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
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

• the present invention relates to an improved fluorescence reporter assay system and its use in screening compounds and combinatorial libraries.
• BACKGROUND OF THE INVENTION Screening of compounds for pharmacological activity is dependent on a means of measuring the effect of the compounds on a pharmacologically relevant process, such as activation or inhibition of specific gene expression.
• a pharmacologically relevant process such as activation or inhibition of specific gene expression.
• this may be done by engineering a DNA construct containing the promoter region of the gene of interest coupled to a readily detectable 'reporter' gene, such as an enzyme or fluorescent protein.
• the construct is transfected into a recipient cell and the effect of compounds on expression of the reporter gene is measured by assaying the level of enzyme or fluorescent protein produced by the cell.
• reporter genes include chloramphenicol acetyltransferase (CAT), firefly luciferase, beta galactosidase (beta-gal), secreted alkaline phosphatase or green fluorescent protein (GFP).
• CAT chloramphenicol acetyltransferase
• beta-gal beta galactosidase
• GFP green fluorescent protein
• CAT beta-gal and luciferase all require cell lysis which precludes their use as selectable markers, making selection of stably transfected cell lines a time consuming process dependent on co-expressed antibiotic resistance markers. Additionally, CAT assays require either chromatographic detection of reaction product or indirect antibody detection which is inconvenient for high throughput screening, while luciferase has a limited duration of signal generation, which in turn limits the time for signal measurement. GFP has sensitivity limitations due to the presence of only one fluorophore per GFP molecule and therefore requires large numbers of cells.
• the GFP molecule is also extremely stable, making it unsuitable for monitoring decreases in gene expression. GFP is also unsuitable for srceening compounds that fluoresce or absorb strongly within its absorption or emission wavelength.
• the latter problem also applies to beta lactamase readouts, while secreted alkaline phosphatase suffers from high backgrounds due to endogenous alkaline phosphatase activity. Multiplexing (allowing for the measurement of expression of more than one gene per cell) is difficult with all of the above readouts.
• This invention relates to a fluorescence reporter assay system.
• This invention also relates to a homogeneous fluorescence reporter assay system.
• This invention also relates to a time resolved fluorescence reporter assay system.
• This invention also relates to a reporter assay system that is compatible with miniaturized assay formats.
• This invention also relates to a method of conducting high throughput screening using the presently invented fluorescence reporter assay system.
• This invention also relates to a method of screening combinatorial libraries using the presently invented fluorescence reporter assay system.
• This invention also relates to compounds identified by the presently invented fluorescence reporter assay system and presently invented methods.
• This invention relates to an improved fluorescence reporter screening system that allows for gene expression in intact cells, has no washing steps that might impede use of the assay in nanowell format, has no endogenous activity in untransfected cells, allows measurement of expression of more than one gene per cell (multiplexing), has a high signal to noise ratio and a long readout duration.
• the presently invented fluorescence reporter assay system uses an extracellular marker capable of generating an optical readout. It can be detected and quantitated without lysing the cells, is unaffected by compound fluorescence, allows multiplexing, requires no washing steps, has no endogenous background in mammalian cells and has a low background and long duration of appearance.
• the presently invented fluorescence reporter assay system comprises a nucleotide sequence encoding a promoter/enhancer region, an extracellular protein (defined herein as either a cell surface or secreted protein) containing sequences coding for one or more binding partners or other molecules capable of generating an optical signal; a recipient cell; accessory molecules necessary to produce and/or enhance the optical signal and an optical detection apparatus.
• the extracellular protein is xenogeneic (defined herein as a protein that is not naturally produced by the recipient cell type).
• the nucleic acid construct is prepared such that expression of the cell surface or secreted protein is driven by the promoter/enhancer region, the latter being derived from a pharmacologically relevant protein target.
• pro-alpha 1 chain of Type 1 collagen which is specifically expressed in osteoblasts and is linked to formation of new bone matrix (Identification of a minimal sequence of the mouse pro-alpha 1 (I) collagen promoter that confers high-level osteoblast expression in transgenic mice and that binds a protein selectively present in osteoblasts. Rossert-JA; Chen-SS; Eberspaecher-H; Smith-CN; de-Crombrugghe-B Proc- Natl-Acad-Sci-USA. 1996; 93(3): 1027-31).
• the extracellular protein is constructed with the optical signal-generating sequences inserted in frame within or appended to the coding sequence and carries additional sequences specifying membrane targeting or secretion (Duffaud GD et al, in: Current Topics in Membranes and Transport, Chapter 2 Vol. 24, Academic Press NY, 1985).
• the optical signal may be generated directly, for example, by inserting sequences encoding for firefly luciferase.
• the optical signal may be generated indirectly, for example, by providing binding sites for exogenous binding partners that contain optically active constituents such as fluorescent dyes or enzymes capable of generating optically detectable products.
• polypeptide sequences that are not naturally present on or produced by the recipient cell are preferred to minimize background.
• An example of a suitable extracellular protein with an indirect optical readout is the epidermal growth factor (EGF) receptor, with two or more defined epitope tags inserted into the extracellular domain.
• EGF epidermal growth factor
• a defined epitope tag is preferably a short non-mammalian polypeptide sequence that is recognized by a specific monoclonal antibody, such as the 11 amino acid epitope from Human Immunodeficiency Virus Type 1 (HIV-1) envelope protein gpl20 (or gpl60) that is recognized by the monoclonal antibody 178.1 (see, e.g., Thiriart et al., J. Immunol., 143: 1832-1836 (1989)), which was prepared by immunization of mice with a yeast-expressed HIV-1 gp 160 molecule from strain BH10 (Ratner et al., Nature. 313: 277-284 (1985)).
• a specific monoclonal antibody such as the 11 amino acid epitope from Human Immunodeficiency Virus Type 1 (HIV-1) envelope protein gpl20 (or gpl60) that is recognized by the monoclonal antibody 178.1 (see, e.g., Thiriart
• DET is the commercially available FLAG tagging system from Eastman Kodak, Rochester NY.
• each molecule of the EGF receptor carries tandem copies of each of two DETs and the copies may be repeated within the molecule to afford additional binding sites.
• the DET sequences may be inserted into a secreted protein such as human growth hormone. It will be appreciated that there are many variations for implementation of such an extracellular protein. All such variations are included within the scope of this invention.
• the minimal essential features of the construct are a promoter/enhancer region, a membrane or secretion-targeting signal sequence(s), a transmembrane or membrane insertion region for cell surface proteins and a polypeptide that is directly or indirectly capable of generating an optically readable signal.
• An additional, preferred, component of this invention is the provision of accessory molecules for generation and or enhancement of the optical signal.
• Such molecules include but are not limited to antibodies labeled with fluorescent dyes, substrates for light-generating enzymes or color-generating reagents for enzymes.
• two exogenous binding partners are provided, each of which recognizes one of the DET sequences that was inserted into the extracellular domain of the extracellular protein.
• one or both exogenous binding partners may recognize spatially discrete regions of a naturally occurring extracellular protein used in the above promoter/enhancer construct and which is not present on or produced by the chosen recipient cell, such that when bound, the two exogenous binding partners are separated by a distance comparable to or less than the energy transfer radius for the chosen fluorophores.
• exogenous binding partners are monoclonal antibodies to DETs. These binding partners are each labelled, preferably with fluorescent dyes capable of resonance energy transfer. Preferably, one of the dyes is capable of time resolved fluorescence (luminescence). Examples of such dyes are terbium chelate/cryptates (Selvin P R and Hearst (J E. Proc. Natl. Acad. Sci. 91: 10024- 10028, 1994) and tetramethyl rhodamine (Molecular Probes, Eugene, OR). Methods for labelling antibodies with fluorescent dyes are well known in the art.
• An additionally preferred component of this invention is a recipient cell.
• This cell may be of eukaryotic or prokaryotic origin and in the former case may be somatic or germline.
• the preferred cell type is mammalian, preferably a human cell line that can be maintained in culture.
• An example of such a cell line is the human embryonic kidney cell line HEK293 which is available from the American Type Culture Collection. Methods for transfection of mammalian cells with nucleic acid constructs are well known to one skilled in the art. Depending on the nature of the promoter, it may be necessary to stimulate the cells with a hormone or other substance to initiate expresion of the extracellular protein.
• An additionally preferred component of this invention is a device capable of detecting and measuring the optical signal.
• a device should produce a numeric or graphical output that is related to the intensity and wavelength of the optical signal. It should be sensitive to the wavelength of the optical signal and may contain a source of electromagnetic radiation to assist in the generation of the optical signal, for example, an excitation source for fluorophores. It may also incorporate a time delay mechanism such that excitation radiation may be applied for a discrete time interval and measurement may be initiated at a specific time after termination of the excitation. Examples of such devices are fluorimeters, fluorescence correlation spectrometers, luminometers, spectrophotometers, CCD cameras, photon counters and fluorescence activated cell sorters. Such devices are readily available from commercial sources.
• the polynucleotide construct is transfected into the chosen recipient cell. If the promoter is active in the cellular environment, it drives expression of the extracellular protein, which leads to display on the cell surface or secretion of the protein carrying the tandem binding partners. Cells that express high levels of a construct utilizing a cell surface protein are selected by addition of the labelled exogenous binding partners, followed by fluorescence activated cell sorting. Cells that produce secreted markers are cloned by limiting dilution and selected by monitoring of the culture media for the optical signal.
• accessory molecules are added to the culture media.
• monoclonal anti-DET antibodies labeled with fluorescent dyes that are capable of resonance energy transfer are added.
• the fluorescent dyes Upon binding to their respective recognition sites on the extracellular protein, the fluorescent dyes are brought within their critical radii for resonance energy transfer and emission from the donor or acceptor dye is used to select cells that express high levels of the extracellular protein. These cells may be subjected to iterative selections as above to generate stable transfectants.
• the optical signal is detected by using a suitable detection device as above.
• the cells are used for screening compound collections or combinatorial libraries.
• Cells are plated in test wells in groups, preferably of more than 100 cells, to average out cell-to cell variations in reporter expression levels.
• Test compounds are added with or without an inducing hormone or other substance as appropriate for the promoter region of interest.
• accessory reagents are added to each well.
• the optical signal is quantitated using a suitable detection device.
• cells are co-transfected with two or more constructs of the same extracellular protein but with different promoter regions driving each construct. For each construct, the optical signal-generating sequences are chosen to give distinct signals.
• peptide binding partners are chosen such that they interact with distinct exogenous binding partners.
• cells may be transfected with promoter 1 linked to DET1-DET2 and with promoter 2 linked to DET1-DET3 or DET3-DET4.
• the accessory molecules are each labelled with a fluorophore of distinct abso ⁇ tion or emission wavelength.
• an antibody to DET1 could be labeled with terbium chelate/cryptate (emission 545 nm), anti-DET2 with TMR (excitation 550 nm emission 575 nm) and anti-DET3 with samarium chelate/cryptate (emission 655 nm) and DET4 with Cy5 (Amersham, Arlington Heights IL) (excitation 650nm emission 670nm).
• Stable transfectants are selected as above for expression of both constructs. Screening of test compounds is done as above but fluorescence emission is monitored both at 575 nm and 670 nm, so that differential effects of compounds on one or other promoter activity can be measured.
• the advantages of this process compared with existing reporter gene systems are 1) it allows the use of time resolving dyes which are insensitive to fluorescence of test compounds or cellular components, 2) it allows unrestricted choice of fluorophores for multiplexing and avoidance of quenching by test compounds, 3) it does not require cell lysis so the selection of stable transfectants is simplified, 4) the signal is present for a long duration so that time-dependent imaging can be used for detection 5) multiple fluorophores can be conjugated to each accessory molecule and multiple binding partner sequences can be added to the extracellular protein for increased sensitivity 6) no washing steps are needed which simplifies high throughput screening 7) the binding partners can be chosen from microbial, viral, fungal, insect or artificial sources so that no such molecules occur endogenously on mammalian cells 8) signal to noise ratios are enhanced by the requirement that two specific binding events must occur in close proximity to one another for signal generation, and by the use of time resolving dyes.

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• 1998
  • 1998-04-22 JP JP54630298A patent/JP2002511744A/ja active Pending
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