WO2003002726A2 - Systeme de detection dependant d'une boucle d'amplification - Google Patents

Systeme de detection dependant d'une boucle d'amplification Download PDF

Info

Publication number
WO2003002726A2
WO2003002726A2 PCT/EP2002/007260 EP0207260W WO03002726A2 WO 2003002726 A2 WO2003002726 A2 WO 2003002726A2 EP 0207260 W EP0207260 W EP 0207260W WO 03002726 A2 WO03002726 A2 WO 03002726A2
Authority
WO
WIPO (PCT)
Prior art keywords
ligand
receptor
lif
promoter
cells
Prior art date
Application number
PCT/EP2002/007260
Other languages
English (en)
Other versions
WO2003002726A3 (fr
Inventor
Xaveer Van Ostade
Peter Ulrichts
Joël VANDEKERCKHOVE
Jan Tavernier
Original Assignee
Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw filed Critical Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
Priority to AU2002317012A priority Critical patent/AU2002317012A1/en
Publication of WO2003002726A2 publication Critical patent/WO2003002726A2/fr
Publication of WO2003002726A3 publication Critical patent/WO2003002726A3/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a loop dependent detection system for ligands, preferably cytokine receptor ligands.
  • the presence of the ligand induces the loop; the loop acts as an amplifier so that minute quantities of the ligand can be detected and/or measured in a quantitative way.
  • the system allows detecting and/or measuring of amounts of ligand that cannot be detected and/or measured in another way.
  • ELISA ELISA
  • FACS cell
  • bioassay bioassay
  • an immune answer can be characterized by a Th1 response (IL-2, IL-12, TNF and IFN ⁇ secretion) or Th2 response (IL-4, IL-5, IL-6 and IL-10 secretion) from which the cytokines are usually present in very low concentrations in primary cell cultures.
  • Th1 response IL-2, IL-12, TNF and IFN ⁇ secretion
  • Th2 response IL-4, IL-5, IL-6 and IL-10 secretion
  • the present invention combines the advantages of the bioassay to a very high sensitivity, by inserting an amplification loop between the signal, induced by the binding of the ligand to be detected to its receptor, and the reporter system.
  • the amplification loop essentially consists of a recombinant gene comprising a promoter, inducible by the signaling pathway of at least one of said receptors, whereby said signaling pathway is activated by binding of a specific ligand (called ligand A) to this receptor and whereby said promoter is operationally linked to a coding sequence encoding ligand A. Binding of the ligand to be detected activates the amplification loop, which on its turn activates the reporter system.
  • the amplification loop essentially consists of a recombinant gene comprising a promoter, inducible by the signaling pathway of a constitutively activated receptor, whereby said promoter is operationally linked to a coding sequence encoding said constitutively activated receptor.
  • It is a first aspect of the invention to provide an eukaryotic cell comprising a) one or more receptors binding a ligand A b) a recombinant gene comprising a promoter, inducible by the signaling pathway of at least one of said receptors, whereby said signaling pathway is activated by binding of ligand A to this receptor and whereby said promoter is operationally linked to a coding sequence encoding ligand A c) a reporter system, inducible by binding of ligand A to at least one of said receptors.
  • a reporter system can be any system that allows the detection and/or the selection of the cells carrying a receptor, binding ligand A, according to the invention. It is clear for the person skilled in the art that several reporter systems can be used.
  • a luciferase gene, an antibiotic resistance gene or a cell surface marker gene can be placed after a promoter that is induced by the signaling pathway.
  • the coding sequence of the reporter gene is preferentially operationally linked to a promoter that is induced upon binding of ligand A to at least one of the receptors.
  • said promoter may be induced in an indirect way too, e.g. by binding of a ligand B to its receptor, whereby the coding sequence of ligand B is operationally linked to a promoter inducible by binding of ligand A to at least one of the receptors (ligand B being a specific ligand, different from ligand A).
  • reporter systems may be used that are based on the change in characteristics of compounds of the signaling pathway, when said pathway is active, such as the phosphorylation and/or dimerisation of such compounds.
  • the receptors used may be any naturally occurring receptor or chimeric receptor, including, but not limited to nuclear receptors such as steroid receptors, G-coupled receptors and multimerizing receptors, such as cytokine receptors.
  • the use of a chimeric receptor has the advantage that the amplification loop may be initiated by the interaction of a ligand B to a chimeric receptor, comprising a ligand B binding domain and a signaling domain of the receptor for ligand A.
  • a preferred embodiment is an eukaryotic cell according to the invention whereby said receptor is a multimerizing receptor, preferably a cytokine receptor.
  • the amplification loop may be initiated by any extracellular or intracellular event that can induce the promoter inducible by binding of ligand A to at least one of the receptors, such as by the induction of the expression of a constitutively activated receptor activating said promoter, or by binding of a ligand B to a receptor, preferably a chimeric receptor, whereby said promoter is activated.
  • the eukaryotic cell according to the invention may be any eukaryotic cell.
  • said eukaryotic cell is a mammalian cell, more preferably it is a HEK293 cell.
  • Another aspect of the invention is an eukaryotic cell according to the invention, whereby the coding sequence encoding ligand A, and operationally linked to a promoter inducible by binding of ligand A to at least one of the receptors is placed downstream or upstream of an IRES sequence.
  • the amplification loop may switch on spontaneously, e.g. by promoter leakage, resulting in ligand A production and amplification.
  • a sequence encoding a conditional lethal gene product is operationally linked to a promoter that is induced upon binding of ligand A to at least one of the receptors, whereby said sequence is followed by an IRES sequence operationally linked to a coding sequence encoding ligand A.
  • ligand A is operationally linked to a promoter that is induced upon binding of ligand A to at least one of the receptors, whereby said sequence is followed by an IRES sequence operationally linked to a sequence encoding a conditional lethal gene product.
  • the product of the conditional lethal gene product is activated, resulting in the death of the cell with a leaking promoter.
  • Activation and/or inactivation of the conditional lethal gene may be an active proces, e.g. by modification of the conditional lethal gene product.
  • the activation or inactivation is switched on by adding a compound to the medium.
  • it may be an activity that can only exert its toxic effect in the presence of another compound.
  • thymidine kinase of the Herpes Simplex Virus can be used as conditional lethal gene; this is toxic in presence of gancyclovir and harmless when no gancyclovir is present in the medium.
  • Another aspect of the invention is a eukaryotic cell according to the invention whereby at least one of the receptors, binding ligand A, or a part of this receptor is encoded by an inducible gene.
  • a part of the receptor as used here, may be one chain of a heteromerising receptor (illustrated in figure 1 ).
  • the gene encoding the receptor is switched on, resulting in a functional receptor binding ligand A, and an induction of the amplification loop in presence of ligand A.
  • the amplification loop can be switched off if needed.
  • the inducible receptor is induced by adding a compound to the medium.
  • Still another aspect of the invention is the use of a eukaryotic cell according to the invention to detect a ligand and/or to measure the concentration of said ligand.
  • Said ligand may be, but is not necessarily ligand A.
  • the amplification loop may be switched on by binding of ligand B to the ligand binding domain of the chimeric receptor, whereby said receptor is inducing a promoter that is normally switched on by binding of ligand A to its receptor.
  • ligand B is ligand binding domain of the chimeric receptor
  • said receptor is inducing a promoter that is normally switched on by binding of ligand A to its receptor.
  • ligand A is ligand A.
  • said ligand is a cytokine.
  • Receptor as used here does not necessarily indicate a single polypeptide, but may indicate a receptor complex, consisting of two or more polypeptides.
  • Recombinant receptor means that at least one of said polypeptides is recombinant.
  • a chimeric receptor is a recombinant receptor whereby the recombinant polypeptide comprises domains that are derived from at least two receptors.
  • Polypeptide as used here means any proteineous structure, independent of the length and includes molecules such as peptides, phosphorylated proteins and glycosylated proteins. Polypeptide as used herein is not necessarily indicating an independent compound but can also be used to indicate a part of a bigger compound, such as a domain of a protein.
  • Ligand means every compound that can bind to the ligand-binding domain of a receptor and that is able to initiate the signaling pathway by binding to said ligand binding domain, or every compound that can mimic this effect.
  • Ligand A and ligand B can be any ligand; however, in one experimental set up all ligands A are identical to each other, and all ligands B are identical to each other, but ligand A is different from ligand B.
  • Signaling pathway as used here does not imply that other compounds than ligand and receptor are involved: the signaling may be caused by translocation of the receptor, which directly induces the transcription of the target genes.
  • Initiating means starting the events that normally directly follow the binding of the ligand to the ligand-binding domain of a receptor, such as, as a non-limiting example, multimerization for a multimerizing receptor.
  • Compound means any chemical or biological compound, including simple or complex organic or inorganic molecules, peptides, peptido-mimetics, proteins, antibodies, carbohydrates, nucleic acids or derivatives thereof.
  • Bind(ing) means any interaction, be it direct or indirect.
  • a direct interaction implies a contact between the binding partners.
  • An indirect interaction means any interaction whereby the interaction partners interact in a complex of more than two compounds.
  • Operationally linked refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner.
  • a promoter sequence "operationally linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the promoter sequence.
  • An IRES sequence operationally linked to a coding sequence means that ribosome entry and initiation of translation for said coding sequence can start from the IRES sequence.
  • FIG. 1 Principle of the amplification loop, illustrated by the doxycyclin-induced cytokine cascade.
  • Cells stabile carrying the TRE2-IFNaR1/gp130 construct will only produce IFNaR1/gp130 after stimulation of the TRE2 promoter with doxycyclin, a process that acts via activation and inhibition of a doxycyclin-dependent transcriptional activator and repressor, respectively (not shown in the figure).
  • IFNaR1/gp130 associates with the endogenous IFNaR2 to form a functional receptor complex, capable of activating the rPAP promoter after IFN ⁇ stimulation.
  • Figure 2 A. Overview of vector cloning. Vectors from which fragments were transferred to the backbone of other vectors by means of PCR or restriction fragment exchange, are shown at the left of the converging arrows.
  • B_ Schematic representation of the final constructs pUTrPAP-TK-IRES-IFN ⁇ or pUTrPAP-TK-l RES-LI F.
  • FIG. 3 Triggering of the LIF (Leukemia Inhibiting Factor) cytokine cascade by a LIF concentration series after co-transfection of the cells with pXP2rPAP-luci.
  • LIF Leukemia Inhibiting Factor
  • Cells were transfected with pUTrPAP-TK-l RES-LI F (gray bars) or pUTrPAP-TK-IRES-IFN ⁇ (black bars) and in both cases co-transfected with pXP2rPAP-luci. The cells were subsequently trypsinized and seeded in 24-well microtiterplates, stimulated with a serial dilution of LIF as indicated and tested for luciferase activity.
  • LIF Leukemia Inhibiting Factor
  • FIG. 4 Triggering of the LIF cytokine cascade by a LIF concentration series in HEKrPAP-luci cells.
  • Cells, stably transfected with pXP2rPAP-luci (HEKrPAP-luci) were transfected with pUTrPAP-TK-l RES-LI F (gray bars) or pUTrPAP-TK-IRES-IFN ⁇ (black bars).
  • the cells were subsequently trypsinized and seeded in 24-well microtiterplates, stimulated with a serial dilution as indicated of LIF and tested for luciferase activity.
  • Figure 5 Triggering of the LIF cytokine cascade by limited LIF incubation times after co-transfection of the cells with pXP2rPAP-luci.
  • Cells were transfected with pUTrPAP- TK-IRES-LIF (gray bars) or pUTrPAP-TK-IRES-IFN ⁇ (black bars) and in both cases co-transfected with pXP2rPAP-luci.
  • the cells were subsequently trypsinized and seeded in 24-well microtiterplates, stimulated with a fixed LIF concentration (1 ng/ml) during different time periods (full bars) or left without LIF (hatched bars), washed, and finally tested for luciferase activity.
  • the plasmids pUTrPAP-TK-l RES-LI F and pUTrPAP-TK-IRES-IFN ⁇ were constructed. For this reason the constructs have inserted the TK (HSV) gene between the rPAP promoter and the cytokine cDNA sequence, allowing for selection of stabile transfected clones with a strictly regulated rPAP promoter.
  • TK HSV
  • These plasmids will in first instance be used in transient transfection experiments for demonstration of the principle of a cytokine amplification loop. The construction of these vectors is schematically shown in figure 2.
  • the resulting plasmid was digested with Xhol and Xbal and ligated in the Xhol-Xbal opened pXP2rPAP-luci vector such that the blasticidin resistance gene was operationally linked to the rPAP promoter.
  • the plasmid was named XP2-rPAP-blasti. After this, the gene for blasticidin resistance was replaced by the TK (HSV) cDNA.
  • a PCR fragment containing the gene for TK was made using oligo's MBU-O-976 and MBU-O-977 and plasmid pPHT-Hyg (BCCM, Belgium) as a template, thereby creating an EcoRI site at the 5' end and Xhol and Notl sites at the 3' end of the gene.
  • the resulting plasmid was digested with EcoRI and Xhol and ligated in the EcoRI-Xhol opened pXP2rPAP-blasti vector such that the TK (HSV) gene was operationally linked to the rPAP promoter.
  • the resulting construct was named pXP2-rPAP-TK.
  • the 6-16 promoter was digested from the plasmid p6-16luci (kindly provided by Dr. S. Pellegrini, Institut Pasteur, Paris) by a Hindlll digest, blunted by Klenow polymerase and ligated into the pCR-blunt vector.
  • a fragment containing the 6-16 promoter was isolated by a Spel and EcoRV digest and inserted upstream the gene for ⁇ -galactosidase in the Spel-Pmll opened pUT-651 vector (Eurogentec, Seraing), resulting in the plasmid pUT6-16- ⁇ gal.
  • TK cDNA sequence The gene for ⁇ -galactosidase was then replaced by the TK cDNA sequence as follows: the plasmid pPHT-Hygro (BCCM, Belgium) was digested with Hindlll, blunted with Klenow polymerase, digested with Hindi I and ligated in pCR-blunt. This plasmid was digested with Spel, blunted with Klenow, digested with Notl and ligated in the Hindll-Notl opened pUT6-16- ⁇ gal plasmid, resulting in the plasmid pUT6-16-TK.
  • pUT-rPAP-TK From the pXP2rPAP-TK plasmid, a fragment containing the rPAP promoter and a short 5' end of the TK cDNA sequence was isolated by digestion with Nhel, blunting the end with Klenow polymerase, and a subsequent digest with BsiWI. This fragment was then transferred to the Spel (also blunted with Klenow polymerase)-BsiWI opened pUT6-16- TK vector such that the gene for TK (HSV) was operationally linked to the rPAP promoter in the pUT backbone. The resulting plasmid was named pUTrPAP-TK.
  • the fragment was then inserted in the pUTrPAP-TK vector, which was first opened with Tth111 (located downstream the TK gene), blunted with Klenow polymerase, and subsequently digested with Notl.
  • Tth111 located downstream the TK gene
  • Klenow polymerase blunted with Klenow polymerase
  • Notl a blunt end-Notl ligation resulted in a plasmid that contained in 5' to 3' direction: the rPAP promoter, the TK gene, the IRES sequence and the gene for puromycin resistance with an Ncol site at its start codon (pUTrPAP-TK-IRES-puro).
  • the forward oligonucleotides MBU-O-1016 (LIF) and MBU-O-1018 (IFN ⁇ ) and reverse oligonucleotides MBU-O- 1017 (LIF) and MBU-O-1019 (IFN ⁇ ) were used.
  • the reactions were performed on cDNA of PMA-stimulated K562 cells for LIF and on the plasmid pATHIFN ⁇ g41 (BCCM, Belgium) for IFN ⁇ .
  • the fragments were cloned in the pCR-blunt vector and named pCR-blunt-LIF and pCR-blunt-IFN ⁇ , respectively.
  • LIF and IFN ⁇ cDNA containing fragments were isolated by an Ncol-Xbal digest.
  • the fragments were then ligated in an Ncol-Xbal opened pUTrPAP-TK-IRES-puro vector that was dephosphorylated by calf intestinal alkaline phosphatase (CIAP; Invitrogen-Life Sciences) because frequently problems arose as a result of intramolecular ligation of this vector.
  • the puromycin gene was exchanged by the cytokine cDNA sequences which were operationally linked to the rPAP-TK-lRES sequence.
  • the corresponding plasmids were named pUTrPAP- TK-IRES-LIF and pUTrPAP-TK-IRES-IFN ⁇ .
  • Hek 293-Flp-ln cells were transfected with pM5neo-mEcoR (kindly provided by Dr. S. Kinoshita, Stanford University, Stanford) according to the calciumphosphate DNA-precipitation technique.
  • This plasmid contained the gene for the ecotropic receptor for moloney murine leukemia virus.
  • the cells were selected in 500 ⁇ g/ml G418.
  • the pool of G418 resistant cells was co-transfected with the plasmids pXP2rPAP-luci and plRES-puro2 (Clontech) at a ratio of 4:1 , again according to the calciumphosphate DNA-precipitation technique.
  • HEKrPAP-luci The strongest inducible clone with a maximal signal/noise ratio was selected and named HEKrPAP-luci.
  • Example 1 Co-transfection with pXP2rPAP-luci and treatment of cells with a LIF concentration gradient.
  • HEK293 cells were transfected with pUTrPAP-TK-l RES-LI F.
  • activation of the rPAP promoter could lead to LIF production and endogenous LIF receptor activation, which on its turn could again activate the rPAP promoter.
  • the secreted LIF will stimulate and activate its own production in the same cell (autostimulation) as well as in surrounding cells (paracrine stimulation), resulting in a cascade of LIF production that ultimately exponentially amplifies the initial signal.
  • Co- transfection with the pXP2rPAP-luci construct or use of cells that have this plasmid stabile integrated allows for detection and quantification of the LIF cascade.
  • To trigger the LIF cascade we used an exogenous stimulus by LIF itself. The intensity of this initial trigger was varied by treating the cells with a LIF concentration gradient or with a fixed LIF concentration during different time periods (figure 3).
  • 6x10 6 HEK293 cells were transfected in a petridish with 18 ⁇ g pUTrPAP-TK-l RES-LI F + 6 ⁇ g rPAP-luci DNA.
  • pUTrPAP-TK-IRES-LIF was replaced by pUTrPAP-TK-IRES-IFN ⁇ .
  • 24 hrs. after transfection cells were trypsinized en transferred to a 24-well plate at a density of 3x10 5 cells/well. After another 24 hrs. incubation, a LIF concentration gradient ranging from 0 to 1 ng was added to the cells. 24 hrs. later, luciferase activity was determined in transfected cells by chemiluminescence.
  • lysis buffer 25 mM Tris, pH 7.8, with H 3 P0 4 ; 2 mM EDTA; 2 mM DTT; 10% glycerol; 1 % Triton X-100 from which 100 ⁇ l was transferred to a 96-well microtiterplate.
  • inducible promoters can be 'leaky' because of the lack of repression factors (e.g. histones, repression factors ...) that function only when the DNA is integrated in the genome. This may account for the high rPAP-luci induction seen in non-treated cells, transfected with rPAP-TK-l RES-LI F where small, background LIF levels may already trigger the LIF cascade.
  • repression factors e.g. histones, repression factors
  • the amount of luciferase, induced by a certain LIF concentration is substantially higher in the pUTrPAP-TK-IRES-LIF transfected cells as compared to the negative control, suggesting that in the first cells, rPAP activation is increased exponentially after triggering.
  • Example 2 transfection of stabile HEK rPAP-luci cells and treatment of the cells with a LIF concentration gradient.
  • rPAP-luci probably implies a lower copy number of this plasmid in the HEK rPAP-luci cells as compared to transient transfection. Revealing activation of the LIF cascade may therefore be less clear in cells containing a lower amount of rPAP- luci replicons.
  • the degree of rPAP activation induced by LIF is much higher in the pUTrPAP-TK-l RES-LI F transfected cells as compared to the negative control.
  • Example 3 co-transfection with pXP2rPAP-luci and treatment of cells with a fixed LIF concentration during different time periods
  • 6x10 6 HEK293 cells were transfected with 18 ⁇ g pUTrPAP-TK-l RES-LI F + 6 ⁇ g rPAP- luci DNA.
  • pUTrPAP-TK-l RES-LI F was replaced by pUTrPAP- TK-IRES-IFN ⁇ .
  • 24 hrs. after transfection cells were trypsinized en transferred to a 24- well plate at a density of 3x10 5 cells/well. After another 24 hrs. incubation, cells were stimulated with 1 ng/ml LIF during varying time periods (5 min. to 8 hrs.) after which the cells were washed twice with medium without LIF. 24hrs. after the onset of stimulation, luciferase activity was determined by chemiluminescence as described above. Results are shown in figure 5.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Genetics & Genomics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Toxicology (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Zoology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un système de détection de ligands dépendant d'une boucle. Les ligands seront de préférence des ligands de cytokine. La présence des ligands induit la boucle, la boucle agit comme un amplificateur, de manière à ce que des quantités minutées du ligand puissent être détectées et/ou mesurées de manière quantitative. Ce système permet de détecter et/ou de mesurer des quantités de ligand qui ne peuvent être détectées et/ou mesurées d'une autre manière.
PCT/EP2002/007260 2001-06-28 2002-06-27 Systeme de detection dependant d'une boucle d'amplification WO2003002726A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2002317012A AU2002317012A1 (en) 2001-06-28 2002-06-27 Cellular reporter gene assay with a ligand amplifying feedback loop

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP01202493.1 2001-06-28
EP01202493 2001-06-28

Publications (2)

Publication Number Publication Date
WO2003002726A2 true WO2003002726A2 (fr) 2003-01-09
WO2003002726A3 WO2003002726A3 (fr) 2003-03-20

Family

ID=8180553

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2002/007260 WO2003002726A2 (fr) 2001-06-28 2002-06-27 Systeme de detection dependant d'une boucle d'amplification

Country Status (2)

Country Link
AU (1) AU2002317012A1 (fr)
WO (1) WO2003002726A2 (fr)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998013513A2 (fr) * 1996-09-24 1998-04-02 Cadus Pharmaceutical Corporation Procedes et compositions pour identifier des modulateurs de recepteur
WO2000006722A1 (fr) * 1998-07-28 2000-02-10 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Clonage par interaction de genes a base de cellules eukaryiotes
WO2000031261A2 (fr) * 1998-11-25 2000-06-02 Cadus Pharmaceutical Corporation Methodes et compositions d'identification d'effecteurs de recepteurs

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998013513A2 (fr) * 1996-09-24 1998-04-02 Cadus Pharmaceutical Corporation Procedes et compositions pour identifier des modulateurs de recepteur
WO2000006722A1 (fr) * 1998-07-28 2000-02-10 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Clonage par interaction de genes a base de cellules eukaryiotes
WO2000031261A2 (fr) * 1998-11-25 2000-06-02 Cadus Pharmaceutical Corporation Methodes et compositions d'identification d'effecteurs de recepteurs

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
GANG G.X. ET AL: "Insulin regulation of beta-cell function involves a feedback loop on SECA gene expression, Ca2+ homeostasis, and insulin expression and secrectin" BIOCHEMISTRY, vol. 39, 2000, pages 14912-14919, XP002181278 *
STORZ P. ET AL: "A cellular reporter assay to monitor insulin receptor kinase activity based on STAT 5-dependent luciferase gene expression" ANALYTICAL BIOCHEMISTRY, vol. 276, 1999, pages 97-104, XP002181277 *
WEST-MAYS J.A. ET AL: "Competence for collagenase gene expresssion by tissue fibroblasts requires activation of an interleukin 1 alpha autocrine loop" PROC NATL ACAD SCI USA, vol. 92, July 1995 (1995-07), pages 6768-6772, XP002181279 *

Also Published As

Publication number Publication date
AU2002317012A1 (en) 2003-03-03
WO2003002726A3 (fr) 2003-03-20

Similar Documents

Publication Publication Date Title
Rossi et al. Recent advances in inducible gene expression systems
Moriggl et al. Deletion of the carboxyl-terminal transactivation domain of MGF-Stat5 results in sustained DNA binding and a dominant negative phenotype
Rossi et al. Tetracycline-regulatable factors with distinct dimerization domains allow reversible growth inhibition by p16
Crispino et al. Use of altered specificity mutants to probe a specific protein–protein interaction in differentiation: the GATA-1: FOG complex
Gingrich et al. Inducible gene expression in the nervous system of transgenic mice
JP4955904B2 (ja) 新規エクジソン受容体／無脊椎動物レチノイドｘ受容体−ベースの誘導性遺伝子発現システム
US9732392B2 (en) Modular sensor architecture for cell based biosensors
Truksa et al. Different regulatory elements are required for response of hepcidin to interleukin‐6 and bone morphogenetic proteins 4 and 9
Abruzzese et al. Ligand-dependent regulation of vascular endothelial growth factor and erythropoietin expression by a plasmid-based autoinducible GeneSwitch system
WO2012116621A1 (fr) Système d'expression génique à contrôle optique
EP1034287A1 (fr) Systeme et procede de regulation de l'expression genique
Rivera Controlling gene expression using synthetic ligands
Imhof et al. A regulatory network for the efficient control of transgene expression
Karzenowski et al. Inducible control of transgene expression with ecdysone receptor: gene switches with high sensitivity, robust expression, and reduced size
Van Craenenbroeck et al. Evaluation of the tetracycline‐and ecdysone‐inducible systems for expression of neurotransmitter receptors in mammalian cells
Yu et al. A Renilla luciferase-Aequorea GFP (ruc-gfp) fusion gene construct permits real-time detection of promoter activation by exogenously administered mifepristone in vivo
EP1498483A1 (fr) Lignees de cellules endocriniennes et procede d'utilisation desdites lignees
WO2003002726A2 (fr) Systeme de detection dependant d'une boucle d'amplification
Lorenz et al. Transcriptional repression mediated by the KRAB domain of the human C2H2 zinc finger protein Kox1/ZNF10 does not require histone deacetylation
Gould et al. Endogenous GATA factors bind the core sequence of the tetO and influence gene regulation with the tetracycline system
US7566685B2 (en) Methods and compositions for screening using diphtheria toxin constructs
JP2003513670A (ja) ジフテリア毒素構築体を用いるスクリーニングのための方法および組成物
Bohl et al. Transcriptional modulation of foreign gene expression in engineered somatic tissues
Konopka et al. hCMV and Tet promoters for inducible gene expression in rat neurons in vitro and in vivo
EP3293263A1 (fr) Expression de gènes inductibles par des fragments d'adn riches linéaires dans des cellules de mammifères et procédés associés

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A2

Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NO NZ OM PH PL PT RO RU SD SE SG SI SK SL TJ TM TN TR TT TZ UA UG US UZ VN YU ZA ZM ZW

AL Designated countries for regional patents

Kind code of ref document: A2

Designated state(s): GH GM KE LS MW MZ SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase in:

Ref country code: JP

WWW Wipo information: withdrawn in national office

Country of ref document: JP