[go: up one dir, main page]

WO2013154429A1 - Dosages de rapporteur multiplexes pour la surveillance de multiples variables - Google Patents

Dosages de rapporteur multiplexes pour la surveillance de multiples variables Download PDF

Info

Publication number
WO2013154429A1
WO2013154429A1 PCT/NL2013/050264 NL2013050264W WO2013154429A1 WO 2013154429 A1 WO2013154429 A1 WO 2013154429A1 NL 2013050264 W NL2013050264 W NL 2013050264W WO 2013154429 A1 WO2013154429 A1 WO 2013154429A1
Authority
WO
WIPO (PCT)
Prior art keywords
reporter
cells
reporters
marker
gluctag
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/NL2013/050264
Other languages
English (en)
Inventor
Rolf Jonas NILSSON
Sjoerd VAN RIJN
Thomas WÜRDINGER
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Vrije Universiteit Amsterdam
Original Assignee
Stichting VU VUMC
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 Stichting VU VUMC filed Critical Stichting VU VUMC
Publication of WO2013154429A1 publication Critical patent/WO2013154429A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Definitions

  • the disclosure relates to multiplex assays for monitoring multiple variables, such as the effects of a biological condition on a biological system. Reporter systems are provided for performing such assays.
  • GFP green fluorescent protein
  • RFP red fluorescent protein
  • near infra-red fluorescent proteins 13 14 which can be multiplexed together to monitor several processes simultaneously using spectral unmixing in conjunction with fluorescence molecular tomography 15 .
  • the disclosure provides improved multiplex screening assays for the detection of multiple reporter signals.
  • the disclosure provides a method for monitoring an effect on a biological system comprising
  • each reporter comprises a marker linked to an epitope tag and each epitope tag is unique to a reporter
  • the biological system is one or more cells, more preferably the cells are tumor cells.
  • said methods further comprise distinguishing said reporter based on the epitope tag.
  • said reporters are provided to said biological system in vitro.
  • said biological system is implanted into an animal.
  • said reporter level is determined in a bodily fluid, preferably in the blood, of said animal.
  • the disclosure provides reporter systems. These reporter systems are ideally suited for use in the methods disclosed herein. Reporter systems are provided comprising at least two reporters,
  • each reporter comprises a marker, preferably a luminescent marker, linked to an epitope tag, and each epitope tag is unique to each reporter.
  • the marker from each reporter is the same.
  • the marker is secreted, preferably wherein the marker is Gaussia luciferase.
  • each reporter comprises a different transcription response element or a different promoter sequence.
  • each reporter comprises a different protease cleavage site.
  • each reporter comprises a different miRNA binding sequence or a different RNA splicing sequence.
  • the epitope tag is selected from Flag, His, HA, AcV5, V5, Glu, Myc, Kt3, Aul, and E2, more preferably wherein said epitope tag is selected from Flag, His, HA, AcV5, V5, and Glu.
  • the reporters are provided in one or more vectors.
  • nucleic acid encoding the reporter system disclosed herein is provided.
  • cells comprising said nucleic are provided.
  • FIG. 1 Gluctag multiplex assay development and validation in vitro, (a) Schematic of lentiviral vector constructs encoding the luciferase reporters, (b) Fluorescence microscopy analysis of a representative U87-FM-Gluctag-CFP cells (using GlucFiag reporter) showing mCherry levels (in red) and CFP expression (in blue; marker for transduction efficiency), (c) Immunostaining against the various tags in U87-FM cells expressing different Gluctag. (d) Glue activity in U87 cells expressing different Gluctag with respect to cell
  • the dashed line represents Glucctri activity, (e-f) Gluctag immunobinding assay versus total Glue activity using serial dilutions of the conditioned medium from one day culture of either individual U87-FM cells expressing a single Gluctag or a mixture of cells expressing all ten different Gluctag (f).
  • (g) Immunostaining for the ten different tags expression in the mixed population of U87-FM cell culture. Size bar (in b,c and g) 100 ⁇ .
  • FIG. 4 GlucTag cloning strategy.
  • the Glue gene is amplified from the parental vector using primers that amplify Glue without the tata-box and adding an Xbal and Xhol restriction site downstream.
  • This modified Glue gene then replaces the original Glue gene in the parental vector to construct the Glucmodified vector. Then, the vector is restricted with Xbal and Xhol to insert an epitope tag of choice.
  • This GlucTag is the final construct.
  • the present invention is based on a reporter system that allows monitoring of multiple variables within a biological system.
  • the system is both sensitive and specific enough to permit real time monitoring of, e.g., mixed populations of tumor cells both in vitro and in vivo (see Figures 1 and 2).
  • the reporters described herein may be used in a variety of different
  • the present invention provides a reporter system comprising both a marker and an epitope tag.
  • each reporter has the same marker and the epitope tag is used to discriminate between reporters.
  • the present reporter system can be used for continuous reporter measurement both in vitro and in vivo. Accordingly, the present disclosure provides a reporter system comprising at least two reporters, wherein each reporter comprises a marker linked to an epitope tag and each epitope tag is unique to each reporter.
  • the reporter system comprises at least three, at least four, or at least five reporters, wherein each reporter comprises a marker linked to an epitope tag and each epitope tag is unique to each reporter.
  • Reporter systems comprising at least 10 different reporters can distinguish biologically relevant differences (see Figures 1 and 2).
  • a reporter refers to a protein comprising a detectable marker and an epitope tag.
  • Preferred markers of the reporter system include luminescent and colorimetric markers.
  • colorimetric labels include compounds which use a chromogenic substrate to produce a color, such as beta-galactosidase, beta- glucouronidase, horseradish peroxidase, and alkaline phosphatase.
  • luminescence refers to the emission of light by energy other than heat and includes bioluminescence, fluorescence, and phosphorescence.
  • Luminescent markers include, e.g., bioluminescent markers, fluorescent markers, and phosphorescent markers.
  • the luminescent marker is a peptide or protein molecule.
  • Suitable fluorescent protein markers include Y66H, Y66F, EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, ECFP, CyPet, Y66W, mKeima- Red, TagCFP, AmCyanl, mTFPl, S65A, Midoriishi Cyan, Wild Type GFP, S65C, TurboGFP, TagGFP, S65L, Emerald, S65T (Invitrogen), EGFP
  • the marker is a bioluminescent marker.
  • Bioluminescence refers to the emission of light from a living organism and is a common trait in deep-sea marine organisms. It also occurs, e.g., in arthropods, fungi, and
  • Bioluminescence is the product of a reaction catalyzed by an enzyme, i.e., luciferase. As used herein, bioluminescence also refers to the emission of light resulting from a reaction catalyzed by a luciferase.
  • Luciferases are proteins which react with a suitable substrate to produce light as one of the reaction products. Luciferases catalyze the oxygen oxidation of an organic molecule, i.e., a luciferin (such as aldehydes, benzothiazoles, imidazolopyrazines, tetrapyrroles and flavins). Luciferases that use
  • coelenterazine an imidazoloyrazine derivative
  • luciferases from the species Renilla, Gaussia, Metridia and Obelia.
  • the amount of light produced by a bioluminescent reaction can be measured and used to determine the presence of or amount of luciferase in a sample.
  • luciferase refers to a naturally occurring or mutant luciferase
  • the marker protein is secreted. This permits the detection of the reporter in, e.g., conditioned cell culture medium in vitro or in a bodily fluid such as blood.
  • Secretion of the marker protein can be achieved by using a naturally secreted marker protein or by engineering a signal peptide into the marker sequence.
  • a signal peptide is a sequence which is present as an N-terminal sequence on the precursor form of an extracellular protein. The function of the signal peptide is to allow the heterologous protein to be secreted to enter the endoplasmatic reticulum. The signal peptide is normally cleaved off in the course of this process.
  • the signal peptide may be heterologous or homologous to the host organism producing the protein.
  • Secreted markers include glucoamylase::green fluorescent protein fusion, (Gordon et al. Microbiology 2000 2:415-426), green fluorescent protein with ecdysteroid UDP-glucosyltransferase signal peptide (Laukkanen et al.,
  • the marker is Gaussia or Metridium luciferase.
  • the marker is Gaussia luciferase (Glue).
  • Glue is highly sensitive, naturally secreted, and can be detected in the conditioned medium of cells in culture as well as in the blood of mice ex vivo, allowing realtime monitoring of cellular processes 5 6 .
  • An exemplary sequence of Glue is provided herein.
  • Glue also includes variants which have been codon optimized for use in prokaryotes or eukaryotes (see, e.g., Wille et al. Applied and
  • Glue also includes variants which have been modified, e.g., for prolonged bioluminescence, increased bioluminescence intensity, or bioluminescence stability (e.g., US Patent Application No. 20120034672, which is hereby incorporated by reference)
  • an epitope tag is an amino acid sequence that may be specifically bound by another moiety, usually another polypeptide, most usually an antibody.
  • An epitope tag also includes any epitope that is experimentally determined to raise a monoclonal response in an animal that results in a high-affinity antibody for a defined peptide epitope.
  • An epitope tag is a peptide sequence heterologous to the marker, i.e., the reporter is a fusion protein of a marker and an epitope tag.
  • Suitable epitope tags include Flag, His, HA, AcV5, V5, Glu, Myc, Kt3 (Martin et al., Science, 255: 192-194 (1992), Aul, E2, glutathione S transferase (GST) and maltose binding protein (MBP), polyoma virus T antigen epitope, and the T7 gene 10 protein peptide tag (Lutz-Freyermuth et al., Proc. Natl. Sci. USA, 87:6363-6397 (1990).
  • larger proteins may be used, e.g., GST and MBP, smaller epitopes are preferred, for example epitopes between 5 to 50 amino acids in length.
  • a skilled person would recognize that other epitope tags are also suitable in the present invention.
  • the epitope tag is selected from Flag, His, HA, AcV5, V5, Glu, Myc, Kt3, Aul, and E2. More preferably, the tag is selected from Flag, His, HA, AcV5, V5, and Glu. These tags showed the highest efficiency in terms of antibody binding in present examples. However, a skilled person recognizes that other tags and/or their respective antibody or tag- binding moieties tags could be optimized to achieve higher sensitivity and are thus encompassed by the invention.
  • the nucleic acid encoding each reporter comprises a transcription factor response element (TRE).
  • a TRE is the nucleic acid sequence that a transcription factor binds to.
  • the TRE is selected from the binding sequence for P53, NF-kB, Hif-la, E2F, Creb, SP-1, AP-1, Stat3, Sox2, Klf4, Nanog, c-Myc, Elk, and Oct4.
  • P53 NF-kB
  • Hif-la Hif-la
  • E2F Creb
  • SP-1 AP-1
  • Stat3, Sox2, Klf4 Nanog
  • c-Myc Elk
  • Oct4 transcription factor response element
  • Binding sequences can also be predicted by a number of computational tools, such as TFSEARCH, TRANFAC, Matlnspector (Cartharius K (2005) Bioinformatics 21, 2933-42). Binding sequences can also be confirmed or determined experimentally using such protocols as DNase I Hypersensitivity, EMSA, and ChlP. Further details for both computational and experimental determination of TREs may be found in Elnitski et al. Genome Research 2006 16: 1455, the contents of which are hereby incorporated by reference in their entirety. Expression of the reporter provides an indication of the effect of a variable on a TRE. Preferably, each reporter comprises a different TRE.
  • TRE sequences may be flanked with restriction sites for cloning. Suitable primer sequences are disclosed herein, although it is understood that different restriction sequences may be used or in some cases the addition of restriction sites may not be necessary.
  • the nucleic acid encoding each reporter comprises a promoter sequence.
  • a promoter sequence is a nucleic acid sequence capable of initiating transcription. Promoters may be constitutive wherein the transcription level is constant and unaffected by modulators of promoter activity, e.g., CMV. Promoters may be inducible in that promoter activity is capable of being increased or decreased, for example as measured by the presence or quantitation of transcripts or translation products. Promoters may also be cell specific wherein the promoter is active only in particular cell types. Expression of the reporter provides an indication of the effect of a variable on the promoter. In some embodiments, a viral promoter is operably linked to the reporter.
  • each reporter Upon infection of the cell by a virus, the viral promoter is activated and detection of the specific reporter provides a measure of viral infection.
  • each reporter comprises a different promoter.
  • each reporter comprises a protease cleavage site.
  • a protease cleavage site is inserted in between the marker and the epitope tag.
  • Suitable cleavage sites include those for caspase proteases; viral proteases, e.g., HIV protease; proteases of bacterial toxins (e.g, botulinum toxin); proteases that process cellular proteins, e.g., secretase processing of beta-amyloid, proteases regulating cell adhesion (e.g., metalloproteases associated with extracellular matrix); proteases involved in blood coagulation, inflammation and would healing; and tumor cell associated proteases.
  • the protease cleavage site is located between the marker and the epitope or within the marker itself.
  • each reporter comprises a different protease cleavage site.
  • the nucleic acid encoding each reporter comprises an miRNA binding sequence.
  • the binding of an miRNA to its respective binding sequence usually leads to translational repression or target degradation.
  • Expression of the reporter, or lack thereof, provides an indication of the effect of a variable on the miRNA binding and silencing process.
  • each reporter comprises a different miRNA binding sequence.
  • the nucleic acid encoding each reporter comprises an RNA splicing sequence.
  • the RNA splicing sequence may be inserted either in the marker or epitope tag or between the two.
  • splicing leads to a fusion protein of the marker and epitope tag.
  • Such reporter systems are useful, e.g., in monitoring the efficiency of a splicing sequence or splicing machinery in different cells types or under different biological conditions.
  • each reporter comprises a different RNA splicing sequence.
  • the reporter systems disclosed herein may be used in additional applications and may also include additional sequences.
  • the reporters are provided as fusion proteins, nucleic acids encoding said fusion proteins, or as vectors comprising said nucleic acids.
  • the reporters are provided such the epitope tag is fused in frame at the N- or C-terminus of the marker sequence.
  • Methods for producing fusion proteins are well-known in the art.
  • nucleic acids are provided comprising a nucleic acid sequence encoding a marker as disclosed herein and a nucleic acid sequence encoding an epitope tag as disclosed herein.
  • the nucleic acids described herein can be prepared using standard recombinant DNA techniques described in, for example, Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1989.
  • each reporter may be provided on a separate nucleic acid or on the same nucleic acid, e.g., a vector may be provided comprising two or more reporters.
  • Said nucleic acids may be operably linked to additional sequences such as promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
  • Promoter sequences encode either constitutive or inducible promoters.
  • the promoters may be either naturally occurring promoters or hybrid promoters.
  • Hybrid promoters which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
  • the promoters are strong promoters, allowing high expression in cells, particularly mammalian cells, such as the CMV promoter, particularly in combination with a Tet regulatory element.
  • the nucleic acid also comprises a sequence encoding a signal peptide.
  • Signal peptides target the protein to the secretory pathway and are well-known in the art.
  • the signal peptide may be an endogenous or exogenous sequence.
  • the nucleic acids are provided in vectors. Accordingly, vectors are provided comprising a nucleic acid sequence encoding a marker as disclosed herein and a nucleic acid sequence encoding an epitope tag as disclosed herein.
  • the vector also comprises one or more additional sequences as described above, such as promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop
  • a “vector” is a recombinant nucleic acid construct, such as plasmid, phase genome, virus genome, cosmid, or artificial chromosome, to which another DNA segment may be attached.
  • the term “vector” includes both viral and nonviral means for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo.
  • Viral vectors include retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr and adenovirus vectors.
  • Vector sequences may also contain one or more regulatory regions, and/or selectable markers useful in selecting, measuring, and monitoring nucleic acid transfer results (transfer to which tissues, duration of expression, etc.).
  • the nucleic acid sequences may also be provided in a delivery complex, e.g., in liposomes, electrically charged lipids (cytofectins), and biopolymers.
  • a delivery complex e.g., in liposomes, electrically charged lipids (cytofectins), and biopolymers.
  • Cells comprising said nucleic acids or vectors comprising nucleic acids are also provided.
  • the method of introduction is largely dictated by the targeted cell type include, e.g., CaPO 4 precipitation, liposome fusion, lipofectin,
  • nucleic acids may stably integrate into the genome of the host cell (for example, with retroviral introduction, outlined below), or may exist either transiently or stably in the cytoplasm (i.e. through the use of traditional plasmids, utilizing standard regulatory sequences, selection markers, etc.).
  • the reporters as described herein may be produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding a reporter polypeptide.
  • Appropriate host cells include yeast, bacteria,
  • mammalian cells Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, Pichia pastoris, etc.
  • said polypeptides are expressed in mammalian cells.
  • Mammalian expression systems are also known in the art, and include retroviral systems. Suitable cell types include tumor cells (such as U87 human glioma cells), Jurkat T cells, NIH3T3 cells, CHO, and Cos cells.
  • said polypeptides are expressed in bacterial systems. Bacterial expression systems are well known in the art.
  • the reporters described herein are particularly useful for monitoring multiple variables, preferably in response to a biological condition. Accordingly, methods are provided for monitoring the effect on a biological system comprising providing said biological system with at least two reporters as described herein, exposing said system to a biological condition; and
  • At least three or at least four reporters are provided.
  • a biological system includes a biological system or a bodily fluid.
  • a biological system includes a cell, a tissue sample, cell extract, a bodily fluid, as well as a viral particle. Any type of cell may be used, such as animal (e.g., mammalian, avian, insect), plant, fungal (e.g., yeast), bacterial cell, or any type of viral infected cell.
  • a bodily fluid, as described herein, includes blood, serum, plasma, amniotic fluid, brain/spinal cord fluid, liquor,
  • a “biological condition” includes in vivo or in vitro growth conditions, administration of a test compound (such as a small molecule, nucleic acid, or protein), infection (e.g., by a virus, parasite, micro-organism) and stress conditions (e.g., heating, cooling, toxins, varying osmotic conditions).
  • a test compound such as a small molecule, nucleic acid, or protein
  • infection e.g., by a virus, parasite, micro-organism
  • stress conditions e.g., heating, cooling, toxins, varying osmotic conditions.
  • the biological system is a cell.
  • Suitable cell types include, but are not limited to, tumor cells of all types (e.g., glioma, melanoma, myeloid leukemia, carcinomas of the lung, breast, ovaries, colon, kidney, prostate, pancreas and testes), cardiomyocytes, endothelial cells, epithelial cells, lymphocytes (T-cell and B cell), mast cells, eosinophils, vascular intimal cells, hepatocytes, leukocytes including mononuclear leukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney, liver and myocyte stem cells, osteoclasts, chondrocytes and other connective tissue cells, keratinocytes, melanocytes, liver cells, kidney cells, and adipocytes.
  • tumor cells of all types e.g., glioma, melanoma, myeloid leukemia, carcinomas of the lung
  • Suitable cells also include known research cells, including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, and Cos cells.
  • the reporter system may be provided to the biological system in vitro, in vivo, or ex vivo.
  • the reporter system may be provided as nucleic acid (e.g., nucleic acid can be provided to cell extracts or transformed into cells or tissues) or as expressed proteins.
  • the biological system may then be exposed to a biological condition, which may include implanting the biological system into an organism, preferably a mammal.
  • the reporter system described herein may be provided to the same biological system (e.g., multiple reporter molecules provided to a single cell) or to different biological system (e.g., a first reporter provided to a first cell and a second reporter provided to a second cell; or a first and second reporter provided to a first cell and a third and fourth reporter provided to a second cell).
  • the second biological system may be a different type of biological system from the first biological system (e.g., the first cell may be a wild-type cell and the second cell may have a mutatation) or may have been subjected to a different condition or treatment, e.g., exposure to a compound or infection.
  • the reporter system When provided to different biological systems, the reporter system has the advantage that the effects between different biological systems (e.g., different cells) can be monitored.
  • cells are exposed to a biological condition and the level of the reporter is then determined in the first cell and the respective level is determined in the second cell.
  • the respective levels of the reporters provides an indication of an effect on the cells.
  • the effect on the cells is preferably due to the effect of a biological condition on a biological system.
  • Said condition may result, e.g., in the increase in RNA transcription, protein translation, cell proliferation, etc., which would generally lead to an increase in the level of a reporter.
  • methods for monitoring an effect on a cell comprising providing said cell with at least two reporters as described herein, exposing said cell to a biological condition; and determining the level of said reporters produced by said cell. If the cells are allowed to proliferate, then the reporter levels can also be used as an indication of proliferation, or a lack thereof (or alternatively cell death).
  • the epitope tag is used to immobilize the reporters on a surface (e.g., well-plate, bead, microsphere, or chip) before marker detection.
  • a surface e.g., well-plate, bead, microsphere, or chip
  • reporters are separated from each other using immunobinding.
  • the surface comprises an epitope tag binding molecule, such as an antibody specific for an epitope tag, which allows the surface, via the binding molecule, to bind the epitope tag, i.e., the reporters are captured on the surface.
  • the epitope tag binding molecules are specific for the epitope tags used in the reporter system.
  • the methods disclosed herein comprise the steps of providing a biological system with at least two reporters as disclosed herein, exposing said biological system to a biological condition; contacting said system with one or more surfaces, and determining the level of marker bound to each solid substrate.
  • Epitope tag binding molecules e.g., an antibody
  • Suitable surfaces include polymers, nylon, microarrays such as protein chips, the well of an assay plate, a filter, a membrane, a chromatographic resin, a bead, or microsphere.
  • each surface with a particular epitope tag binding molecule is separated such that the identity of each reporter can be distinguished based on the epitope tag (and its binding properties).
  • the marker is used to quantitate the amount of reporter.
  • each well of an assay plate or each spot on a chip or filter may have a different epitope tag binding molecule.
  • the reporter system described herein is provided to cells (either the same or different cells as described herein) and said cells are then introduced into an organism, preferably an animal such as, e.g., a mouse, rat, rabbit, or human.
  • said reporter system may be provided to cells or tissues within an organism.
  • one or more viral vectors encoding a reporter system described herein may be provided to a tumor within an individual. Bodily fluid for said individual may be collected in order to detect the level of the respective reporters.
  • the body fluid is blood, plasma or serum.
  • the reporter system may also comprise additional elements such as a transcription response element, promoter sequence, a protease cleavage site, miRNA binding sequence or RNA splicing sequence. These additional elements may be the same or different between the reporters. Preferably, the elements are the same when the reporter system is provided to different cells.
  • the elements are different between each reporter when the reporter system is provided to the same cells.
  • the detection of the reporters as described herein may be carried out by any of the well-known methods known in the art. Fluorescent, luminescent and colorimetric labels and methods of detecting and measuring quantities with them are well-known and readily understood by those skilled in the art.
  • buffering agents such as Tricine, HEPPS, HEPES, MOPS, Tris, glycylglycine, or a phosphate salt may be present to maintain pH and ionic strength; a proteinaceous material, such as a mammalian serum albumin (preferably bovine serum albumin) or lactalbumin or an ovalbumin, that enhances the activity of luciferases in the luciferase- luciferin reaction, may be present; EDTA or CDTA
  • cyclohexylenediaminetetraacetate or the hke
  • metal-containing proteases or phosphatases that might be present in systems (e.g., cells) from which luciferase to be assayed is extracted and that could adversely affect the luciferase or the ATP.
  • Glycerol or ethylene glycol which stabilize luciferases, might be present.
  • An exemplary assay is provided in the examples.
  • a coelenterazine type luciferase for example Glue or Mluc
  • the conditions described in US Application No. 20110256564 and hereby incorporated by reference may be used.
  • to comprise and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded.
  • verb "to consist” may be replaced by "to consist essentially of meaning that a compound or adjunct compound as defined herein may comprise additional component(s) than the ones specifically identified, said additional component(s) not altering the unique characteristic of the invention.
  • an element means one element or more than one element.
  • lentiviral vectors consisting of the Glue cDNA fused to ten different epitope tags at its C-terminus, under the constitutively active cytomegalovirus (CMV) promoter resulting in GlucFiag, GlucHis, GIUCHA, G1UCACV5, Glucv5, GIUCGIU, GlucMyc, GlucKt3, GlucAui, G1UCE2, and the control reporter construct without tag, Glucctri.
  • CMV cytomegalovirus
  • This lentiviral vector also expresses cerulean fluorescent protein (CFP) separated from Gluctag by an internal ribosomal entry site (IRES) element as a marker for transduction efficiency (Fig. la and Online Methods section).
  • CFP cerulean fluorescent protein
  • Glue activity As a control, the same amount of conditioned medium was assayed directly for Glue activity (total Glue).
  • total Glue we determined that six of the ten individual Gluctag constructs, i.e. GlucFiag, GlucHis, GIUCHA, G1UCACV5, G1UCV5, GIUCGI U , reported with high-efficiency after immunobinding in the antibody- coated wells, as compared to total Gluctag activity (Fig. le).
  • the GlucMyc, GlucKt3, GlucAui, and G1UCE2 reporters showed lower activity upon
  • Gluctag reporter system for real-time multiplex application and demonstrated its ability in monitoring individual cell populations in a mixed cell cultures in vitro, and in a single tumor in vivo both in subcutaneous model and in deeper tissues such as the brain.
  • the individual epitope tags could also be employed to localize expression by immunostaining.
  • this tag library could be extended to virtually hundred different tags allowing the possibility of high-throuhput multiplexing applications.
  • antibody binding of the low-efficiency tags in microtiter wells could be further optimized to achieve higher sensitivity since these different tags (except for GIUCAUI) were readily detected, by immunostaining.
  • Real time monitoring of individual cells in a heterogenous mixture may allow, for instance, multiplexed RNAi screening or measurement of drug responses of multiple cell populations in parallel.
  • the Gluctag multiplex system could be extended to monitor different variables in a single cell type. For instance, engineering each individual Gluctag under the control of a different transcription response element for multiplex transcription factor activity measurement; constructing miRNA-binding sequences in the 3'-UTR of the Gluctag constructs for multiplex miRNA activity monitoring; or by inserting different protease cleavage sites into the Gluctag gene for multiplex protease activity measurement.
  • This reporter assay provides a valuable tool to study complex processes with different variables in systems biology.
  • glioblastoma cell line U87 stably co-expressing Firefly luciferase and mCherry fluorescent protein.
  • Cell lines were maintained in DMEM high glucose complemented with sodium pyruvate, stable glutamine, 10% FBS and pen/strep (all PAA), incubated under standard cell culture conditions of 37°C and 5% CO2.
  • Plasmid DNA was transformed in XL- 10 Gold ultracompetent cells (Agilent Technologies), grown overnight on LB agar containing 50 ⁇ g/ml Ampicillin. We picked colonies to grow overnight and isolated DNA using a DNA plasmid mini kit (Qiagen) and verified successful transformation by Xbal restriction analysis. The Glue construct was then digested with Xbal and Xhol to insert an epitope tag. The epitope tags were designed with a Xbal site upstream, a stop codon and Xhol site downstream.
  • annealing buffer 100 mM Tris-HCl pH 7.5, 1 M NaCl and 10 mM EDTA
  • the epitope tag was then inserted into the vector using T4 DNA ligase and transformed in XL- 10 Gold ultracompetent cells. Bacteria were cultured and DNA was isolated. We verified the Gluctag constructs by sequencing using BigDye Terminator v3.1 Cycle Sequencing kit (Life Technologies).
  • the Gluctag construct was then co-transfected with a third generation lentiviral packaging mix (pMDLg/pRRE, pRSV-Rev and pMD2.G) in HEK293T cells using Lipofectamine 2000 (Life Technologies). Virus was harvested 2 and 3 days after transfection and cell debris were spun down for 5 minutes at 1,000 x g. U87 cells were transduced overnight with lentivirus using a multiplicity of infection of 100 transducing units per cell in the presence of 8 ⁇ g/ml polybrene in standard culture conditions.
  • pMDLg/pRRE pRSV-Rev
  • pMD2.G Lipofectamine 2000
  • Endogenous peroxide was blocked with 0.3% H2O2 in methanol for 30 minutes. After rinsing with water, antigens were retrieved with citrate buffer (pH 6) with 0.05% Tween 20 using a microwave (5 minutes 100%, 10 minutes 50% power). After slowly cooling, tissues were washed 3x with PBS and incubated with primary antibody (10 ⁇ g/ml), for one hour at room temperature. After washing again, tissues were incubated with envision anti-mouse and DAB stained as above (both Dako). Tissue sections were dehydrated with ethanol series as before and fixed in xylene. Cells and sections were imaged and photographed by light microscopy (Leica).
  • Glue activity assay In vitro Glue activity assay. For Gluctag activity over time, 50,000 cells were plated in a 24 well plate and incubated overnight. 10 ⁇ of conditioned medium were harvested from cells and Glue activity was measured by adding 50 ⁇ (5 ⁇ g/ml) coelenterazine (Nanolight Technologies) in PBS and 0.1% Triton X-100). Before addition to the sample, the substrate was incubated at room
  • Gluctag activity in medium was determined as described above. Gluctag immunobinding assay. White goat-anti-mouse-coated 96-well plates
  • lidocaine 5 mg/ml in PBS
  • X 0.5 mm
  • Y 2 mm
  • Z -2 mm from Bregma
  • a small drill was used to drill a hole into the skull.
  • a total of 2 x 10 5 cells in 3 ⁇ of DMEM were injected vertically.
  • tumor size was monitored using a calliper (for subcutaneous) and bioluminescence in vivo Flue imaging, by injecting D-luciferin (100 mg/kg) intraperitoneally. Imaging was performed with an IVIS CCD camera and analyzed with Living Image software (Cahper Life Sciences).
  • CMV-Gluc sequence restriction sites are indicated in bold. [BamHI] [CMV promoter]
  • HA 1 CTAGATATCCGTATGATGTGCCGGATTAT Hemaggl Abeam
  • V5 1 CT AGAGGC AAGC CT ATC C CT AAC C CT CT G V5 tag Abeam
  • Glu-Glu 1 CTAGATGCGAGGAAGAGGAATACATGCCT Glu-Glu Abeam
  • Myc 1 CTAGAGAACAAAAACTCATCTCAGAAGAG Anti-c- Sigma- GAT CTGTGAC Myc Aldrich
  • Kt3 1 CT AGAAAGC CTC C AAC AC CT C C AC CT GAG KT3 tag Abeam
  • Aul 1 CT AG AG AC AC CTAC AGAT AC ATCT GAC Covance
  • GTC GAC ATTTC C GT AAATC GTC GA
  • GTC GAC ATTTC C GT AAATC GTC GA ATATA G-3'
  • NF-kB (5NF, Chris Badr et al. Mol. Imaging, 2009)
  • the CSCW-Gluc-IRES-CFP lentiviral vector was used to demonstrate the multiplex method using Gaussia luciferase.
  • This lentiviral vector co- expresses the Glue bioluminescent reporter and the cerulean fluorescent protein (CFP) control.
  • the internal ribosomal entry site (IRES) allows co- expression of both proteins using the same cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • Glucmodified gene (633 bp) in thermal cycler by 1: denature the plasmid DNA for 2 minutes at 95°C. Then 2: denature for 30 seconds at 95°C, 3: anneal primers for 30 seconds at 62°C and 4: elongate DNA for 1 minute at 72°C and cycle sequence 2, 3, 4 for 35 times. Allow 5: final elongation of DNA for 2 minutes at 72°C. 2.
  • Glucmodified gene and CSCW lentiviral backbone restriction mixtures from (2.) and load both the samples into a separate gel well. Also load a separate well with a DNA molecular weight marker to identify the product and confirm product sizes. Run the gel in 1 x TAE buffer at 100 volts until the Glucparental gene (718 bp) has separated properly from the CSCW lentiviral backbone (9408 bp).
  • oligonucleotide 100 ⁇
  • 20 ⁇ of the antisense oligonucleotide 100 ⁇
  • HEK293T and U87 cells are cultured in DMEM complete culture medium at 37°C and 5% C02.
  • the cells are diluted 1/10 when the culture vessel is ⁇ 90% confluent.
  • Detach the cells by adding lx Trypsin + EDTA, enough to just cover the surface of the culture vessel.
  • DMEM complete culture medium and culture the cells in 37°C and 5% C02.
  • Temgesic (Buprenorfinehydrochloride, 0.1 mg/kg) in PBS analgesia and anesthetize the mouse using an isoflurane anaesthesia and fix the mouse in a small animal stereotaxic frame.
  • a high fidelity proof reading DNA polymerase is used for the amplification of the Glue reporter gene. This minimizes optimization and the risk of copy errors due to incorrect basep airing.
  • high fidelity restriction enzymes for restriction of the DNA vectors are used. This increases restriction efficiency and user simplicity since most high fidelity enzymes are optimised in the same restriction reaction buffer. Also, most high fidelity restriction enzymes are optimised to have less star activity, increasing ligation efficiency in later steps of the protocol.
  • ethidium bromide such as SYBR safe DNA gel stain (Life Technologies). Besides being less hazardous, these stains can also be used to visualize DNA with non-UV- light, decreasing damage of the DNA due to UV exposure.
  • white microplates are used to prevent crossover detection of photons between wells.
  • the white goat anti- mouse IgG coated microplates (Thermo Scientific) are pre-coated to improve assay stability but it is also possible to manually coat microplates (e.g. ELISA microplates).
  • the antibodies we used are all mouse monoclonal IgG antibodies.
  • the coated microplates used (see note 5) are optimised to use with mouse IgG antibodies. Using monoclonal antibodies over polyclonal antibodies improves assay stability.
  • the capillary collection and sample container (Sarstedt) we used combines a capillary blood collector with an EDTA coated sample container. This enables fast and easy sample collection and storage. If you collect mouse blood using other methods, make sure you add EDTA to the blood sample to prevent clotting.
  • virus titers are obtained using other methods of transfection of the plasmids and vectors. Using Lipofectamine 2000 (Life Technologies) or Fugene (Promega) can be beneficial for virus production titres.
  • the most stable method of measuring would be to measure Glue signal directly after coelenterazine substrate addition.
  • a plate reader with a substrate injector would be optimal but it is also an option to use a multichannel pipette. Measuring a 96-well microplate for 0.1 second per well would take about 10 seconds, so the time difference as a result of measuring the first well and the last well would be well within 10% deviation as a result of the Glue signal degradation.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Urology & Nephrology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Analytical Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • General Physics & Mathematics (AREA)
  • Toxicology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des dosages multiplexes pour la surveillance de multiples variables, tels que les effets d'un état biologique sur un système biologique. L'invention concerne des systèmes rapporteurs pour la mise en œuvre de tels dosages.
PCT/NL2013/050264 2012-04-11 2013-04-11 Dosages de rapporteur multiplexes pour la surveillance de multiples variables Ceased WO2013154429A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP12163785 2012-04-11
EP12163785.4 2012-04-11

Publications (1)

Publication Number Publication Date
WO2013154429A1 true WO2013154429A1 (fr) 2013-10-17

Family

ID=48227520

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/NL2013/050264 Ceased WO2013154429A1 (fr) 2012-04-11 2013-04-11 Dosages de rapporteur multiplexes pour la surveillance de multiples variables

Country Status (1)

Country Link
WO (1) WO2013154429A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10544472B2 (en) * 2014-01-18 2020-01-28 Attagene, Inc. Multiplexing transcription factor reporter protein assay process and system

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003031591A2 (fr) * 2001-10-10 2003-04-17 Superarray Bioscience Corporation Detection de cibles a l'aide de marqueurs uniques d'identification de nucleotides
WO2010059253A2 (fr) * 2008-11-24 2010-05-27 Massachusets Institute Of Technology Procédés et compositions pour la délivrance localisée d'agents
US20110256564A1 (en) 2007-10-29 2011-10-20 Perkinelmer Health Science B.V. Methods, reagents and kits for luciferase assay
US20120034672A1 (en) 2009-04-17 2012-02-09 Sungbae Kim Superluminescent luciferase variant with prolonged bioluminescence

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003031591A2 (fr) * 2001-10-10 2003-04-17 Superarray Bioscience Corporation Detection de cibles a l'aide de marqueurs uniques d'identification de nucleotides
US20110256564A1 (en) 2007-10-29 2011-10-20 Perkinelmer Health Science B.V. Methods, reagents and kits for luciferase assay
WO2010059253A2 (fr) * 2008-11-24 2010-05-27 Massachusets Institute Of Technology Procédés et compositions pour la délivrance localisée d'agents
US20120034672A1 (en) 2009-04-17 2012-02-09 Sungbae Kim Superluminescent luciferase variant with prolonged bioluminescence

Non-Patent Citations (26)

* Cited by examiner, † Cited by third party
Title
BADR, C.E.; HEWETT, J.W.; BREAKEFIELD, X.O.; TANNOUS, B.A.: "A highly sensitive assay for monitoring the secretory pathway and ER stress", PLOS ONE, vol. 2, 2007, pages E571
BADR, C.E.; TANNOUS, B.A.: "Bioluminescence imaging: progress and applications", TRENDS IN BIOTECHNOLOGY, vol. 29, 2011, pages 624 - 33, XP028112001, DOI: doi:10.1016/j.tibtech.2011.06.010
BHANG, H.-EUN C.; GABRIELSON, K.L.; LATERRA, J.; FISHER, P.B.; POMPER, M.G.: "Tumor-specific imaging through progression elevated gene-3 promoter- driven gene expression", NATURE MEDICINE, vol. 17, 2011, pages 123 - 9, XP055214896, DOI: doi:10.1038/nm.2269
BHAUMIK, S.; GAMBHIR, S.S.: "Optical imaging of Renilla luciferase reporter gene expression in living mice", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 99, 2002, pages 377 - 82, XP002977218, DOI: doi:10.1073/pnas.012611099
CARTHARIUS K, BIOINFORMATICS, vol. 21, 2005, pages 2933 - 42
CONTAG, C.H.; ROSS, B.D.: "It's not just about anatomy: in vivo bioluminescence imaging as an eyepiece into biology", JOURNAL OF MAGNETIC RESONANCE IMAGING: JMRI, vol. 16, 2002, pages 378 - 87
DELIOLANIS, N.C. ET AL.: "In vivo tomographic imaging of red-shifted fluorescent proteins", BIOMEDICAL OPTICS EXPRESS, vol. 2, 2011, pages 887 - 900
ELNITSKI ET AL., GENOME RESEARCH, vol. 16, 2006, pages 1455
FILONOV, G.S. ET AL.: "Bright and stable near-infrared fluorescent protein for in vivo imaging", NATURE BIOTECHNOLOGY, vol. 29, 2011, pages 757 - 61
G. WANG ET AL: "Detection of the Potential Pancreatic Cancer Marker MUC4 in Serum Using Surface-Enhanced Raman Scattering", ANALYTICAL CHEMISTRY (AMERICAN CHEMICAL SOCIETY), vol. 83, no. 7, 1 April 2011 (2011-04-01), Washington DC USA, pages 2554 - 2561, XP055071395, ISSN: 0003-2700, DOI: 10.1021/ac102829b *
GORDON ET AL., MICROBIOLOGY, vol. 2, 2000, pages 415 - 426
GROSS, S.; PIWNICA-WORMS, D.: "Spying on cancer: molecular imaging in vivo with genetically encoded reporters", CANCER CELL, vol. 7, 2005, pages 5 - 15, XP002418389, DOI: doi:10.1016/S1535-6108(04)00373-3
LAUKKANEN ET AL., BIOCHEM AND BIOPHY RES COMM, vol. 3, 1996, pages 755 - 761
LEE, J.Y. ET AL.: "Development of a dual-luciferase reporter system for in vivo visualization of MicroRNA biogenesis and posttranscriptional regulation", JOURNAL OF NUCLEAR MEDICINE: OFFICIAL PUBLICATION, SOCIETY OF NUCLEAR MEDICINE, vol. 49, 2008, pages 285 - 94
LUTZ-FREYERMUTH ET AL., PROC. NATL. SCI. USA, vol. 87, 1990, pages 6363 - 6397
MARTIN ET AL., SCIENCE, vol. 255, 1992, pages 192 - 194
MCMILLIN, D.W. ET AL.: "Tumor cell-specific bioluminescence platform to identify stroma-induced changes to anticancer drug activity", NATURE MEDICINE, vol. 16, 2010, pages 483 - 9
PITTET, M.J.; WEISSLEDER, R.: "Intravital Imaging", CELL, vol. 147, 2011, pages 983 - 991, XP028597184, DOI: doi:10.1016/j.cell.2011.11.004
S. VAN RIJN ET AL: "Functional multiplex reporter assay using tagged Gaussia luciferase", SCIENTIFIC REPORTS, vol. 3, 1046, 10 January 2013 (2013-01-10), Basingstoke UK, pages 1 - 5, XP055071382, DOI: 10.1038/srep01046 *
SAMBROOK, J. ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR PRESS
SHANER, N.C.; STEINBACH, P.A.; TSIEN, R.Y.: "A guide to choosing fluorescent proteins", NATURE METHODS, vol. 2, 2005, pages 905 - 9
TANNOUS, B.: "a Gaussia luciferase reporter assay for monitoring biological processes in culture and in vivo", NATURE PROTOCOLS, vol. 4, 2009, pages 582 - 91
TANNOUS, B.A.; KIM, D.-E.; FERNANDEZ, J.L.; WEISSLEDER, R.; BREAKEFIELD, X.O.: "Codon-optimized Gaussia luciferase cDNA for mammalian gene expression in culture and in vivo", MOLECULAR THERAPY: THE JOURNAL OF THE AMERICAN SOCIETY OF GENE THERAPY, vol. 11, 2005, pages 435 - 43, XP004757251, DOI: doi:10.1016/j.ymthe.2004.10.016
TIAN, W. ET AL.: "High-Throughput Functional MicroRNAs Profiling by Recombinant AAV-Based MicroRNA Sensor Arrays", PLOS ONE, vol. 7, 2012, pages E29551
WILLE ET AL., APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 78, 2012, pages 250 - 257
WURDINGER, T. ET AL.: "A secreted luciferase for ex vivo monitoring of in vivo processes", NATURE METHODS, vol. 5, 2008, pages 171 - 3, XP002696521, DOI: doi:10.1038/NMETH.1177

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10544472B2 (en) * 2014-01-18 2020-01-28 Attagene, Inc. Multiplexing transcription factor reporter protein assay process and system

Similar Documents

Publication Publication Date Title
EP0656954B1 (fr) Procede de production de genes, produits de transcription et proteines marques
US8252535B2 (en) RNA interference tags
US9506073B2 (en) Blue-light inducible system for gene expression
CN104004098A (zh) 一种利用BiFC指示细胞中Wnt信号活性状态的载体组合物及应用
US20250327117A1 (en) Crispr-based imaging system and use thereof
US10202656B2 (en) Dividing of reporter proteins by DNA sequences and its application in site specific recombination
EP2776579B1 (fr) Protéines de fusion propeptide-luciférase et leurs procédés d'utilisation
US20180356408A1 (en) Methods and materials for sensitive detection of target molecules
WO2013154429A1 (fr) Dosages de rapporteur multiplexes pour la surveillance de multiples variables
EP3037534B1 (fr) Polypeptide présentant des propriétés fluorescentes et son utilisation
US20230031446A1 (en) Split-enzyme system to detect specific dna in living cells
WO2008094316A2 (fr) Nouvelles protéines fluorescentes dérivées du genre branchiostoma
WO2021253126A1 (fr) Lieurs clivables pour rapport de traduction de protéines
JP7585015B2 (ja) 核酸構築物のセット、キット、検出方法及び薬剤効果予測方法
JP6436908B2 (ja) 外因性遺伝子発現ベクター、形質転換体判別マーカー及び形質転換体
US20230357870A1 (en) Coronavirus replicons for antiviral screening and testing
WO2024199219A1 (fr) Transposase isolée et son utilisation
JP2008182905A (ja) ヒト由来プロレニン受容体の製造方法、ヒト由来プロレニン受容体阻害剤のスクリーニング方法、プロレニン濃度の測定方法及び抗ヒト由来プロレニン受容体抗体
KR20250004513A (ko) Bret-기반 rab11a 활성 바이오센서 및 이를 포함하는 세포주
US20250270522A1 (en) Isolated transposase and use thereof
US20250051759A1 (en) METHODS AND MOLECULES FOR RNA INTERFERENCE (RNAi)
KR101460246B1 (ko) 사상성 자낭균류에서 생체내 단백질 상호작용 분석용 벡터 및 이를 이용한 스플릿 루시퍼라아제 상보성 분석방법
CN101514340B (zh) 人自吞噬基因Beclin 1启动子序列及其应用
US20220220568A1 (en) Nucleic acid construct, kit, detection method, and therapeutic effect prediction method
Kondo et al. A cryptic promoter in the exon of HKR1 drives expression of a truncated form of Hkr1 in Saccharomyces cerevisiae

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13719629

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13719629

Country of ref document: EP

Kind code of ref document: A1