a stabilizing factor and as a specific inducer of p21.
  • cyclins e.g., cyclin E
  • Ras Ras
  • Raf the MAP kinase family
  • Src Jak, Jun, Fos, pRB, Mek2, EGF, TGF, PDGF
  • a polynucleotide that is antisense to a tumor suppressor gene e.g., p27 anti-sense expression has been shown to stimulate proliferation of quiescent fibroblasts and enable growth in serum-free medium (Rivard et al , 1996, J. Biol. Chem. 271: 18337-18341.) and nedd5 which is known as positive growth controlling gene (Kinoshita et al , 1997, Genes Dev. 25 11: 1535-1547).
  • this aspect of the invention is illustrated by way of a non-limiting example using the combined installation of tetracycline-dependent and pristinamycin-responsive gene regulation in the same cells.
  • a double regulation vector pDuoRexl was constructed which contains both tetracycline-dependent and
  • Still another aspect of the invention are multipurpose expression vectors, as well as
  • the method entails incubating the host cells of the invention, the host cells containing a P ptr -linked reporter gene and a sequence encoding a P ptr -binding protein, in the presence of a test compound, wherein a change in the transcription of the reporter gene indicates that the test compound is a candidate antibiotic.
  • ⁇ _ For example, detection of streptogramins is based on addition of metabolic libraries of
  • pMF206 contains the following PIT2 coding sequence:
  • plasmid pMF153 was amplified by PCR using oligos OMF62:CTTGTACGGTGTACGAGTATCTTCCCGTACACCGTACAAGGAGCCTGCAGGgagta ccctcgaccgccg (SEQ ID NO:5) and OMF57: GATCCATCGATTGATCAGGCGC (SEQ ID NO: 6) and cloned in anti-Z ⁇ cZ orientation into the pCR2.1-TOPO vector (Invitrogen) to give plasmid pMF153.
  • the premature P PIR sequence contained in pMF153 was subjected to a second round of PCR using primers OMF69:
  • the P PIR -containing fragment was excised from pMF161 by Aatll andEcoRI and ligated to the corresponding sites (AatTUEcoRI) of the GFP expression vector pMF104, thereby replacing the T ⁇ T-responsive promoter P hCM v * - ⁇
  • pTWIN contains both PIT and tTA in a P sv40 -driven dicistronic configuration.
  • PIT was first transferred from pMF156 as an EcoRI/HindlU fragment to the corresponding sites of pSBC-1 (Ecd UHind ⁇ l; resulting in pMF167) before the PIT expression unit could be fused to the tTA expression unit contained on pSBC2-tTA (Fussenegger et al, 1997, supra) via SspllNotl sites.
  • pTWIN contains the dicistronic Psv 4 o-PIT-IRES-tTA-pA expression cassette.
  • Transient transfected cells were routinely analyzed after 48 h for GFP or SEAP expression using fluorescence microscopy and p-nitrophenolphosphate-based light absorbance timecourse, respectively, as described before (Fussenegger et al, 1998, Nat. Biotechnol. 16, 468-472;
  • CHO-PIT 1 -SEAP was constructed by cotransfection of pMF172 and pZeoSN2 (Invitrogen).
  • CHO-PITl-SEAP were cultured at cell densities of 150,000/ml for 48 at various PI concentrations.
  • the mixed populations were cloned using FACS-mediated single- cell-sorting (FACStar* 11115 ; Beckton Dickinson). Gel retardation assay were performed as described (Salah-Bey et al, 1995, supra) using Pip purified from an overproducing strain of E. coli. Pip (0.02pmol) was titrated with a range of antibiotic concentrations in a reaction volume of 20 ⁇ l. Protein solutions were pre-incubated at room temperature for 15 min. in the absence or presence of PI or its derivative quinupristin in a buffer containing 10 mM Tris (pH 7.8), 10 mM MgCl 2 , 300mM ⁇ acl, 2mM DTT and 10% glycerol.
  • PyostacinTM pills 500 mg were ground in a mortar and solubilized in DMSO or water at a stock concentration of 50 mg/ml.
  • SynercidTM (RP 59500; Lot Nr. CB06253) was provided by Aventis in injection-ready vials containing 500 mg lyophilized antibiotic which was reconstituted in 5 ml of 5% glucose solution and frozen at -
  • Antibiotic discs containing 15 ⁇ g pristinamycin or virginiamycin (bioMerieux, ref.
  • Nirginiamycin consists of virginiamycin Sj (VS_), the PI analogue, and virginiamycin M_, which is identical in structure to PII (Barri re et al, 1994, supra). 2 ⁇ g/ml of streptogramin B was routinely used for regulation studies in cell culture. The concentration of individual streptogramin components was calculated based on their fixed 70:30 ratio (w/w;
  • PII/NM dalfopristin PI/NS/quinupristin
  • Table 2 Regulation potential of pristinamycin (Pyostacin 0 ) and the pristinamycin group B (PI) and group A (PII) compounds.
  • CHO-Kl cells were simultaneously transfected with pMF156 (PIT) and pMF172 (P prR -SEAP) in the presence or absence of the inducer indicated.
  • Reporter gene expression (SEAP) was assayed 48 hr later.
  • pMF206 (PIT2; Pip-p65) was cotransfected withpMF172 (P PIR -SEAP) and the SEAP readout in the absence and presence of PI (2 ⁇ g/ml) was directly compared to CHO-Kl cells cotransfected with pMF156 (PIT; Pip-NP16) and pMF172 (P PIR -SEAP).
  • PIT2 mediates higher overall SEAP expression levels compared to the pMF156/pMF172 configuration in the absence of pristinamycin but basal expression levels in the presence of pristinamycin remain significantly higher.
  • CHO-PIT 1 and CHO-PIT2 Two representative clones, CHO-PIT 1 and CHO-PIT2, were chosen at random among 11 PIT-expressing CHO cell clones stably transfected with a constitutive PIT expression construct (pMF156). Both cell lines show no unusual cell morphologies and display similar growth behavior compared to wild-type CHO-Kl cells, indicating that sustained constitutive PIT expression does not have obvious deleterious physiological effects on CHO cells.
  • This characteristic is necessary to achieve fluctuating daily dosing regimes optimal for many therapeutic proteins such as insulin.
  • SEAP production values are normalized by SEAP activities under the antibiotic-free, active promoter conditions as follows: pMF172 in CHO- TWIN1 95 : 1.7mU/ml; in CHO-TWTNl 108 : 1.8mU/ml. pMFl ll in CHO-TWTNl 95 : 18.6mU/ml; in CHO-TWIN1 108 : 19.2mU/ml.
  • the PI and the TET systems can be used for combined therapeutic applications requiring different control modalities for different transgenes since they are compatible and regulated by different therapeutic antibiotics for which large sets of clinical and
  • OMF92 GATCGATATCACTAGTCGCCACCatgcccaagaagaagcc (S ⁇ Q ID ⁇ O:10) and OMF93: GATCGGATCCACGCGTtcagatgctggcagcgtg (S ⁇ Q ID NO:ll) ⁇ and the 1.3 kb fragment was cloned into pcDNA3.1 /V5/His-TOPO under control of the P CMN promoter to give pMF192.
  • MeklDD other genetic determinants exerting positive control of mammalian cell cycle such as cyclins (e.g. cyclin E) or E2F could also be envisioned.
  • cyclins e.g. cyclin E
  • double regulation vectors analogous to pMF195 (which contained p27-MeklDD) were constructed in which the MeklDD gene coding sequence was replaced by sequences encoding the adenoviral large T antigen, the adenoviral small T antigen, and the human papillomavirus E7 protein (all under control of the P PIR promoter).
  • pMF195 which contained p27-MeklDD
  • pMF195 which contained p27-MeklDD
  • the MeklDD gene coding sequence was replaced by sequences encoding the adenoviral large T antigen, the adenoviral small T antigen, and the human papillomavirus E7 protein (all under control of the P PIR promoter
  • PIT4 which is simply Pip expressed in a eukaryotic configuration, bind to PIR3 in front of P sv40 and block transcription of this promoter. Besides sterical transcription blocking the silencing domain of PIT3 can additionally downregulate P SV40 activity.
  • the P PIR promoter was excised from pMF164 by SspVEc ⁇ RI and the P PIR ON promoter was excised from pMF222 by SspVEcoRI.
  • P pP i R! and P pPIR2 respectively (PIR3-TATA-box; Figure 10B).
  • the P pPIR -driven GUS expression unit harbors a polyadenylation signal (pA 35S ) derived from the cauliflower 35S gene (Vancanneyt et al., 1990, Mol. Gen. Genet. 220: 245-250).
  • PIPpON pristinamycin-inducible plant gene regulation systems
  • pMF310 was restricted with EcoRUBglR and the PIT-IRES-rtTA-CITE*-zeo cassette was inserted into the retroviral vector pMSCVneo cut with EcoRUBamHl which resulted in pMF312 (5'LTR- ⁇ + -PIT-mES-rtTA-CITE*-zeo-3'LTR) ( Figure 14).
  • Replication-incompetent pMF311 - and pMF312-derived refroviruses were produced using the panfropic retroviral packaging system (GP-293; Clontech).

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Abstract

L'invention concerne un nouveau système destiné à la régulation des gènes dans des cellules eucaryotes, ainsi que des procédés d'utilisation de celui-ci dans la production de protéines, dans le génie tissulaire et dans la thérapie génique. L'invention concerne, notamment, un nouveau système destiné à une expression génique régulée par antibiotique dans des cellules eucaryotes en fonction des séquences issues de promoteurs de résistance aux antibiotiques Actinomycètes, de polypeptides se liant à ceux-ci d'une manière sensible aux antibiotiques, et des nucléotides codant de tels polypeptides. De plus, l'invention concerne des nouveaux procédés sensibles de criblage d'antibiotiques candidats.
PCT/IB2001/001963 2000-10-20 2001-10-19 Systeme de regulation des genes a base d'antibiotique WO2002033104A2 (fr)

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WO2002033104A3 WO2002033104A3 (fr) 2003-10-02

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017134137A1 (fr) * 2016-02-05 2017-08-10 Polygene Ag Système de régulation de gènes à base de glucocorticoïdes

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000065080A1 (fr) * 1999-04-23 2000-11-02 Fussenegger, Martin Systeme de regulation genique par antibiotiques

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000065080A1 (fr) * 1999-04-23 2000-11-02 Fussenegger, Martin Systeme de regulation genique par antibiotiques

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
AUGUST P R ET AL: "BIOSYNTHESIS OF THE ANSAMYCIN ANTIBIOTIC RIFAMYCIN: DEDUCTIONS FROM THE MOLECULAR ANALYSIS OF THE RIF BIOSYNTHETIC GENE CLUSTER OF AMYCOLATOPSIS MEDITERRANEI S699" CHEMISTRY AND BIOLOGY, CURRENT BIOLOGY, LONDON, GB, vol. 5, no. 2, 13 February 1998 (1998-02-13), pages 69-79, XP001084972 ISSN: 1074-5521 *
BLANC V ET AL: "Molecular characterization and transcriptional analysis of a multdrug resistance gene cloned from the pristinamycin-producing organism, Streptomyces pristinaespiralis" MOLECULAR MICROBIOLOGY, BLACKWELL SCIENTIFIC, OXFORD, GB, vol. 17, no. 5, 1995, pages 989-999, XP008009341 ISSN: 0950-382X cited in the application *
FOLCHER MARC ET AL: "A transcriptional regulator of a pristinamycin resistance gene in Streptomyces coelicolor." JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 2, 12 January 2001 (2001-01-12), pages 1479-1485, XP002184465 ISSN: 0021-9258 *
KIM C-G ET AL: "3-amino-5-hydroxybenzoic acid synthase, the terminal enzyme in the formation of the precursor of mC7N units in rifamycin and related antibiotics" JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY OF BIOLOGICAL CHEMISTS, BALTIMORE, MD, US, vol. 273, no. 11, 13 March 1998 (1998-03-13), pages 6030-6040, XP002145258 ISSN: 0021-9258 *
SALAH-BEY K ET AL: "STRESS-ACTIVATED EXPRESSION OF A STREPTOMYCES PRISTINAESPIRALIS MULTIDRUG RESISTANCE GENE (PTR) IN VARIOUS STREPTOMYCES SPP. AND ESCHERICHIA COLI" MOLECULAR MICROBIOLOGY, BLACKWELL SCIENTIFIC, OXFORD, GB, vol. 17, no. 5, 1995, pages 1001-1012, XP008009342 ISSN: 0950-382X *
SALAH-BEY K ET AL: "UNUSUAL REGULATORY MECHANISM FOR A STREPTOMYCES MULTIDRUG RESISTANCE GENE, PTR, INVOLVING THREE HOMOLOGOUS PROTEIN-BINDING SITES OVERLAPPING THE PROMOTER REGION" MOLECULAR MICROBIOLOGY, BLACKWELL SCIENTIFIC, OXFORD, GB, vol. 17, no. 6, 1995, pages 1109-1119, XP008009340 ISSN: 0950-382X cited in the application *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017134137A1 (fr) * 2016-02-05 2017-08-10 Polygene Ag Système de régulation de gènes à base de glucocorticoïdes

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