WO1999060371A2 - Procede et appareil de detection d'agents presentant un danger pour la sante - Google Patents

Procede et appareil de detection d'agents presentant un danger pour la sante Download PDF

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Publication number
WO1999060371A2
WO1999060371A2 PCT/US1999/010770 US9910770W WO9960371A2 WO 1999060371 A2 WO1999060371 A2 WO 1999060371A2 US 9910770 W US9910770 W US 9910770W WO 9960371 A2 WO9960371 A2 WO 9960371A2
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cells
sample
toxic
response
cell
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PCT/US1999/010770
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WO1999060371A3 (fr
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Thomas Okarma
Joseph Gold
James Murai
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Geron Corporation
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Priority to AU41885/99A priority Critical patent/AU4188599A/en
Publication of WO1999060371A2 publication Critical patent/WO1999060371A2/fr
Publication of WO1999060371A3 publication Critical patent/WO1999060371A3/fr

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    • 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
    • G01N33/5014—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 for testing toxicity

Definitions

  • the present invention relates to the detection of biologically active agents. More specifically, the present invention provides methods and apparatus for detecting chemical and biological toxins.
  • the present invention has applications in the fields of epidemiology, environmental safety, counter-terrorism, and chemical/biological warfare. THE RELATED ART Humans are exposed to a wide variety of deleterious substances daily. Our natural environment is filled with animals, plants, and microorganisms that produce natural substances that are toxic to humans. For example, many plants and animals produce substances toxic to humans to ward off predators. In addition, the rise of modern industrial economies has provided additional exposure to non-natural toxic substances such as industrial pollutants, pesticides, radiation, and the like.
  • CBW chemical and biological warfare
  • Some biosensors detect target molecules either by binding the target to a specific immobilized molecule (e.g., an antibody or receptor directed to the target), or by the interaction between the target and an enzymatic pathway.
  • binding of the target to the immobilized antibody molecule triggers a change in one of a variety of measured properties (e.g., by measuring changes in surface plasmon resonance, fluorescence, luminescence, mass, or calorimetry).
  • the binding of a target that is antigenic to a detector comprising mast cell-bound IgE antibody specific for the target triggers the activation of the mast cells. This leads to the generation of heat, which is registered using a microcalo ⁇ miter.
  • the presence of a target molecule is detected by its capacity to serve as a substrate for an enzymatic reaction or by virtue of its ability to inhibit a reaction whose progress is being monitored
  • immobilized enzyme-based detection systems include those used for the detection of organophosphates such as the nerve agent Sarm (methylphosphonofluo ⁇ dic acid 1-methylethyl ester) and VX (methylphosphonothioic acid S-[2-[b ⁇ s(l-methylethyl)am ⁇ no]ethyl] O-ethyl ester).
  • organophosphates such as the nerve agent Sarm (methylphosphonofluo ⁇ dic acid 1-methylethyl ester) and VX (methylphosphonothioic acid S-[2-[b ⁇ s(l-methylethyl)am ⁇ no]ethyl] O-ethyl ester).
  • organophosphate nerve agents inhibit the enzyme acetylcholinesterase
  • acetylcholinesterase immobilized on a membrane or electrode can be used to generate a signal due to such parameters as the pH change occurring during hydrolysis of its normal substrate or by generation of a current following oxidation of the thiochohne formed by cleavage of butyrylthiocholme.
  • presence of an organophosphate is detected by inhibition of the reaction.
  • both types of sensor are limited to the detection of known, well-characterized agents for which either an antibody or receptor has been identified and purified.
  • Recombinant E. coli has also been used to detect organophosphates.
  • Cryo-immobilized bacteria engineered to express organophosphate hydrolase were exposed to solutions of organophosphates, and the presence of the compound was detected as a pH change caused by the production of protons during the hydrolysis reaction
  • Immobilized enzymes whether present as purified molecules or incorporated into bacteria, allow for a high degree of specificity in detection, provide signal amplification, and are frequently robust.
  • the biochemical pathway of action must be known for the agent to be detected
  • individual enzyme sensors must be designed for every agent to be detected Novel agents will therefore escape detection.
  • the present invention provides cell- and tissue-based sensors for detecting chemical or biological agents in which exposure of the sensor to a toxin produces a measurable perturbation in the function of a normal cell or tissue
  • Such perturbations include, for example, the loss of electrical activity in a neuron, hyperstimulation of a muscle cell, or disruption of a cellular biochemical process.
  • the devices of the present invention will not only allow the identification of known agents but will also provide advance warning of the presence of novel toxic agents.
  • such devices can be used to provide a profile of the cellular and gene expression responses to unknown agents; thereby facilitating the development of counter-agents such as vaccines
  • the present invention provides an apparatus for determining the presence of a toxic agent, such a toxic chemical, bacterium, or virus
  • the apparatus of the invention includes a sampler that comprises a means for extracting a sample from the environment of the apparatus, such as the ambient atmosphere, water, or soil
  • the sampler can include additional pre-processing means to isolate particular elements of the sample or put the sample in better condition for assay.
  • Samples are passed from the sampler to a cell chamber that includes cells that, when exposed to sample, respond to the presence of toxic agents.
  • the cell chamber is configured to support the growth and maintenance of the cells
  • the cell chamber further includes detection means to detect and, optionally, quantitate, the response(s) of the cells to any toxic agent(s) in the sample.
  • detection can include fluorescence, conductive, and colo ⁇ metic detection
  • the cells are derived from mammalian, and, more particularly human, embryonic stem (“ES”) or embryonic germ (“EG”) cells that have been modified to include one or more elements that produce a detectable, and optionally quantitative, response to toxic insult.
  • the cells further are further configured to express telomerase and thereby have extended, possibly indefinite, hfespans.
  • the apparatus further includes a sample storage chamber to store the collected sample for further analysis in addition to data processing means for analyzing data and communications means for transmitting the collected data and/or analysis to remote locations.
  • Figure 1 illustrates one embodiment of a system for detecting chemical and biological agents in accordance with the present invention.
  • DESCRIPTION OF SOME EMBODIMENTS OF THE INVENTION The present invention provides highly specific and robust methods and apparatus for detecting deleterious chemical and biological agents using a variety of cell-based detection techniques.
  • the cells employed in the detectors of the present invention can be any type of human or animal (preferably mammalian, more preferably primate) cell.
  • the use of human or animal cells for detecting chemical or biological toxic agents has the advantage of being less prone to false negative signals, as toxic agents can show species specificity.
  • the cells used for the detection of dangerous substances include cells from tissues most likely to be exposed to, or be affected by, a toxic agent.
  • those cells most likely to be exposed to an environmental toxic agent include, but are not limited to, bronchial and gastric epithelial cells, neuronal cells, cardiomyocytes, and muscle cells.
  • the cells can be used in a substantially unaltered state, or the cells may include one or more modifications. Examples of such modifications include the inclusion of reporter constructs and lifespan extension by the expression of telomerase. Still other modifications will be apparent to those of skill in the art. PREPARATION OF CELLS
  • the cells used in the methods and apparatus of the invention can be obtained from various natural sources, i.e., in a fully differentiated form, or derived from cells of earlier developmental lineage by induced, or uncontrolled, differentiation.
  • the cells used in the methods and apparatus of the invention are derived from embryonic stem (“ES") or embryonic germ (“EG”) cells.
  • ES and EG cells have the advantage of exhibiting normal characteristics (e.g., maintain a normal karyotype through prolonged culture) unlike virally transformed cells, for example.
  • the ES or EG cells used in the invention are obtained from primate sources (e.g., Rhesus monkey cells).
  • human ES and EG cells are used.
  • the source of the ES or EG cells need only be one effective to provide cells having a morphology suitable for the detection of deleterious environmental agents
  • ES and EG cells can be obtained from a variety of sources and cultured using known methods and materials.
  • the ES and EG cells are of primate, more particularly of
  • ES and EG can be differentiated to form cells of any lineage using a variety of methods
  • differentiation to specific, desired cells types can be done using methods and materials described in the US patent and co-pending U.S. Patent Applications incorporated by reference above.
  • An example of a broadly applicable method of obtaining substantially pure populations of specific cell types by the differentiation of ES cells includes the use of a cell-type specific promoter effective to drive a selectable marker gene (e g , one providing resistance to an otherwise toxic drug)
  • a selectable marker gene e g , one providing resistance to an otherwise toxic drug
  • the cells derived from ES- or EG-cells can include one or more genetic modifications.
  • modifications include, but are not limited to, modification of the cells to provide telomerase expression (e.g , using the materials and methods desc ⁇ bed in Bodnar et al., 1998, as well as those methods and materials described in UK Patent GB2317891 and co-pending
  • ES- or EG-de ⁇ ved cardiomyocytes are purified further by the use of cardiomyocyte specific promoters driving a selectable marker, e g., the ⁇ -cardiac mvosm heavy chain (MHC) promoter fused to the aminoglycoside phosphotransferase (neomycm resistance) gene.
  • a selectable marker e g., the ⁇ -cardiac mvosm heavy chain (MHC) promoter fused to the aminoglycoside phosphotransferase (neomycm resistance) gene.
  • MHC ⁇ -cardiac mvosm heavy chain
  • Undifferentiated ES cells can be transfected with the ⁇ -MHC/neo r construct, grown as embryoid bodies as detailed above, then plated onto tissue culture dishes in the presence of the drug G418 Under these conditions, essentially pure populations of cardiomyocytes are isolated (Klug et al , 1996) Thus, by transfecUon of undifferentiated ES cells as described above, large quantities of substantially pure, fully functional cardiomyocytes can be derived
  • cardiomyocytes can be isolated based on different patterns of gene expression.
  • at ⁇ al (but not ventricular) cardiomyocytes express myosin light chain (MLC) 2a.
  • MLC myosin light chain
  • at ⁇ al cardiomyocytes can be produced preferentially to other subtypes of cardiomyocytes.
  • ventricular cardiomyocytes expressing MLC-2v have a pattern of expression complementary to at ⁇ al cardiomyocytes and can be cultured by analogy to the methods just described (Klug et al, 1996).
  • neuroepithelial stem cells are derived from ES or EG cells that are allowed to replicate in msulin-transfe ⁇ n-selenium-fibronectin ("ITSN")-supplemented medium, a medium which is effective m inducing neuronal differentiation in embryonal carcinoma cells
  • ITSN msulin-transfe ⁇ n-selenium-fibronectin
  • bFGF basic fibroblast growth factor
  • the ability to transfect undifferentiated embryonic stem cells also permits a genetic approach to neuroepithelial precursor cell derivation and expansion
  • the use of cell- type specific promoters driving drug resistance genes allows the selection of specialized cells during ES cell differentiation. Accordingly, if the undifferentiated ES cells are stably transfected with a nestin promoter/neo r construct, the use of the culture conditions described above combined with drug selection will provide a significant enrichment for neuroepithelial cell precursors.
  • genetic approaches allow the potential for greatly expanding the pool of neuroepithelial precursors by intervening in the normal differentiation of the precursors.
  • Idl a gene denoted a gene denoted Idl which is repressed upon differentiation (Duncan et al., 1992).
  • This hehx-loop-helix protein is a member of a gene family which antagonizes the differentiation of several cell types when overexpressed (Jen et al , 1992; Kreider et al., 1992; Shoji et al., 1994).
  • a selectable marker driven by a neurotransmitter- synthetic enzyme promoter can be used to isolate a desired cell type.
  • the tyrosine hydroxylase promoter driving the neo r gene will select for doparmne- producmg neurons over other types of neurons. HUMAN INTESTINAL EPITHELIAL CELLS.
  • NHIE Normal human intestinal epithelial cells
  • ATCC American Type Culture Collection
  • FHs 74 Int were isolated from a 3-4 month old fetus in 1976 by R. Owens of the Naval Research Laboratory in Oakland, CA
  • the cell line retains a normal diploid karyotype, does not form colonies in soft agar, is contact inhibited, and does not induce tumors in immunosuppressed mice
  • Normal human intestinal epithelial cells can also be isolated from tissues by established methods. (Owens et al, 1976. Smith, 1979). HUMAN BRONCHIAL EPITHELIAL CELLS. Normal human bronchial epithelial cells (NHBE) can be purchased commercially (e.g., from
  • NHBE cells are derived from normal tissue by established techniques
  • NHBE cells can also be directly isolated from tissues by established methods (Yao, et al. 1997, Lechner er ⁇ / , 1981, Breza-Squibam et ⁇ /., 1996, Franklin et al , 1996) SYSTEMS FOR DETECTING TOXIC CHEMICALS AND BIOLOGICAL AGENTS
  • the present invention includes systems for detecting toxic substances.
  • a sampler 104 provides an interface with the environment external to system 100.
  • the sampler comprises one or more means for extracting materials from the environment proximal to system 100.
  • Such means can include means for extracting atmospheric samples, soil samples, water samples.
  • Sampler 104 can further include means for pre-processing samples such as, but not limited to, water and/or organic solvent extractors, heaters, coolers, homogenators, and the like as will be familiar to those of skill in the art
  • Cell chamber 108 includes cells used for the detection of toxic agents, such as those cell types described above, in addition to systems and materials necessary to maintain the cells in a substantially functional state Examples of such materials and systems include growth and/or nutrient media, means for removing and adding such growth and/or nutrient media, reagents for performing tests, and means for introducing the sample to the cells (e g., one or more injectors), as well as means for detecting the response of the cells to the sample.
  • Sample chamber 108 can include only chamber or can comprise several chambers that may, or may not, be hermetically sealed from each other
  • the cells are maintained in tissue culture wells in the multiwell plates including HEPES -buffered tissue culture medium that is maintain at a pH of 7 4
  • the multiwell plates are sealed and maintained at 37° C in a mobile tissue culture incubator.
  • the culture system will require a mechanism to periodically exchange media to maintain cell viability Once placed in the field, air will be sampled at the site and delivered to the multiwells by in j ection
  • sample materials collected and/or pre-processed by sampler 104 are passed to cell chamber 108 so that the cells contained in cell chamber 108 are exposed to the sample material.
  • Putative toxic agents in the sample material are detected and identified by a series of assays.
  • Toxic agents include, without limitation, chemicals, biologicals and radiation.
  • a first assay detects changes in electrical activity of cell membranes in response to the putative toxic agents. In one embodiment, these cell are the above-described neuronal and cardiomyocyte cells.
  • a second assay monitors fluorescence emissions in a simple live-dead assay to detect cellular lethality.
  • the cells used for the second assay are the above-described telomerase- transfected human bronchial and intestinal epithelial cells.
  • a final set of assays include the above- described indicator cells to provide information regarding the presence of toxic biological organisms such as bacteria (e.g., anthrax) and viruses (e.g., Ebola or Marburg) in the sample.
  • Cell chamber 108 is coupled with Sample Storage chamber 110.
  • the cells upon detection of possible toxic substance in the sample, portions of the sample and the medium bathing the cells in Cell Chamber 108 is taken and stored in Sample Storage Chamber 110 for later recovery and chemical analysis
  • the cells are frozen in situ, e g , using liquid nitrogen in stepwise aliquot fashion over a period of about 12 hours, to facilitate their analysis using gene expression chip arrays as described below, to monitor alterations in gene expression induced by the putative toxic agent.
  • Such a system will allow for the putative toxic agent to be "fingerprinted" and correlated with corresponding chemical analysis of the putative agent from recovered samples and cell medium captured and stored in the field.
  • detector 112 Coupled with cell chamber 108 is detector 112 that includes detection means to monitor and, optionally, quantitate the response(s) of the cells in cell chamber 108 to the sample
  • the detection means used will depend on the type(s) of s ⁇ gnal(s) produced by the cells in response to a toxin.
  • Detector 112 can optionally be coupled with an analyzer 116 that processes signals gathered by detector 112. Such analysis can include waveform and time-series analysis of the signals gathered by detector 112. The analysis can further include pattern recognition to identify pathogenic substances.
  • the apparatus further includes a controller 118, such as a microprocessor or other multipurpose computer, to operate the various elements described above. The controller can further be coupled to a transmitter 120 that relays data and/or analysis results to remote locations at which the information so relayed can be further processed or reviewed.
  • normal cells obtained from differentiation of primate ES cells as described and telomerase-transfected cells having extended life spans, also prepared as described above, are plated on biosensors typically located at the bottom of a multiwell plate.
  • the biosensors are configured to detect electrical changes in membranes of the cells in response to exposure of the cells to a putative toxic agent. In one embodiment, these changes are recorded as a function of time for further analysis. Such analysis can be performed on-site or at a remote site (following transmission of the recorded data) and can be performed manually (e.g., by reading charts or lists of values including or derived from the recorded data) or with the aid of a machine such as a multipurpose computer.
  • the plated cells comprise the above-described cardiomyocytes, and the measured electrical activity includes electrical depolarizations associated with each cardiomyocyte contraction.
  • Recorded parameters for this embodiment can include, e.g., the amplitude, frequency, and/or wave form characterizing the electrical behavior of the cells in response to the putative toxin as a function of time.
  • Such data provides a sensitive measure of cardiomyocyte physiology.
  • neurons are coupled with the biosensor to detect membrane depolarizations, an event associated with toxic insult to such cells.
  • the biosensors are coupled with the above-described bronchial and gastric epithelial cells for detection of toxic agents by the live/dead assay as described below. "LIVE/DEAD" ASSAY
  • the detection system of the present invention includes a "live/dead” assay.
  • Such assays distinguish living cells from diseased or dead cells, for example, by their response to cellular uptake of a dye, such as ethidium homodimer (“EthD”)
  • EthD ethidium homodimer
  • This dye is impermeable to intact cell membranes but permeable once the integrity of the cell membrane is compromised.
  • Ethidium homodimer free in solution, exhibits low fluorescence; thus, exposure of normal, healthy cells to EthD will provide a low level of fluorescence upon interrogation of the sample.
  • EthD enters cells having compromised membranes (i.e , unhealthy or dead cells) and intercalates with the cellular DNA
  • the resulting fluorescence is dramatically enhanced Ethidium homodimer is stable in solution (if protected from light) and is not cytotoxic to the cells and can be added as a standard component to the medium
  • This assay is simple, rapid and reliable and will give a real-time readout of the effect of the CBW agent on the cells
  • Such assays are available commercially (Molecular Probes, Eugene, OR).
  • an air sample is incubated with telomerase transfected bronchial and/or gastric epithelial cells in a medium containing EthD to expose thereby the cells to the suspected toxic agent
  • Fluorescence readings are taken periodically using an excitation wavelength of 530 nm and an emission wavelength of 645 nm, (e.g., once every minute) and the resulting data coOllected and analyzed
  • An increase detection of a "red" signal indicates that the cells' membranes are damaged (i.e , the dye has entered the cells and stained their cellular DNA).
  • embryonic stem cells are plu ⁇ potent. Embryonic stem cells have the ability to develop into any cell derived from the three germ cell layers or an embryo itself.
  • an ES cell line When injected into SCID mice, an ES cell line will differentiate into the cells derived from all three embryonic germ layers including: bone, cartilage, smooth muscle, st ⁇ ated muscle, and hematopoietic cells (mesoderm); liver, primitive gut and respiratory epithelium (endoderm), neurons, g a cells, hair follicles, and tooth buds (ectoderm)
  • a normal ES cell in contrast to a compromised ES cell (i.e., an ES cell affected by a toxic agent), a normal ES cell has the ability to participate in normal development Developmental potential can be evaluated by injecting approximately 0.5-1.0 xlO 6 primate ES cells into the rear leg muscles of 8-12 week old male SCID mice.
  • ES cells injected into the hind leg muscles of SCID mice in this way contribute to normal differentiated tissues derived from all three embryonic germ layers and to germ cells, when analyzed 15 weeks later.
  • An agent having a toxic effect on ES cells can impair
  • the presence and identity of toxic agents is detected using cells transfected with a sensitive reporter gene linked to specific promoter sequences.
  • the choice of promoter dictates the stimulus to which the reporter gene responds.
  • the reporter system is one having the following properties: a) high sensitivity; b) ability to be assayed in living cells; c) utilizes a substrate that is easy to load within cells; and d) is capable of providing real-time responses to the presence of stimuli.
  • the promoter is one that is activated in the presence of one or more toxins or viruses.
  • the cells used in the detection system of the present invention are derived from primate ES or EG cells that have been stably transfected with the reporter gene/promoter sequence just described.
  • the Gram negative bacterium Vibrio cholerae produces cholera toxin, an enterotoxin producing the characteristic massive diarrhea which can cause death within 18 hours of infection.
  • the toxin is cholera and the promoter comprises a cyclic AMP response element ("CRE").
  • CRE is an octamer consisting of the sequence 5'-
  • TGACGTCA-3' This sequence can confer cAMP inducibility upon minimal promoters by virtue of its ability to bind members of the cAMP response element binding protein family (Tinti et al, 1996).
  • Promoters which contain cAMP responsive elements (CREs) show large increases in activity when intracellular cAMP levels are raised, either by the direct addition of cAMP to cultured cells or by stimulation of adenylate cyclase by molecules such as cholera toxin or forskolin (Gabellini et al, 1991).
  • a vector comprising a reporter gene under the control of a basal promoter (containing a TATA and CAAT sequence) and comprising multiple copies of the canonical CRE within similar distance of the start site, as described above.
  • This construct when introduced into cells, will serve as an indicator of the presence of cholera toxin, since the reporter gene will be induced by the resulting elevated cAMP.
  • Such cells can be grown on a biologically compatible surface connected to a sensitive detector of the reporter gene product, e.g., fluorescence or chemiluminescence.
  • the reporter gene is a LacZ gene.
  • the LacZ gene encoding E-coli ⁇ - galactosidase, has been widely used as a reporter gene in eukaryotic cells.
  • the development of lipophilic fluorescinated-derivatives of di- ⁇ -D-galactopyranoside allows sensitive, real-time measurement of ⁇ -galactosidase activity in living cells (Zhang et al, 1991).
  • These substrates commercially available as ImaGene Green and ImaGene Red from Molecular Probes, Inc., Eugene, OR
  • these molecules Prior to cleavage by ⁇ -galactosidase, these molecules are non-fluorescent and pass freely through cellular membranes. Once the glycosidic linkage is cut, the product fluoresces and the lipophilic cleavage product is retained within the cell (Zhang et al, 1991).
  • These substrates demonstrate very low toxicity to cultured cells. For example, LacZ-expressing NIH3T3 cells grown for several days in the presence of typical concentrations of these substrates retained normal morphology and continued to divide (Hoagland and Johnson, 1993); NIH3T3 cells and CRE BAG 2 cells grown in medium containing ImM (20- fold excess) ImaGene Green showed normal morphology and viability (Zhang et al, 1991).
  • the reporter used is the E-coli ⁇ -lactamase coding sequence in conjunction with a family of membrane-permeable, fluorogenic substrates (Zlokarnik et al, 1998).
  • a family of membrane-permeable, fluorogenic substrates Zlokarnik et al, 1998.
  • One such substrate is CCF2/AM a non-polar, non-fluorescent molecule that can penetrate cell membranes. Once inside the cell, nonspecific esterases hydrolyze the molecule's ester groups, leaving the polar CCF2 substrate trapped within the cell.
  • CCF2 contains two distinct fluo ⁇ hores in close proximity, separated by a target site for ⁇ -lactamase cleavage. Due to the overlapping spectra of the two fluorophores, their fluorescence is quenched by fluorescent resonance energy transfer (FRET).
  • FRET fluorescent resonance energy transfer
  • Classical examples of the operation of such systems include responses to heat shock, UV damage, xenobiotic challenge, and viral pathogens. These responses can be monitored by measuring and evaluating changes in gene expression patterns, e.g., by measuring changes in rnRNA expression. However, alterations in gene expression patterns have thus far relied on low throughput methods, such as Northern blot analysis and RT-PCR, that typically track only well-characterized genes.
  • High density DNA arrays have proven to be a powerful means of monitoring mRNA expression patterns. These technologies allow thousands of independent genes to be monitored simultaneously. This is accomplished by depositing or synthesizing target gene DNA onto glass or silicon surfaces at a density that can su ⁇ ass 10,000 elements per square centimeter. For example, once the DNA array has been designed and fabricated, the process of expression monitoring begins by converting a population of mRNA molecules into a fluorescent probe which is then hybridized directly to the DNA array. Subsequently, the unhybridized probe is removed by stringent washing and the remaining signal is detected with a fluorescence monitor; the signal at any element of the array is proportional to the relative abundance of an mRNA species in the starting RNA population.
  • a modification of this general procedure is used to perform a comparison of the relative expression levels between two independent RNA populations.
  • the two populations are converted into probe using separate labeling fluors that can be distinguished on the basis of their emission spectra.
  • the two probes When the two probes are applied to a single array, they compete for hybridization to the array elements.
  • the relative contribution of the two probes can be quantified, thus defining the relative abundance of a specific mRNA in the two populations. Since much of the interest in monitoring gene expression lies in determining relative changes in expression, as opposed to absolute abundance, this technique has wide application.
  • an array comprising at least about 1 ,000, more particularly at least about 10,000, and, still more particularly, at least about 100,000, independent genes that survey widely the major regulatory systems of the human cell is provided in the above-described detection system.
  • genes include, but are not limited to, tumor suppressors, oncogenes. receptors and growth factors, cell cycle components, stress response, and toxicology genes.
  • the arrays include genes related to additional targets of any origin, such as additional human viral or bacterial genes.
  • cells prepared as described above are exposed to specific agents (such as viral toxins, xenobiotics and radiation and RNA). The expression patterns of the genes in the cells is determined using the arrays just described arrays and compared to a control. Changes in expression pattern relative to the control are used to determine the presence of a toxic agent.
  • telomere activity is reduced by the telomeric repeat amplification protocol (TRAP assay) (Kim, et al, 1994).
  • Bodnar A.G.. et al. Science 279:349-352 1998. Breckenridge, L.J., et al. J. Neurosci Res 42:266-276, 1995.

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Abstract

L'invention se rapporte à un procédé et à un appareil permettant de déceler la présence d'un agent toxique, tel qu'un produit chimique toxique, une bactérie ou un virus. Dans une réalisation, l'appareil comporte un échantillonneur comprenant un organe conçu pour extraire un échantillon de l'environnement dans lequel se trouve ledit appareil, par exemple un échantillon d'atmosphère ambiant, d'eau ou de sol. Ledit échantillonneur peut éventuellement comporter un organe supplémentaire de prétraitement conçu pour isoler des éléments particuliers de l'échantillon ou pour placer l'échantillon dans de meilleures conditions d'analyse. L'appareil et le procédé de cette invention peuvent permettre la détection d'une variété de toxines chimiques et biologiques, qu'il s'agisse de toxines naturelles ou non, et notamment de toxines chimiques et biologiques de composition et d'origines inconnues.
PCT/US1999/010770 1998-05-15 1999-05-14 Procede et appareil de detection d'agents presentant un danger pour la sante WO1999060371A2 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6447995B1 (en) 2000-10-04 2002-09-10 Genvec, Inc. Utilizing intrinsic fluorescence to detect adenovirus
EP1271145A1 (fr) * 2001-06-25 2003-01-02 Cardion AG Procédé et populations cellulaires pour l'identification et la validation de séquences génomiques et pour le criblage de médicaments
EP1423506A2 (fr) * 2001-08-06 2004-06-02 Vanderbilt University Appareil et procedes permettant de discriminer un agent a l'aide de matiere biologique
US7445930B2 (en) 1996-11-20 2008-11-04 Introgen Therapeutics Inc. Method for the production and purification of adenoviral vectors

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5660986A (en) * 1987-06-05 1997-08-26 Us Health Immortalized human cell lines containing exogenous cytochrome P450 genes
US5661035A (en) * 1995-06-07 1997-08-26 The Regents Of The University Of California Voltage sensing by fluorescence resonance energy transfer

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5660986A (en) * 1987-06-05 1997-08-26 Us Health Immortalized human cell lines containing exogenous cytochrome P450 genes
US5661035A (en) * 1995-06-07 1997-08-26 The Regents Of The University Of California Voltage sensing by fluorescence resonance energy transfer

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE ON DIALOG, 09317177 CAMILLERI ET AL: 'Validity of a Model of cultured Myocardial Cells for Assessment of Cardioplegia' & CELLULAR BIOLOGY AND TOXICOLOGY vol. 13, no. 6, October 1997, pages 435 - 444 *
HARLE-BACHOR ET AL: 'Telomerase Activity in the Regenerative Basal Layer of the Epidermis in Human Skin and in Immortal and Carcinoma-Derived Skin Keratinocytes' PROC. NATL. ACAD. SCI. USA, vol. 93, June 1996, pages 6476 - 6481, XP002923264 *
SEHLMEYER ET AL: 'Primordial Germ Cell-derived Embryonic Germ Cells of the Mouse-In Vitro Model for Cytotoxicity Studies with Chemical Mutagens' TOXICOLOGY IN VITRO. vol. 10, 1996, pages 755 - 763, XP002923261 *
STEGMANN ET AL: 'Membrane Fusion Activity of Influenza Virus. Effects of Gangliosides and Negatively Charged Phospholipids in Target Liposomes' BIOCHEMISTRY vol. 28, 1989, pages 1698 - 1704, XP002923263 *
WOBUS ET AL: 'In Vitro Cellular Models for Cardiac Development and Pharmacotoxicology' TOXICOLOGY IN VITRO. vol. 9, no. 4, pages 477 - 488, XP002923262 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7445930B2 (en) 1996-11-20 2008-11-04 Introgen Therapeutics Inc. Method for the production and purification of adenoviral vectors
US7510875B2 (en) 1996-11-20 2009-03-31 Introgen Therapuetics, Inc. Methods for producing purified adenoviral vectors
US6447995B1 (en) 2000-10-04 2002-09-10 Genvec, Inc. Utilizing intrinsic fluorescence to detect adenovirus
US6630299B2 (en) 2000-10-04 2003-10-07 Genvec, Inc. Fluorescence detection
EP1271145A1 (fr) * 2001-06-25 2003-01-02 Cardion AG Procédé et populations cellulaires pour l'identification et la validation de séquences génomiques et pour le criblage de médicaments
WO2003001202A1 (fr) * 2001-06-25 2003-01-03 Cardion Ag Methodes et populations cellulaires convenant pour l'identification et la validation de cibles genomiques, et pour le criblage de medicaments
EP1423506A2 (fr) * 2001-08-06 2004-06-02 Vanderbilt University Appareil et procedes permettant de discriminer un agent a l'aide de matiere biologique
EP1423506A4 (fr) * 2001-08-06 2008-08-06 Univ Vanderbilt Appareil et procedes permettant de discriminer un agent a l'aide de matiere biologique
US7704745B2 (en) 2001-08-06 2010-04-27 Vanderbilt University Apparatus and methods for monitoring the status of a metabolically active cell
US7713733B2 (en) 2001-08-06 2010-05-11 Vanderbilt University Device and methods for detecting the response of a plurality of cells to at least one analyte of interest
US7981649B2 (en) 2001-08-06 2011-07-19 Vanderbilt University Device and methods for monitoring the status of at least one cell

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