WO2008150802A1 - Analyses pour la détection de mutagenèse - Google Patents

Analyses pour la détection de mutagenèse Download PDF

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WO2008150802A1
WO2008150802A1 PCT/US2008/064985 US2008064985W WO2008150802A1 WO 2008150802 A1 WO2008150802 A1 WO 2008150802A1 US 2008064985 W US2008064985 W US 2008064985W WO 2008150802 A1 WO2008150802 A1 WO 2008150802A1
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cell
del
cells
culture
assay
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Robert H. Schiestl
Jin Aubrecht
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The Regents Of The University Of California
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Priority to US12/627,985 priority Critical patent/US9193983B2/en

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    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/5076—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 involving cell organelles, e.g. Golgi complex, endoplasmic reticulum

Definitions

  • the invention relates to colorogenic assays and to cells and kits useful therein.
  • Mutagens are agents that cause an increase in the rate of mutation, i.e. detectable and heritable structural changes in the genetic material of an organism. Such changes may include the addition or deletion of a whole chromosome, a structural change to a chromosomes (e.g., a translocation) and a structural change to a portion of the genomic sequence (e.g., point mutations, mutations to multiple sequential nucleotides and deletions of portions of the genomic sequence) . Because genetic changes can damage or otherwise interfere with the action of genes, mutagens are characterized as genotoxins, i.e. agents that are toxic to genes.
  • the disclosure provides a method for characterizing a test agent, comprising: treating a eukaryotic cell comprising a DEL selection marker with a test agent; and measuring mitochondrial activity following treating the eukaryotic cell with the test agent.
  • the disclosure uses an MTT or MTS reagent to detect mitochondrial activity.
  • the cell is a yeast cell.
  • the disclosure provides a method for characterizing a test agent, comprising: providing a eukaryotic cell culture comprising a DEL selection marker; treating the eukaryotic cell culture with or without a test agent; measuring the mitochondrial activity of a treated portion of the cell culture in the presence of a suitable selection medium; and measuring the mitochondrial activity of an untreated portion of the cell culture in the presence of said selection medium.
  • the disclosure uses an MTT or MTS reagent to detect mitochondrial activity.
  • the cell is a yeast cell.
  • the disclosure also provides a kit comprising a cell comprising a DEL selection marker and a mitochondrial activity detection agent.
  • Figure 1 shows the DEL assay was simulated by adding different dilutions of RS112 His + revertants to 100,000 background RS112 cells in -His media. 12, 18, and 24 hour time points are charted. At 12 hours, RS112 His + additions corresponding to 25, 50, and 100 DEL events per 10,000 cells were discernibly significant. By both 18 and 24 hours, as few as 2.5 DEL events per 10,000 cells were significantly detectable. Yet at 24 hours, whilst 25-1000 RS112 cells are still significantly different than background, growth in -His media becomes saturated and response pattern is lost. The experiment was carried out using at least 6 repeats for each treatment group, and the results are presented as means ⁇ SD. Significance * (p ⁇ 0.05).
  • Figure 2A-C show well-based DEL assay evaluating the cytotoxicity and genotoxicity of 13 carcinogens by measuring O. D. (490 nm) 14h after the addition of MTS. Cytotoxicity in treated samples is represented by diminished growth in +13 media compared to untreated yeast labeled "RS112.” Genotoxicity is represented by increased growth in -HIS media compared to untreated yeast.
  • panel A compounds were previously tested using the DEL assay;
  • panel B crosslinking agents previously uncharacterized by DEL assay;
  • panel C crosslinking agents previously uncharacterized by DEL assay.
  • Bioluminescence means light emission in a living cell wherein the light emission is dependent upon and responsive to metabolic activity.
  • Bioluminescent marker means a nucleotide sequence that, when incorporated into a cell and expressed, causes bioluminescence during metabolic activity of the cell.
  • colorogenic refers to a composition that generates a colored composition or a colored composition that exhibits a change in its absorption spectrum upon interacting with another substance, for example, upon binding to a biological compound or metal ion, upon reaction with another molecule or upon metabolism by an enzyme. In some aspects, colorogenic labels result in a detectable precipitate.
  • Gene means a chromosomal fragment that expresses a specific protein, including regulatory sequences preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence. The term gene includes endogenous genes in their natural location in the genome or foreign genes that are not normally found in the host organism, but are introduced into the host organism by recombinant molecular biology techniques.
  • Mutagens are agents that cause an increase in the rate of deletion or gene recombination. Mutagens may have genotoxic effect by damaging or otherwise interfering with the action of genes.
  • “Mutation” is a detectable and heritable structural change in the genetic material of an organism, and may include the addition or deletion of a whole chromosome, a structural change to a chromosomes (e.g., a translocation) and a structural change to a portion of the genomic sequence (e.g., point mutations, mutations to multiple sequential nucleotides and deletions of portions of the genomic sequence) .
  • DEL selection marker means a disrupted genetic locus wherein: (1) the disruption comprises an insertion of a heterologous polynucleotide within the genetic locus; (2) said heterologous polynucleotide comprises one duplication of a portion of the genetic locus; (3) the head-to-tail (i.e., 5' end to 3' end) orientation of the duplicated portion of said heterologous polynucleotide is the same as that of the genetic locus; and (4) the genetic locus is useful for phenotypic selection of the cell when grown on suitable selection media.
  • DEL selection markers are described below .
  • Selection medium means a composition which can be used for phenotypic selection of cells.
  • a nutrient composition which lacks histidine can be used to selectively screen for yeast cells that are able to produce histidine.
  • a nutrient composition which contains the antibiotic G418 can be used to selectively screen for cells that have the neo resistance gene.
  • Other commonly used selection medium will be readily apparent to one of skill in the art.
  • Suitable selection medium refers to a selection medium having a composition that can be used for phenotypic selection of cells based upon the selection marker. Typically such a medium will comprise a composition that results in positive or negative selection of an appropriate cell types.
  • the DEL assay also known as the intrachromosomal recombination assay, first described by Schiestl et al . (1988) using Saccharomyces cerevisiae, measures deletions of parts of [0027] Schiestl et al. (1988) reported a positive selection system for intrachromosomal recombination in the yeast, Saccharomyces cerevisiae, by integration of a plasmid containing an internal fragment of the HIS3 gene at the HIS3 locus resulting in two copies of the gene with terminal deletions at the 3' end of one and 5' end of the other.
  • the target gene sequences used in the DEL assay are genes whose function has been disrupted by the integration of an exogenous DNA fragment.
  • Schiestl et al . (1988) describes the use of a strain of S. cerevisiae designated "RSY6" (available from the ATCC, deposit number 201682), in which the HIS3 gene is disrupted by the integration of an exogenous DNA fragment.
  • the resulting his-yeast strain requires histidine in its growth medium in order to grow. In histidine-free medium, a very small number of cells will spontaneously revert to his + . However, when the cells are treated with a mutagen, the reversion rate increases beyond the normal background level.
  • a mutagen causes the formation of double-stranded DNA breaks.
  • a cell's own repair mechanism may result in removal of the exogenous DNA and repair of the sequence (e.g., by single-strand annealing) , thus the assays of the disclosure select for a deletion by recombination between a repeated sequence and reversion of the gene to its wild-type form.
  • the DEL assay has certain advantages over other mutagenicity assays. For example, it has been reported that the DEL assay has better predictability of carcinogenicity than the more commonly used Salmonella reverse mutation Ames assay. Many carcinogenic compounds which give negative results using the Ames assay are positive by the DEL method (Bishop and Schiestl, 2000) .
  • DEL assay is its impracticality for large scale and automated screening of potential mutagens (i.e., high throughput screening) .
  • the current assay requires that cells be given enough time to grow into visible colonies in order to determine whether a test compound is a potential carcinogen.
  • the current assay cannot be miniaturized, for example, into a multi-well plate system, which would enable a reduction in the amount of test agent necessary.
  • the disclosure overcomes this problem through the design of a liquid version of the DEL assay.
  • Yeast growth can be identified in a non-clonogenic quantitative colorimetric assay, which measures the ability of proliferating cells to reduce MTT (3-4, 5-dimethylthiazol-2- yl) -2 , 5-diphenyl tetrazolium bromide), a yellow tetrazolium salt, into a purple formazan precipitate. This reaction however, requires quenching and solubilizing the cells in order to measure the formazan precipitate.
  • MTT [3- (4,5-dimethylthiazol-2-yl) -2,5- diphenyltetrazolium bromide] assay, first described by Mosmann in 1983, is based on the ability of a mitochondrial dehydrogenase enzyme from viable cells to cleave the tetrazolium rings of the pale yellow MTT and form a dark blue formazan crystals which is largely impermeable to cell membranes, thus resulting in its accumulation within healthy cells. Solubilization of the cells by the addition of a detergent results in the liberation of the crystals which are solubilized. The number of surviving cells is directly proportional to the level of the formazan product created. The color can then be quantified using a simple colorimetric assay. The results can be read on a multiwell scanning spectrophotometer (ELISA reader) .
  • ELISA reader multiwell scanning spectrophotometer
  • An improvement upon the MTT assay can be made by substitution with the MTS tetrazolium compound [3- (4,5- dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4- sulfophenyl) ] -2H-tetrazolium, inner salt and an electron- coupling reagent PES (phenazine ethosulfate) .
  • the MTS compound is reduced by cells into a colored formazan product soluble in tissue culture medium. This reaction need not be quenched and cell proliferation can be directly measured by recording absorbance at 490nm. Cell proliferation is proportional to the quantity of formazan product.
  • the MTS assay is suitable as a colorimetric assay that can be used for high-throughput applications.
  • MTS is used to construct a liquid based version of the yeast DEL assay capable of scoring DNA deletions on both 96 and 384-well plate formats.
  • MTS tetrazolium compound is reduced to a colored formazan product in metabolically active cells; it is therefore an assay of viable cell number. A decline in values implies cell killing.
  • the disclosure provides a method and system for determining the mutagenic properties of an agent.
  • a recombinant cell comprising a DEL selectable marker is contacted with an agent suspected of being mutagenic under conditions wherein the agent and the cell interact.
  • the viability of the cells are then measured in a selectable medium.
  • the cells are assayed for viability using a colorgenic MTT or MTS assay or other mitochondrial cell viability assay.
  • a mutagenic agent will provide improved recombination and survival in a selectable medium.
  • cell viability can be measured by adding an MTS solution to wells and incubating the cells with the MTS solution during which a colored formazan product is generated in viable cells.
  • MTS assay One advantage of the MTS assay is that the formazan product is soluble in tissue culture medium which avoids the solubilization step of the MTT assay.
  • Viable cell number is then measured by reading absorbance at 490 nm in a Dynex microtitre plate reader. Cell viability is represented as the ratio of absorbance at time "x" (post drug addition) minus "blank” readings (medium with drug without cells) over absorbance at time zero (prior to drug addition) minus blank readings (medium without drug or cells), expressed as a percentage.
  • the mutagenecity is directly proportional to the amount of DEL increase.
  • DEL is a ratio: growth of yeast on the selective medium (-HIS) compared to growth on complete medium.
  • one of the benefits of the DEL assay of the disclosure is that you can measure two endpoints with one assay; one can measure cytotoxicity (e.g, the amount of cell killing an agent causes a cell) and genotoxicity (the amount of mutagenesis an agent causes a cell) .
  • cytotoxicity e.g, the amount of cell killing an agent causes a cell
  • genotoxicity the amount of mutagenesis an agent causes a cell
  • the disclosure is based, in part, on the discovery of DEL-type methods that use colorogenic MTT or MTS assays as a positive indicator of mutagenicity as DEL recombination events. By this disclosure, one may expeditiously and economically test agents of unknown carcinogenic potential for DEL recombination in a manner that was previously unavailable. [0038]
  • the disclosure involves the use of cells having, as a component a disrupted DEL-type selection marker. The marker is used to select those cells that undergo recombination events induced by a test mutagen.
  • the rate of deletion or gene recombination will be increased relative to a control conferring to the cell increased survival in a selection medium.
  • the viability of the cells is measured using a colorogenic assay comprising mitochondrial enzymatic activity (e.g., MTS or MTT assay) .
  • MTS or MTT assay mitochondrial enzymatic activity
  • the methods and cells of the disclosure which are based on colorogenic detection (e.g., MTT or MTS reduction) of revertant cells on selection medium which allows for multi- well high throughput screening techniques for testing agents.
  • the methods and cells of the disclosure enable a significant reduction in the amount of test agent necessary for mutagenicity testing. This can be a significant advantage where test agents are only available in small quantities.
  • the use of MTT and MTS reduction allows the use of plate reader devices which can measure absorbance of the colorogenic signal.
  • any suitable eukaryotic cells may be used in the practice of this disclosure.
  • the cells may originate from vertebrate organisms, such as mammals, birds, fishes, reptiles and amphibians as well as invertebrates (e.g., insects, nematodes) and single-celled eukaryotes.
  • the cells may be derived from any organ or tissue, including blood, endothelium, thymus, spleen, bone marrow, liver, kidney, heart, testis, ovary, heart and skeletal muscle, and can be primary cells or cells derived from immortalized cell lines.
  • Typical cells include human lymphoblastoid cell lines such as GM6804 (see, for example, Monnat, R. J. et al . (1992) and Aubrecht, J. et al . (1995)) and yeast cells, for example, of the species, Saccharomyces cerevisiae .
  • Cells and cell lines for use in the methods of this disclosure may be obtained, for example, from the ATCC, Manassas, Va. 20110-2209.
  • a DEL selection marker means a disrupted genetic locus wherein: (1) the disruption comprises an insertion of a polynucleotide within the genetic locus; (2) said polynucleotide comprises one duplication of a portion of the genetic locus; (3) the head-to-tail (i.e., 5' end to 3' end) orientation of the duplicated portion of said polynucleotide is the same as that of the genetic locus; and (4) the genetic locus is useful for phenotypic selection of the cell.
  • suitable DEL selection markers based upon such a genetic locus can encompass the sequences A-B-C-B- C-D-E-F-G.
  • any suitable phenotype selection marker may be used for the DEL selection marker in the practice of the disclosure. It will be further appreciated that the type of selection marker used may, in part, depend upon the types of cells used in the practice of the disclosure.
  • the DEL selection marker comprises a disruption of the function of a nutrient marker gene, such that the cell requires, as a result of this disruption, a specific nutrient in order to maintain its viability, metabolic activity or growth.
  • agents may be tested for their ability to cause reversion of the nutrient marker to its non-disrupted form, thus enabling cells to thrive in media lacking the corresponding nutrient.
  • An exemplary nutrient marker includes the his3 in yeast cells which alters cellular requirements for histidine. Other nutrient markers will be apparent to those with skill in the art based upon the present disclosure.
  • the DEL selection marker is a gene that conveys resistance to specific physical or chemical agents that would otherwise be toxic to the cell (i.e., hinder viability, metabolic activity or growth) .
  • Such "resistance markers” confer resistance to the cell against chemical agents, including, for example, antibiotics, antimetabolites or herbicides.
  • a disruption of the function of the resistance marker gene causes toxicity to the cell when exposed to the toxic agent.
  • this embodiment comprises the testing of agents for their ability to cause reversion of the gene to its non-disrupted form, thereby enabling the cells to thrive in media containing the toxic substance.
  • Exemplary resistance markers include dhfr (dihydrofolate reductase) which confers resistance to methotrexate; neo, which confers resistance to the aminoglycosides, neomycin and G418; and als and pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (see, Wigler, M. et al . (1980); Colbere-Garapin, F. et al . (1981)).
  • Other resistance markers are known to those with skill in the art or will be apparent to them based upon the present disclosure. Transfection or transformation of a cell with a disrupted resistance marker can be performed using techniques known to those of skill in the art.
  • DEL selection markers may also encompass a non- disrupted nutrient or resistance marker that is controllable by a secondary genetic element, wherein the function of the secondary genetic element is disrupted.
  • Such secondary genetic element may include a gene which encodes a transcriptional activator protein which binds to an activation domain, thereby initiating or accelerating the rate of transcription of the nutrient or resistance marker.
  • agents may be tested for their ability to cause reversion of the secondary genetic element to its functional form, thereby enabling the expression of the nutrient marker or resistance marker gene.
  • an exemplary transcriptional activators and activation domain sequence combination includes the Tet-controlled transactivator which is part of the BDTM Tet-Off Gene Expression System (BD Biosciences, Palo Alto, Calif.). Other transcriptional activators and activation domain sequences are known to those with skill in the art or will apparent to them based upon this disclosure. [0046] As will be further appreciated by those with skill in the art based upon the disclosure, the DEL selection markers may also encompass a non-disrupted negative selectivity marker gene that is controllable by a transcriptional repressor genetic element, wherein the function of the transcriptional repressor is disrupted. When active, the negative selectivity marker is toxic to the cell.
  • agents may be tested for their ability to cause reversion of the transcriptional repressor to its functional form, thereby enabling the expression of the negative selectivity marker gene.
  • An exemplary negative selectivity marker is the herpes simplex virus gene, thymidine kinase, which causes cytotoxicity in the presence of the drug, gancyclovir (Moolton (1986)) .
  • Other negative selectivity markers include Hprt (cytotoxicity in the presence of 6-thioguanine or 6-thioxanthine) , and diphtheria toxin, ricin toxin, and cytosine deaminase (cytotoxicity in the presence of 5-fluorocytosine) .
  • a transcriptional repressor genetic element would, when expressed, repress expression of the negative selectivity marker.
  • An exemplary transcriptional repressor is through the use of RNA interference (RNAi) using methods, for example, described in Fire et al . (1998), in Brummelkamp et al . (2002) and by other methods known to those with skill in the art.
  • RNAi RNA interference
  • the disrupted gene or genetic element that makes up a DEL selection marker used in the methods and cells of this disclosure may be an endogenous gene or genetic element or it may be an exogenous gene or genetic element introduced into a progenitor cell by recombinant methods that are well known to those with skill in the art based upon the disclosure.
  • the cells used in this disclosure may be either haploid, having one copy of each type of chromosome, or diploid, having two copies of each chromosome-type.
  • diploid cells when diploid cells are used in the methods and cells of this disclosure and the disrupted gene or genetic element that makes up a DEL selection marker is an endogenous gene or genetic element, or when there is otherwise more than one copy of an endogenously existing gene or genetic element, typically all copies of the gene or genetic element will be disrupted for the practice of methods and use of cells of the disclosure.
  • the DEL selection marker for use in Saccharomyces cerevisiae yeast cells comprises a HIS3 gene which is disrupted by insertion of the plasmid pRS6 as described in Schiestl et al . (1988) and which is contained in the S. cerevisiae strains RSY6 and RS112 as described in U.S. Pat. No. 4,997,757, (all of which are incorporated herein by reference in their entirety.
  • the disclosure uses a colorogenic detectable signal based on mitochondrial enzymes to detect cell viability.
  • a number of other enzymatic colorogenic assays can be used in the methods of the disclosure.
  • a combination of detectable signals can be used.
  • a combination of bioluminescence and colorogenic methods can be used. It will be appreciated by those with skill in the art, based upon the disclosure, that any suitable bioluminescent marker may be used in the practice of the disclosure. It will be further appreciated that the type of bioluminescent marker used may, in part, depend upon the types of cells used in the practice of the disclosure.
  • An exemplary bioluminescent marker for use in yeast cells is the firefly luciferase (luc) gene (GeneBank accession number AAA89084) driven by a constitutive glyceraldehydes-3-phosphate dehydrogenase (GPD) promoter.
  • the bioluminescence catalyzed by the luc gene requires the substrate (luciferin) and energy in the form of endogenous ATP. So long as the medium in which the cells grow contains luciferin as a supplement, the bioluminescence of yeast cells is exclusively dependent on the availability of intracellular ATP. Since the intracellular ATP concentration is dependent on energy metabolism, the bioluminescent output represents the level of metabolic activities of yeast cell.
  • a test compound which causes a deletion recombination event to restore function of a DEL selection marker allows the cells to maintain metabolic activities and multiply in the absence of the applicable nutrient or the presence of a potentially cytotoxic substance.
  • bioluminescent markers that may be used in the methods and cells of this disclosure are known to those with skill in the art or will be apparent to them based upon the present disclosure.
  • Bronstein et al . (1994) describe bioluminescent markers that may be used in this disclosure.
  • the bioluminescent markers and DEL selection markers that are used in the methods and cells of the disclosure may be incorporated into a cell by inserting the polynucleotide encoding such markers into an appropriate vector.
  • Such vectors may be designed so that they are stably incorporated into the chromosomal DNA of a cell or they may be designed to express the applicable marker without chromosomal integration.
  • Expression vectors containing the necessary elements for transcriptional and translational control of the inserted coding sequence in a cell may be used to incorporate into a cell a biologically active enzyme (for generation of a colorogenic signal), a bioluminescent marker or a DEL selection marker that will become biologically active upon reversion following treatment with a test agent.
  • the transcriptional and translational control elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding the applicable marker. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding the markers.
  • Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • sequences encoding a marker and its initiation codon and upstream regulatory sequences are inserted into the appropriate expression vector, no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
  • Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al . (1994)).
  • a typical method of incorporating a DEL selection marker is through homologous recombination.
  • Homologous recombination methods for incorporating engineered gene constructs into the chromosomal DNA of cells are well known to those skilled in the art and/or those that will be further apparent to them based upon the disclosure.
  • a DEL selection marker targeting vector is introduced into a cell having the undisrupted target gene.
  • the introduced vector targets the gene using a nucleotide sequence in the vector that is homologous to the target gene.
  • the homologous sequence facilitates hybridization between the vector and the sequence of the target gene. Hybridization causes integration of the vector sequence into the target gene through a crossover event, resulting in disruption of the target gene.
  • cloning vectors may be used as vector backbones in the construction of the DEL selection marker targeting vectors of the disclosure, including pBluescript-related plasmids (e.g., Bluescript KS+11), pQE70, pQE60, pQE-9, pBS, pDIO, phagescript, phiX174, pBK Phagemid, pNH8A, pNHl ⁇ a, pNH18Z, pNH46A, ptrc99a, pKK223- 3, pKK233-3, pDR540, and pRIT5 PWLNEO, pSV2CAT, pXTl, pSG (Stratagene) , pSVK3, PBPV, PMSG, and pSVL, pBR322 and pBR322- based vectors, pMB9, pBR325, pKH
  • pBluescript-related plasmids e.g., Bluescript
  • vectors are available from a variety of commercial sources (e.g., Boehringer Mannheim Biochemicals, Indianapolis, Ind. ; Qiagen, Valencia, Calif.; Stratagene, La Jolla, Calif.; Promega, Madison, Wis.; and New England Biolabs, Beverly, Mas.) .
  • any other vectors e.g. plasmids, viruses, or parts thereof, may be used as long as they are replicable and viable in the desired host.
  • the vector may also comprise sequences which enable it to replicate in a host cell whose genome is to be modified. The use of such a vector can expand the interaction period during which recombination can occur, increasing the efficiency of targeting (see Ausubel et al (2003), Unit 9.16, FIG. 9.16.1).
  • the specific host cell employed for propagating the targeting vectors of the disclosure is not critical. Examples include E. coli K12 RRl (Bolivar et al . , (1977)), E. coli K12 HBlOl (ATCC No. 33694), E. coli MM21 (ATCC No. 336780), E. coli DHl (ATCC No. 33849), E. coli strain DH5a, and E. coli STBL2.
  • host cells such as C. cerevisiae or B. subtilis can be used.
  • the above-mentioned exemplary hosts, as well as other suitable hosts are available commercially (e.g., Stratagene, La Jolla, Calif.; and Life Technologies, Rockville, Md. ).
  • the targeting constructs for disruption of target gene also include an exogenous nucleotide sequence encoding a resistance marker protein.
  • a resistance marker conveys resistance to specific physical or chemical agents that would otherwise be toxic to a cell.
  • the resistance marker gene is positioned between two flanking homology regions so that it integrates into the target gene following the crossover event in a manner such that the resistance marker gene is positioned for expression after integration. By imposing the selectable condition, one may isolate cells that stably express the resistance marker- encoding vector sequence from other cells that have not successfully integrated the vector sequence on the basis of viability.
  • a resistance marker does not distinguish between cells that have integrated the vector by targeted homologous recombination at the target gene locus rather than by random, non-homologous integration of vector sequence into any chromosomal position. Therefore, when using a replacement vector for homologous recombination to make the cells of the disclosure, it is also useful to include a polynucleotide encoding a negative selectivity marker protein. As described above regarding various possible types of DEL selection markers, negative selectivity marker is a protein that when expressed is toxic to a cell. The nucleotide sequence encoding a negative selectivity marker is positioned outside of the two homology regions of the replacement vector.
  • cells will only integrate and stably express a negative selectable marker if integration occurs by random, non-homologous recombination; homologous recombination between the target gene and the two regions of homology in the targeting construct excludes the sequence encoding the negative selectable marker from integration.
  • cells that have integrated the targeting vector by random, non-homologous recombination lose viability.
  • Vectors containing a colorogenic enzyme maker and/or bioluminescent markers and a DEL selection marker may be introduced into a cell according to standard methods well known to those with skill in the art or those that will be apparent to them based upon the disclosure.
  • the transformation protocol chosen will depend upon, for example, the cell type and the nature of the gene of interest, and can be chosen based upon routine experimentation. Several transformation protocols are reviewed in Kaufman (1988). Methods may include electroporation, calcium-phosphate precipitation, retroviral infection, microinjection, biolistics, liposome transfection, DEAE-dextran transfection, or transferrinfection (see, e.g., Neumann et al .
  • a typical method in the practice of the disclosure for introducing foreign DNA into a yeast cell involves the use of lithium acetate/PEG, as described in Gietz and Woods (2002) .
  • Cells to be used in the practice of the methods of the disclosure may be stored and cultured according to methods well known to those with skill in the art based upon the present disclosure.
  • mammalian cells may be cultured according to methods described in Bonifacino et al . (2003), Chapter 1.
  • Yeast cells may be cultured according to general methods described in Ausubel et al. (2003), Chapter 13.
  • the treatment of cells with a test agent may be employed according to methods known by those with skill in the art based upon the disclosure.
  • the method used will depend upon many variables, including the types of cells used, characteristics of the DEL selection marker and characteristics of the test agents used.
  • yeast cells Sacharomyces cerevisiae having a disruption of the his gene as the DEL selection marker are treated with test agents in 25ml tubes and then plated for about 48h at 30 0 C. Following treatment the cells are washed, for example, with PBS, and sonicated to assure dissociation of the cells into a single-cell suspension. The cells are then plated at an appropriate dilution (see below) onto medium lacking histidine as well as standard medium containing histidine. The histidine-lacking medium is used to determine recombination frequency. Standard medium (medium containing histidine) is used to determine the overall toxicity of the test agent.
  • the cells may be counted using a cell counting device (e.g., using a Coulter Particle Counter, Coulter Corp., Miami, FIa.) .
  • a cell counting device e.g., using a Coulter Particle Counter, Coulter Corp., Miami, FIa.
  • Ten fold serial dilutions are then prepared (D 0 -D 5 , wherein D 0 is the initial cell culture) .
  • the optimal cell dilution is such that there are sufficient cells to be able to measure: (a) the toxicity of the test agent; (b) the baseline recombination frequency of the cells (without treatment) ; and (c) the level of DEL recombination following treatment.
  • a typical dilution when using S. cerevisiae cells is IxIO 5 to IxIO 7 cells per mL.
  • cells may be plated on multi-well plates (e.g., 12, 24 or 48, 96, or 384 wells). The cells are then incubated for a sufficient time to enable revertant colonies to grow, typically 96 or 384-well plates for about 17 hours at 30 0 C.
  • revertant colonies are detected using an MTT or MTS assay.
  • the MTT or MTS assay measures reduction of MTT or MTS by a mitochondrial enzyme.
  • the mitochondrial enzyme will be present in viable cells thus providing an indication of living cells in the selection medium.
  • the detection may be performed by measuring both the colorogenic signal produced by enzymes in the mitochondria in the presence of MTT or MTS as well as a bioluminescence assay. Bioluminescence may be visualized using any light detection device, for example, a Lumi-Imager® Fl photon-counting device (Roche Diagnostics, Indianapolis, Ind. ) that may be used to identify colonies in multi-well plates.
  • the reversion frequency may be expressed as the number of revertant cells per the total number of cells that survive treatment with the test agent. For example, for S. cerevisiae having the his ' DEL selection marker the following formula may be used to calculate reversion frequency:
  • Any statistically significant increase in the reversion frequency as compared to a control will be indicative of a test agent having potential genotoxic and/or carcinogenic properties.
  • the determination of statistical significance is well known to those with skill in the art or will be apparent based upon the disclosure.
  • a typical positive result will yield a p-value that is no more than 0.05, more typically no more than 0.01 (Brownlee (I960)).
  • the determination in a cell population of reversion frequency as compared to a control requires correction for secondary effects of a test agent.
  • certain test agents that cause increased reversion frequency may also reduce the rate of growth and/or division of cells.
  • the number of revertant cells in untreated control cells may grow faster than those in the treated cell population such that the total population in the control exceeds those in the treated cells.
  • a method for correcting such secondary effects is by immobilizing populations of individual treated and control cells, e.g., using selection media which is solid or semisolid, such that the cells form individual colonies. The reversion frequency would then be determined based upon the number of detectable colonies or micro-colonies.
  • the above-described assay methods are for illustrative purposes only. Those with skill in the art will appreciate based upon the disclosure that a variety of assay formats may be utilized in the practice of this disclosure. Variations may be made based upon the types of cells, DEL selection markers, colorogenic markers, the combination of colorogenic and bioluminescence markers and test agents used, methods of treating and culturing cells and methods of detection of revertants.
  • DNA rearrangements including DNA deletions are involved in carcinogenesis.
  • An assay screening for DNA deletions in yeast can detect Salmonella/Ames assay negative as well as positive carcinogens.
  • Salmonella/Ames assay negative as well as positive carcinogens Among 58 compounds (mostly false negatives and false positives with the Salmonella assay) , the DEL assay correctly identified the carcinogenic activity of 86% compared with 36% that were correctly identified in the Salmonella assay.
  • carcinogens have also been reported to induce DNA deletions in related assays in vitro with human cells and in vivo with mice .
  • the RS112 yeast DEL assay tester strain of Saccharomyces cerevisiae contains a plasmid with an internal fragment of the HIS3 gene integrated at the genomic HIS3 locus, yielding an integrative disruption of the HIS3 gene. This disruption results in two copies of the HIS3 gene, each copy having one terminal deletion. Recombination between the two his3 deletion alleles results in reversion to HIS3 + and growth in the absence of histidine. This recombination event leads to a 6 kb DNA deletion comprising the integrated plasmid leading to deletion (DEL) events.
  • DEL deletion
  • the assay utilizing DEL events involves overnight growth of a single colony of the RS112 strain and subsequent subculture with the presence or absence of the chemical being tested for 17 hours at 30 0 C under constant shaking. Yeast are then plated onto SC medium to determine the number of survivors (individual colonies are counted) and onto SC-HIS medium to score for DEL events.
  • the traditional DEL assay is very powerful if one is testing a limited number of chemicals but becomes impractical for screening large numbers of chemicals and chemical libraries.
  • actinomycin D (CAS No. 50-76-0) dissolved in 0.2% acetone, ethyl methanesulfonate (CAS No. 62-50-0), camptothecin (CAS No. 7689-03-4), 4-nitroquinoline-l-oxide (CAS No. 56-57-5) dissolved in 0.2% DMSO, mitomycin C (CAS No. 50-07-0), CrC13 (CAS No. 10025-73-7), K2Cr2O7 (CAS No. 1333-82-0), benzene (CAS No. 71-43-2), methylmethane sulfonate (CAS No. 66-27-3), cyclophosphamide monohydrate (CAS No.
  • the diploid S. cerevisiae strain RS112 was used to determine the frequency of DEL recombination: MATa/MAT ⁇ ura3- 52/ura3-52 leu2-3, 112/leu2- ⁇ 98 trp5-27/TRP5 arg4-3/ARG4 ade2- 40/ade2-101 ilvl-92/ILVl HIS3 : :pRS6/his3 ⁇ 200 LYS2/lys2-801.
  • a mock experiment was setup to measure the sensitivity of the well-based DEL assay.
  • wells containing 100,000 RS112 yeast cells in 100 ⁇ l SC-HIS media were supplemented in six-plicate with 5 ⁇ l of RS112 His+ revertant cells ranging from 0 cells to 1000 cells.
  • 20 ⁇ l MTS was added to each well, plates were incubated at 30 0 C, and 490 nm absorbance was read hourly between 12 and 24 hrs using a Molecular Devices SpectraMax M5 microplate reader (Sunnyvale, CA) .
  • the assay was most sensitive when absorbance was measured 18 hours post incubation at which time as few as 25 RS112 His + revertant cells (corresponding to 2.5 DEL events/10, 000 cells) could be significantly differentiated from spontaneous background levels in 100,000 RS112 cells. 250 RS112 His + revertant cells were significantly detected 12 hours after dispersement into microwells (Fig. 1) suggesting that strong inducers of DEL recombination which induce 25-250 or more DEL events/10 , 000 cells are rapidly discernable using the well-based DEL assay.
  • FoId DEL increase was calculated by dividing the DEL induction measured for the respective compound concentration by that of the controls performed on the same plate. The concentration listed is the final concentration treated in each well . Each experiment was repeated at least 3 times on separate plates and similar results were attained in each measurement.
  • Cytoxicity was calculated by averaging the absorbance across the four treated wells in SC media and dividing that by the average absorbance of control wells .
  • Chromium III did not induce any genotoxic events at concentrations of 0.2 (-32 ⁇ g/ml) or 0.7 mM (-111 ⁇ g/ml), yet a significant increase in DEL recombination was observed at 1.4 mM (-222 ⁇ g/ml) .
  • Chromium VI showed a very potent induction of the DEL assay and severely decreased survival with increased dose between 0.2 and 1.4 mM (-20-140 ⁇ g/ml) .
  • benzene concentrations 0.25 and 0.40 mg/ml (-250 and 400 ⁇ g/ml, respectively) significant DEL induction was observed; no DEL fold-increase was observed with cyclophosphamide up to 200 ⁇ M.
  • chemotherapeutic agent mitomycin C a cross linking agent previously uncharacterized by the yeast DEL assay, caused significant increases in genotoxicity (HIS + growth) in yeast at concentrations of 20 ⁇ M (-6.7 ⁇ g/ml) and above whereas cytotoxicity was only significantly observed at 40 ⁇ M (Fig 2b and Table 1) .
  • Chlorambucil also a drug used for chemotherapy, caused significant genotoxicity and cytotoxicity at concentrations as low as l ⁇ M (-0.3 ⁇ g/ml), while carmustine caused more pronounced genotoxic and cytotoxic effects at concentrations above 30 ⁇ M (-2.1 ⁇ g/ml).
  • HCL-methanol used as a solvent for chlorambucil, by itself induced no DEL events compared to control treated yeast.
  • Cisplatin another widely used chemotherapeutic agent, caused significant cytotoxicity and genotoxicity at concentrations 0.3 and 1 mM (-27 and 300 ⁇ g/ml, respectively) .
  • the disclosure provides a method and system for rapid determination of DEL recombination effects. It has been micro-scaled to 96 or 384-well format using the colorimetric agent MTS. To validate the assay compounds thoroughly studied with the traditional agar plate-based DEL assay were used. Strong and medium genotoxic compounds are readily distinguished with the well-based assay. In as little as 10- 12h after the addition of MTS all of the high and moderate genotoxic treatments (comparable to >25 DEL events induced per 10,000 cells as quantified by the plate based assay) were readily distinguishable from controls. Data herein is reported from experiments done with 384-well plates, yet the same measurements were also done on 96-well plates giving comparable results.
  • crosslinking agents can produce DNA deletion events in yeast, and that the DEL assay is capable of detecting carcinogens whose main mechanism of carcinogenesis is through DNA crosslink production.
  • the well-based assay is economically superior to the plate-based assay and substantially less labor intensive. To perform the plate-based assay, four to five days are needed to perform the entire assay and score colonies. In the well-based assay, significant results can be collected in as few as 10-12 hours, and moreover there is no requirement to count and score colonies.
  • the well-based assay is intended as an easy method to determine the binary presence or lack of genotoxicity .
  • In some tests exposing yeast to extremely high cytotoxic treatments of nongenotoxic compounds can yield a false-positive report of genotoxicity. Such high cytoxic treatments can cause so much yeast killing that the absorbance measured in both +13 and -HIS wells is reduced near to background levels; thus when the ratio of growth in -HIS to +13 is taken, it nears unity.
  • the well-based version of the DEL assay is amenable to multiple formats. For example, yeast could be treated in 5 ml liquid cultures for 17 hours (as done for the plate-based assay) and then afterward scored in +13 and -HIS liquid media using MTS. When this format was used, a similar qualification of genotoxicity was measured for each of the compounds.
  • the well-based assay is also adaptable for high-throughput screening. Generally high-throughput screens use one compound per well. Also, the toxicity of many compounds is only discernable within a specific dosage range. Thus if a high- throughput screen is preformed at a single concentration for each compound, the genotoxicity of some compounds may be overlooked.
  • the DEL assay was micro-scaled for use in a 96 or 384-well format, adept for high-throughput screen-based assays. This format is sensitive enough to detect at least 2.5 DEL events per 10,000 cells and was used to assess the genotoxicity of 13 different compounds tested at various concentrations. Crosslinking agents previously uncharacterized with the DEL assay were strong inducers of DNA deletions using this assay.
  • the well-based DEL assay described here is ergonomically superior and can report genotoxicity much more rapidly than the traditional plate-based assay.
  • the well-based DEL assay is well suited for rapidly qualifying the genotoxicity of a large number of compounds and is amenable to automation in its current format for high-throughput purposes.
  • the assay can be used to detect aneugenic activity. For example, the effects induced by aneugenic agents on chromosome segregation are manifold. Because the assays of the disclosure measure the deletion or recombination of segments of DNA conferring survival to a cell, measuring DNA loss of a cellular toxin can be detected.

Abstract

L'invention propose des procédés, des systèmes et des kits pour analyser les propriétés mutagènes d'un agent. Les procédés, systèmes et kits utilisent un marqueur sélectionnable par DEL et un système de détection colorogène. Sont également inclus des procédés, des systèmes et des kits qui utilisent un marqueur sélectionnable par DEL et un réactif qui détecte une activité mitochondriale.
PCT/US2008/064985 2007-05-29 2008-05-28 Analyses pour la détection de mutagenèse WO2008150802A1 (fr)

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