WO1998036081A3 - Hybrid molecules for optically detecting changes in cellular microenvironments - Google Patents

Hybrid molecules for optically detecting changes in cellular microenvironments Download PDF

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Publication number
WO1998036081A3
WO1998036081A3 PCT/US1998/002774 US9802774W WO9836081A3 WO 1998036081 A3 WO1998036081 A3 WO 1998036081A3 US 9802774 W US9802774 W US 9802774W WO 9836081 A3 WO9836081 A3 WO 9836081A3
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WO
WIPO (PCT)
Prior art keywords
exocytosis
detecting changes
optically detecting
hybrid molecules
microenvironments
Prior art date
Application number
PCT/US1998/002774
Other languages
French (fr)
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WO1998036081A9 (en
WO1998036081A2 (en
Inventor
Gero Miesenbock
James E Rothman
Angelis Dino A De
Original Assignee
Sloan Kettering Inst Cancer
Gero Miesenbock
James E Rothman
Angelis Dino A De
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 Sloan Kettering Inst Cancer, Gero Miesenbock, James E Rothman, Angelis Dino A De filed Critical Sloan Kettering Inst Cancer
Priority to EP98905065A priority Critical patent/EP0981633A2/en
Priority to JP53592998A priority patent/JP2001512316A/en
Priority to CA002281648A priority patent/CA2281648A1/en
Publication of WO1998036081A2 publication Critical patent/WO1998036081A2/en
Publication of WO1998036081A3 publication Critical patent/WO1998036081A3/en
Publication of WO1998036081A9 publication Critical patent/WO1998036081A9/en
Priority to US10/676,428 priority patent/US7094888B2/en

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    • 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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • 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
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0069—Oxidoreductases (1.) acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0393—Animal model comprising a reporter system for screening tests
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/055—Fusion polypeptide containing a localisation/targetting motif containing a signal for localisation to secretory granules (for exocytosis)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Toxicology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Veterinary Medicine (AREA)
  • Cell Biology (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention relates to methods and compositions which utilize the emission of light to monitor changes in microenvironments involving cells. The invention is especially useful for monitoring exocytotic activity such as detecting quantal release of synaptic vesicles. Fusion proteins of Cypridina luciferase and synaptotagmin-I or VAMP/synaptobrevin-2 were targeted to synaptic vesicles and, upon exocytosis, formed light-emitting complexes with luciferin present in the extracellular medium. Photon emissions in the presence of a depolarizing stimulus can be observed with these systems. pH-sensitive mutants of green fluorescent protein are also provided, which are useful for visualizing exocytosis and for imaging and measuring the pH of intracellular compartments.
PCT/US1998/002774 1997-02-13 1998-02-13 Hybrid molecules for optically detecting changes in cellular microenvironments WO1998036081A2 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP98905065A EP0981633A2 (en) 1997-02-13 1998-02-13 Hybrid molecules for optically detecting changes in cellular microenvironments
JP53592998A JP2001512316A (en) 1997-02-13 1998-02-13 Mixed components that optically detect changes in the cellular microenvironment
CA002281648A CA2281648A1 (en) 1997-02-13 1998-02-13 Hybrid molecules for optically detecting changes in cellular microenvironments
US10/676,428 US7094888B2 (en) 1997-02-13 2003-09-30 Hybrid molecules and their use for optically detecting changes in cellular microenvironments

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US3817997P 1997-02-13 1997-02-13
US3680597P 1997-02-14 1997-02-14
US60/036,805 1997-02-14
US60/038,179 1997-02-14

Publications (3)

Publication Number Publication Date
WO1998036081A2 WO1998036081A2 (en) 1998-08-20
WO1998036081A3 true WO1998036081A3 (en) 1999-01-14
WO1998036081A9 WO1998036081A9 (en) 1999-02-18

Family

ID=26713523

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1998/002774 WO1998036081A2 (en) 1997-02-13 1998-02-13 Hybrid molecules for optically detecting changes in cellular microenvironments

Country Status (5)

Country Link
US (2) US6670449B1 (en)
EP (1) EP0981633A2 (en)
JP (1) JP2001512316A (en)
CA (1) CA2281648A1 (en)
WO (1) WO1998036081A2 (en)

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US6124128A (en) * 1996-08-16 2000-09-26 The Regents Of The University Of California Long wavelength engineered fluorescent proteins
US5976796A (en) * 1996-10-04 1999-11-02 Loma Linda University Construction and expression of renilla luciferase and green fluorescent protein fusion genes
DE19727962A1 (en) 1997-07-02 1999-01-14 Juergen Hescheler Fluorescent proteins as cell type-specific reporters
US6140132A (en) * 1998-06-09 2000-10-31 The Regents Of The University Of California Fluorescent protein sensors for measuring the pH of a biological sample
WO2000036106A2 (en) * 1998-12-17 2000-06-22 Xenogen Corporation Non-invasive evaluation of physiological response in a mammal
WO2001009177A2 (en) * 1999-07-29 2001-02-08 Whitehead Institute For Biomedical Research Affinity fluorescent proteins and uses thereof
WO2001018225A1 (en) * 1999-09-03 2001-03-15 Xenogen Corporation Targeting constructs and transgenic animals produced therewith
GB9927331D0 (en) 1999-11-18 2000-01-12 Fluorescience Ltd Assay for measuring enzyme activity in vivo
AUPQ422399A0 (en) * 1999-11-24 1999-12-16 University Of New South Wales, The Method of screening transformed or transfected cells
US8062874B2 (en) 2001-07-06 2011-11-22 The Board Of Trustees Of The University Of Illinois GFP fusion proteins and their use
CA2467084A1 (en) * 2001-12-26 2003-07-17 Clontech Laboratories, Inc. Mutant chromophores/fluorophores and methods for making and using the same
US7691394B2 (en) * 2002-05-28 2010-04-06 Botulinum Toxin Research Associates, Inc. High-potency botulinum toxin formulations
GB0314856D0 (en) 2003-06-25 2003-07-30 Unitargeting Res As Protein expression system
ES2265201B1 (en) * 2003-07-25 2008-01-01 Univ Miguel Hernandez NEW THERAPEUTIC TARGETS AND ITS USE FOR THE TREATMENT OF PAIN.
DK1740945T3 (en) 2004-04-07 2019-01-21 Ncardia Ag KKE-INVASIVE, IN-VITRO FUNCTIONAL TISSUE TEST SYSTEMS
EP1745144B1 (en) 2004-05-11 2010-12-01 Axiogenesis Ag Assay for drug discovery based on in vitro differentiated cells
GB2452457B (en) * 2006-06-12 2011-05-25 Nat Inst Of Advanced Ind Scien Mutant luciferase
WO2008074100A1 (en) * 2006-12-21 2008-06-26 Gene Stream Pty Ltd Bioluminescent assays utilising secreted luciferases
US8417011B2 (en) * 2008-09-18 2013-04-09 Molecular Devices (New Milton) Ltd. Colony detection
US10246492B2 (en) 2009-10-16 2019-04-02 Biomadison, Inc. Botulinum assay with synaptobrevin substrate moiety
ES2576747T3 (en) 2009-10-16 2016-07-11 Biomadison, Inc. Resonance energy transfer assay with a synaptobrevin substrate residue
US10517557B2 (en) * 2016-06-20 2019-12-31 General Electric Company Systems and methods for molecular breast imaging
GB201702500D0 (en) 2017-02-16 2017-04-05 Univ Sheffield Stable vamp reporter assay
CN113501881B (en) * 2017-09-27 2023-07-28 北京大学 Fusion proteins

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Publication number Publication date
US6670449B1 (en) 2003-12-30
EP0981633A2 (en) 2000-03-01
US20040137611A1 (en) 2004-07-15
CA2281648A1 (en) 1998-08-20
US7094888B2 (en) 2006-08-22
WO1998036081A2 (en) 1998-08-20
JP2001512316A (en) 2001-08-21

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