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WO2005078069A1 - Procede d'identification de cibles cellulaires - Google Patents

Procede d'identification de cibles cellulaires Download PDF

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Publication number
WO2005078069A1
WO2005078069A1 PCT/US2003/014788 US0314788W WO2005078069A1 WO 2005078069 A1 WO2005078069 A1 WO 2005078069A1 US 0314788 W US0314788 W US 0314788W WO 2005078069 A1 WO2005078069 A1 WO 2005078069A1
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WIPO (PCT)
Prior art keywords
expression
cdna
cell
nucleic acid
operably linked
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PCT/US2003/014788
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English (en)
Inventor
Albert J. Erives
Barry D. Starr
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Codegrok, Inc., Dba Auilix Biopharma
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Application filed by Codegrok, Inc., Dba Auilix Biopharma filed Critical Codegrok, Inc., Dba Auilix Biopharma
Priority to PCT/US2003/014788 priority Critical patent/WO2005078069A1/fr
Priority to AU2003304710A priority patent/AU2003304710A1/en
Publication of WO2005078069A1 publication Critical patent/WO2005078069A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1086—Preparation or screening of expression libraries, e.g. reporter assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention is directed to nucleic acid constructs and their use in identifying cellular factors that function in various cellular processes involving gene expression. These factors may be the targets of known therapeutic agents, novel targets for a test compound, or amenable to altered expression to modulate cellular processes.
  • Cell-based assays are said to be open-ended or "black-box" assays. This refers to the fact that a chemical compound applied to the cell-based assay may interact with any number of protein targets, many of which are unknown. Thus, a useful compound identified by such an approach may interact with any number of unknown targets.
  • the positive attribute of such a system is that one can screen against many potential targets without first identifying them.
  • Another positive attribute is that the assay is conducted within the physiologically meaningful context of a cell.
  • these types of assays suffer from two negative attributes. The first negative attribute is that it is difficult to optimize the medicinal chemistry properties of a lead compound without first knowing the structure of the target. Also related to not knowing the exact target protein binding the drug, the mechanism of action remains poorly understood.
  • the second negative attribute relates to the non-specificity of the many hits identified by these screens. This latter attribute is partially addressed by building more discrete cell-based reporter assays, such as those described in U.S. Patent Application 09/989,993, filed November 21, 2001 and entitled, "Regulatory Nucleic Acid Assay for Diagnostic and Library Screens", which is hereby incorporated by reference as if fully set forth. [0004] Citation of documents herein is not intended as an admission that any is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.
  • the present invention provides nucleic acid constructs and methods of using them to identify unknown cellular factors and the molecules with which they interact.
  • the identified cellular factors may be used in assays to screen for compounds that target the factors to produce a biological effect. Such compounds affect the activities of the identified cellular factors to modulate cellular processes in which the factors function.
  • the invention identifies factors involved in regulating gene expression in various cellular processes.
  • the invention provides marker/reporter nucleic acid constructs, the expression of which undergoes a detectable change as part of a cellular process.
  • a marker/reporter construct generally comprises a nucleic acid molecule containing a regulatory module operably linked to a basal promoter operably linked to a coding sequence, the expression of which may be readily detected.
  • the regulatory module may be all or part of a naturally occurring regulatory region of a cellular gene. Alternatively, the regulatory module may be a composite of regulatory elements from different regulatory regions. In preferred embodiments of the invention, the regulatory module comprises one or more enhancer or silencer sequences that control activation of the operably linked basal promoter.
  • the coding sequence preferably encodes a selectable (positive or negative) marker, a detectable reporter, or alternatively, and in a 5' to 3' orientation, encodes a reporter followed by a selectable marker.
  • the sequences encoding both the reporter and the selectable marker are operably linked to the basal promoter.
  • the sequence encoding the selectable marker is also operably linked to an internal ribosome entry site (IRES) situated between the coding sequences for the reporter and the selectable marker.
  • IRS internal ribosome entry site
  • the nucleic acid molecule containing an operably linked combination of a regulatory module, basal promoter, and coding sequence is flanked at the 5', and/or 3', end by an insulator sequence.
  • the marker/reporter nucleic acid constructs of the invention may further comprise a second nucleic acid molecule containing a second operably linked combination of a second basal promoter and second coding sequence, optionally with an operably linked second regulatory module.
  • the first and second regulatory modules are preferably not identical.
  • the first and second basal promoters may be the same or different but are preferably the same.
  • the first and second coding sequences are preferably different, with the second coding sequence encoding a positive or negative selection marker, a detectable reporter, or a combination of a detectable reporter and a selection marker in an orientation as described above.
  • the invention also provides a set of expression library constructs capable of expressing nucleic acid molecules of an expression library in combination with the above described marker/reporter constructs.
  • the expression library constructs contain cDNA or genomic sequences such that they may be (individually) expressed in the same cell as that containing a marker/reporter construct as described above.
  • the library constructs contain cDNAs prepared from the cell type (cell specific cDNAs) into which the library and marker/reporter constructs are introduced.
  • the library constructs may contain cDNAs prepared from cells that are heterogeneous relative to the cells into which the library and marker/reporter constructs are introduced.
  • the cDNAs may encode a particular type of functionality, such as a receptor, coupling protein, effector, or second messenger.
  • the cDNAs may be artificially modified forms of naturally occurring sequences or from cells of a heterologous organism. Use of diverse sources of cDNAs in the present invention permits the identification of a variety of coding sequences able to affect control of the regulatory module of a marker/reporter construct as described herein.
  • the cDNA of a library construct is operably linked, and thus its expression is controlled by, a constitutive or inducible promoter.
  • the combination of a marker/reporter and one or more library constructs may be referred to as an expression system of the invention.
  • Cells containing a marker/reporter and an expression library construct of the invention may be assayed for a change in the expression of the marker and/or reporter due to the expression of the library construct. Stated differently, a change in expression of a selectable marker and/or detectable reporter in cells containing an expression system of the invention may be detected and used to identify a library construct as encoding a cellular factor that participates in the control of the regulatory module of the marker/reporter construct.
  • the library construct or the inserted coding sequence may also be isolated from the cell and the sequence of the coding region determined.
  • the factor may also be identified as participating in the control of the cellular gene(s) from which the regulatory module of the marker/reporter construct was derived.
  • the factor may further be identified as a target for controlling the regulatory module, and thus cellular gene(s).
  • the combination of a marker/reporter construct and an expression library construct in a cell may thus be referred to as an assay system of the invention.
  • the assay system permits the identification of a cellular factor without the need for prior knowledge as to its identity. It is only necessary to have a marker/reporter construct comprising a regulatory module affected by the factor.
  • the use of the regulatory module to control expression of a selectable marker allows the identification of cellular factors to be based upon a rapid assay based upon cell viability.
  • use of the regulatory module to control a detectable reporter permits rapid identification of cellular factors to be based upon isolation of cells expressing the reporter by fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • the optional presence of a second nucleic acid molecule under the control of a different regulatory module allows for the rapid exclusion of cellular factors that non-specifically increase gene expression in a cell.
  • the assay systems of the invention are used in methods to identify protein factors involved in a signaling pathway that control expression of one or more cellular genes.
  • the methods can be generalized to identify one or more other proteins that interact in a signaling pathway. These protein factors may act directly on a regulatory module, such as by binding directly to it, or indirectly by interacting with one or more other cellular factors that participate in a signaling pathway that controls the regulatory module. Protein factors that activate, or block activation of, a regulatory module such as an enhancer may be identified by detecting expression of a positive or negative selection marker, respectively, or by affecting expression of a detectable reporter. Protein factors that deactivate, or block deactivation of, a regulatory module such as a silencer may be identified by detecting expression of a positive or negative selection marker, respectively, or by affecting expression of a detectable reporter.
  • the assay systems of the invention may also be used in combination with one or more compounds known to affect, or under consideration as affecting, the control of a regulatory module.
  • cellular factors are identified by their ability to reverse, or modulate, the action of a compound in an assay system of the invention.
  • a cell containing an expression system of the invention may be contacted with a compound that prevents activation of the regulatory module of the marker/reporter construct such that the cells are not viable (or the reporter is not detectable) unless the expression construct provides a factor that counteracts the inhibitory effect of the compound.
  • This is readily accomplished by use of a coding sequence in the marker/reporter construct that encodes a product that imparts cell viability or is readily detectable upon expression.
  • a cell containing an expression system of the invention may be contacted with a compound that activates the regulatory module of the marker/reporter construct such that the cells are not viable (or the reporter is not detectable) unless the expression construct provides a factor that counteracts the activating effect of the compound.
  • a compound that activates the regulatory module of the marker/reporter construct such that the cells are not viable (or the reporter is not detectable) unless the expression construct provides a factor that counteracts the activating effect of the compound.
  • a coding sequence in the marker/reporter construct that encodes a product that causes cell death (or is readily detectable) upon expression.
  • a cell containing an expression system of the invention may be contacted with a compound that modulates activation of the regulatory module of the marker/reporter construct such that the cells display a detectably change in viability when the expression construct provides a factor that counteracts the effect of the compound.
  • the amount of cytotoxic agent can be titrated such that increases or decreases in cell survival may be detected relative to a change in expression from the marker/reporter construct.
  • a compound that inhibits activation of the regulatory module of the marker/reporter construct factors encoded and expressed by an expression construct that tend to decrease (down modulate) the inhibitory effect of the compound may be identified by an increase in cell survival.
  • factors that increase (up modulate) the inhibitory effect of the compound may be identified by a decrease in cell survival. All that is necessary is that the amount of cytotoxic agent be titrated so that there is a detectable level of cell survival in the presence of a compound (and absence of expression from an expression construct).
  • the present invention may be practiced with use of a coding sequence in the marker/reporter construct that encodes a product that imparts cell lethality in the presence of an agent, such as the combination of a thymidine kinase and gancyclovir. The amount of the agent can be titrated such that increases or decreases in cell survival may be detected relative to a change in expression from the marker/reporter construct.
  • factors encoded and expressed by an expression construct that tend to decrease (down modulate) the inhibitory effect of the compound may be identified by an decrease in cell survival.
  • factors that increase (up modulate) the inhibitory effect of the compound may be identified by an increase in cell survival. All that is necessary is that the amount of the agent be titrated so that there is a detectable level of cell survival in the presence of the compound and any expression from the marker/reporter construct (and absence of expression from an expression construct).
  • a compound known to affect, or under consideration as affecting, the control of a regulatory module may be used in combination with combination with expression library constructs to broadly identify factors that alter the activity of said compound.
  • a compound that causes cell death by inhibiting a signaling pathway that activates expression of an essential cellular gene may be used in combination with the expression library constructs of the invention to identify library constructs encoding factors that reverse the inhibition of the pathway and/or the effects of the compound.
  • the factors encoded by these identified constructs may be the object of further study or compared to the factors identified by use of methods comprising the marker/reporter constructs described above.
  • Figure 1 is a schematic representation of a transcription unit, such as a gene, comprising various regulatory components.
  • Figure 2 is a schematic representation of the transcription unit of Figure 1 with signaling pathways A through D that affect the activity of different enhancers present in the unit.
  • Figure 3 is a schematic representation of a generalized signal transduction pathway and four variations thereof.
  • Ligands can be extracellular and recognized by a cell surface receptor or membrane permeable such that they interact directly with an intracellular receptor (see pathway 1). Examples of such ligands include steroid hormones which bind to a cytoplasmic receptor. Pathway 2 shows how some receptors, such as certain growth factor receptors, can directly regulate intracellular proteins.
  • Pathway 3 shows how some receptors have intrinsic "effector" capacity to directly produce second messengers.
  • Pathway 4 shows how some second messengers act pleiotropically and interact with a number of target proteins to produce an integrated response.
  • Non-limiting examples of ligands include protease targets, and non-limiting examples of transmembrane receptors include G-protein coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs).
  • GPCRs G-protein coupled receptors
  • RTKs receptor tyrosine kinases
  • a marker/reporter construct of the invention comprises a nucleic acid molecule containing a regulatory module operably linked to a basal promoter operably linked to a coding sequence.
  • operably linked refers to a functional linkage between nucleic acid sequences such that a regulatory module functionally regulates the linked basal promoter which functionally controls expression of the coding sequence.
  • marker/reporter constructs containing a number of different regulatory modules and different markers and reporters may be prepared and used in the practice of the invention. Marker/reporter constructs containing different regulatory modules and selectable markers can also be used in combination to permit multiplex analysis of the effect of one cellular factor against two different regulatory modules.
  • the markers are preferably heterologous (not normally found in nature) in combination with the operably linked regulatory modules.
  • the construct contains sequences derived from a vector to assist in the propagation and manipulation of the nucleic acid molecule.
  • the nucleic acid molecule is stably integrated into the genome of a cell in which the invention is to be practiced.
  • the invention includes such cells which may be maintained as a cell line for use in the practice of the invention.
  • the invention may be practiced with use of a marker/reporter construct that is not integrated.
  • Non-limiting examples include stably maintained episomal vectors, such as circular vectors derived from Epstein Barr Virus (EBN), or artificial chromosomes.
  • the regulatory module generally comprises one or more cis-regulatory sequences that function to regulate the activity of a promoter (or control module). Two general classes of regulatory sequences are enhancers and silencers.
  • Enhancers are an sequence that is present in the genomes of higher eukaryotes and various animal viruses, which can increase the transcription of genes into messenger R ⁇ A. Enhancers are often found 5' to the start site of a gene, and when bound by a specific transcription factor, enhance the levels of expression of the gene, but are not sufficient alone to cause expression. Enhancers can function in either orientation and at various distances from a promoter. Like an enhancer, a silencer can act at a distance from a promoter. But when bound by a trans-acting factor, a silencer suppresses transcription of genes into messenger R ⁇ A. Regulatory modules that can be used in the present invention are described below. Additionally, methods for the identification and isolation of additional regulatory sequences and regulatory modules are known in the art.
  • a promoter is a nucleic acid sequence involved in the binding of R ⁇ A polymerase to begin transcription. Some promoters contain a consensus region referred to as the TATA box, which is located 5' from the transcriptional start site of a gene. As used herein, promoter is also referred to as a control module. Examples of control modules for use in the present invention are described below.
  • the coding sequence preferably encodes a selectable marker, such as, but not limited to, hygromycin resistance, neomycin resistance, Herpes simplex virus thymidine kinase (HSN tk), diptheria toxin A, and aminotriazole (ATZ).
  • HSN tk Herpes simplex virus thymidine kinase
  • ATZ aminotriazole
  • Selectable markers for use in the practice of the invention may be generally divided into two groups.
  • the first are positive selection markers that provide resistance (and thus cell viability) against a toxic agent, such as a drug that retards cell growth or is cytotoxic (e.g. hygromycin and neomycin resistance).
  • the second are negative markers that are lethal to a cell upon expression in the presence of an otherwise non-toxic agent (e.g. HSN tk in combination with gancyclovir or a histidine biosynthesis gene in combination with ATZ).
  • the coding sequence encodes a reporter, which is an assayable product upon expression. Examples of reporters for use in the present invention are provided below.
  • the coding sequence encodes both a reporter and a selectable marker, where the reporter and marker are operably linked to an internal ribosome entry site (IRES) situated between the coding sequences for the reporter and the selectable marker such that both may be translated.
  • IRES internal ribosome entry site
  • the basal promoter is followed by the coding sequence for a reporter, an IRES, and the coding sequence for a selectable marker.
  • the term "5"' (five prime) generally refers to a region or position in a polynucleotide 5' (upstream) from another region or position in the same polynucleotide.
  • a nucleic acid molecule containing an operably linked combination of a regulatory module, basal promoter, and coding sequence is optionally flanked at the 5', and/or 3', end by an insulator sequence.
  • Insulators limit the range of action of regulatory sequences such as enhancers and silencers. Examples of insulators that can be used in the practice of the invention are provided below. Additionally, methods for the identification and isolation of additional insulator sequences are known in the art. The present invention may be practiced with sequences and modules identified by such methods.
  • the marker/reporter nucleic acid constructs of the invention may further comprise a second nucleic acid molecule containing a second operably linked combination of a second basal promoter and second coding sequence.
  • the second nucleic acid molecule is also operably linked to the insulator sequence.
  • These second nucleic acid molecules may be used to eliminate changes in transcription of the first nucleic acid molecule that are not specific to the first regulatory module. For example, and without limiting the invention, if a factor expressed by an expression construct of the invention generally increases transcription, such as by being a transcription factor utilized in most or all of a cell's transcription units (e.g.
  • Expression library constructs of the invention may be prepared by methods known in the art.
  • the library constructs may be prepared with use of a variety of vectors and control modules not limited to those described herein.
  • the control modules may be constitutive or inducible promoters such that the members (or clones) of the library may be introduced into cells transformed (optionally stably transformed) with a marker/reporter construct of the invention.
  • cDNAs prepared from the cells in which the invention will be practiced increases the likelihood of identifying cDNAs encoding cellular factors that normally participate in controlling the activity of a regulatory module.
  • use of cDNAs from heterologous sources or containing modified sequences permits the identification of coding sequences encoding factors that can substitute for naturally occurring cellular factors that participate in controlling the activity of a regulatory module.
  • Methods for the introduction of nucleic acid constructs into cells are known in the art and include, optionally, the stable integration of the expression construct into a cell.
  • the expression constructs are introduced into a population of cells containing a marker/reporter construct of the invention such that on average, each cell contains one expression construct.
  • individual constructs of an expression library are introduced into a population of cells such that each cell, on average, contains one construct of said library.
  • Induction of expression of the coding sequence in the expression construct and contacting the cells with the appropriate selection agent (such as, but not limited to, hygromycin, neomycin, ATZ or gancyclovir) for the marker/reporter construct used permits the identification of coding sequences as participating in control of the regulatory module by simple detection of surviving cells. This may also be viewed as determining whether an individual cell is viable to identify a viable cell as containing an expression construct of interest.
  • the amount of the selection agent must be sufficient to result in cell death, and such amounts are either known or readily determined by the skilled person without undue experimentation.
  • the coding sequences in the expression constructs in surviving cells may be isolated and identified from the cells by a variety of methods well known in the art. Non-limiting examples include simple PCR mediated by known (expression vector) sequences flanking the coding region in the expression constructs; and isolation of expression library constructs from cells. The coding sequences may also be sequenced by methods known in the art.
  • the expression constructs of the invention preferably contain cDNAs prepared from the cells used in the assay system. Alternatively, the constructs may contain cDNAs of known cellular factors having a particular functionality, such as a receptor protein, a coupling protein, a proteinaceous effector or second messenger, or a nucleic acid molecule.
  • constructs capable of expressing known cellular factors are advantageously used in embodiments of the invention where cells are contacted with compounds known, or thought, to target a particular type of cellular functionality.
  • the use of a compound known, or thought, to inhibit a signaling pathway by inhibiting a kinase molecule may be combined with the use of expression constructs containing coding sequences for various kinases. This provides the ability to identify the actual kinase that is inhibited, if unknown, and/or the ability to identify kinases that functionally substituted for the kinase activity that is inhibited.
  • the invention can be practiced with the use of expression constructs encoding factors, such as second messengers, thought to act downstream of the inhibited kinase activity. This provides the ability to identify factors that act in the same pathway as, or interact with, the inhibited kinase activity.
  • a cellular factor identified by use of the present invention may be used in assays to screen for compounds that target the factor.
  • the screen may simply utilize a marker/reporter construct of the invention comprising a regulatory module that is responsive to changes in activity of the factor. Such a construct is introduced into a cell and the cell is contacted with test compounds to determine whether they affect the expression of the coding sequence in the construct.
  • Compounds that activate or inactivate the factor may be identified by use of the appropriate marker, and the compounds may thus be used to modulate the cellular pathway(s) in which the factor functions to produce a biological effect in a cell.
  • the compounds may be used to modulate cellular gene expression controlled by signaling pathways in which the factor functions.
  • the assay systems of the invention may also be used in combination with one or more compounds known to affect, or under consideration as affecting, the control of a regulatory module. Of course the amount of the compound must be sufficient to affect gene expression controlled by said regulatory module, and such amounts are either known or readily determined by the skilled person without undue experimentation.
  • a compound known to inhibit transcription regulated by a regulatory module may be used in combination with an expression system of the invention to identify members (or clones) of an expression library encoding factors that suppress the inhibition of transcription.
  • This is readily accomplished by use of a positive selection marker the expression of which is needed for cell viability in the presence of a selective agent.
  • a positive selection marker the expression of which is needed for cell viability in the presence of a selective agent.
  • expression constructs encoding a factor that relieves the inhibitory effect of the compound will result in the expression of the positive selection marker and viability of the cells.
  • Expression constructs encoding other factors will not relieve the inhibitory effect and the cells containing them will die.
  • a detectable reporter or a combination of a reporter and a marker, may be used to permit rapid isolation (e.g.
  • the invention may be used in combination with one or more compounds known to activate transcription regulated by a regulatory module.
  • Cells containing a marker/reporter construct comprising a negative selection marker may be contacted with these compounds such that an expression construct expressing a factor which suppresses the activating effect of the compound is needed for cell viability in the presence of an agent that kills the cells when the negative selection marker is expressed. In the absence of such a factor, the compound would activate expression of the negative selection marker and the cells will die.
  • a detectable reporter or a combination of a reporter and a marker may also be used in this case to permit rapid isolation (e.g.
  • the invention may be practiced with a compound known or suspected to have an effect on the control of a regulatory module but without the use of a marker/reporter construct such that expression constructs containing sequences encoding factors that alter the activity of the compound may be identified. This provides a means of broadly identifying factors that affect a compound that affects the control of a regulatory module.
  • vectors refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • One type of vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication.
  • Other vectors are capable of autonomous replication and/expression of nucleic acids to which they are linked.
  • Vectors may also be used to deliver nucleic acid molecules into a cell for integration into the cellular genome.
  • Vectors capable of directing the expression of genes to which they are operably linked are referred to herein as "expression vectors.”
  • expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids” which refer to circular double stranded DNA loops which, in their vector form are not bound to the chromosome.
  • plasmid and vector are used interchangeably.
  • the invention is intended to include other forms of vectors which serve equivalent functions and which become known in the art subsequently hereto.
  • Vectors can be used for the expression of polynucleotides and polypeptides.
  • such vectors comprise cis-acting control regions effective for expression in a host operably linked to the polynucleotide to be expressed.
  • Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector, or supplied by the vector itself upon introduction into the host.
  • the vectors provide for specific expression.
  • Such specific expression may be inducible expression, expression only in certain types of cells, or both inducible and cell-specific.
  • Vectors can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
  • a variety of vectors such as constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, are well known and employed routinely by those of skill in the art.
  • vectors include, but are not limited to, chromosomal, episomal, virus-derived (e.g. retroviral, lentiviral, baculoviral, papovaviral, such as SV40, vaccinia virus, adenoviral, fowl pox viruses, and pseudo-rabies virus) vectors, vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids.
  • virus-derived e.g. retroviral, lentiviral, baculoviral, papovaviral, such as SV40, vaccinia virus, adenoviral, fowl pox viruses, and pseudo-rabies virus
  • vectors derived from bacterial plasmids from bacteriophage
  • yeast episomes from yeast chromoso
  • any vector suitable to maintain, propagate or express polynucleotides in a host may be used.
  • the following vectors which are commercially available, are provided by way of example.
  • vectors for use in bacteria are pQE70, pQE60, and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, ⁇ NH18A, pNH46A, available from Stratagene; and ⁇ trc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • Eukaryotic vectors available are pWLNEO, pSV2CAT, pOG44, pXTl, and pSG available from Stratagene; and pSVK3, pBPV, pMSG, and pSVL available from Pharmacia. These vectors are listed solely by way of illustration of the many commercially available and well known vectors that are available to those of skill in the art for use in accordance with the present invention. It will be appreciated that any other plasmid or vector suitable for, for example, introduction, maintenance, propagation, and/or expression of a polynucleotide or polypeptide of the invention in a host may be used in this aspect of the invention.
  • the appropriate DNA sequence may be inserted into the vector by any of a variety of well-known and routine techniques.
  • a DNA sequence for expression is joined to a vector by cleaving the DNA sequence and the vector with one or more restriction endonucleases and then joining the restriction fragments together using T4 DNA ligase.
  • Procedures for restriction and ligation that can be used are well known and routine to those of skill in the art. Suitable procedures in this regard, and for constructing vectors using alternative techniques, which also are well known and routine to those skilled in the art, are set forth in great detail in Sambrook et al. cited elsewhere herein.
  • the sequence in the vector is operably linked to appropriate expression control sequence(s), including, for instance, a promoter to direct mRNA transcription.
  • appropriate expression control sequence(s) including, for instance, a promoter to direct mRNA transcription.
  • the choice and/or design of the vector may depend on such factors as the choice of the host cell to be transformed and/or the type of protein(s) desired to be expressed.
  • the vector's copy number, the ability to control that copy number, and the expression of any other proteins encoded by the vector, such as antibiotic markers should also be considered.
  • Expression vectors can be used to transfect cells and thereby replicate regulatory sequences and produce proteins or peptides, including those encoded by nucleic acids as described herein.
  • a regulatory module can comprise an enhancer, silencer, scaffold-attachment region, negative regulatory element, transcriptional initiation site, regulatory protein binding site, any combination or multiplicity of these sequences, and any other regulatory sequence which has a transcription-rate modifying function when placed adjacent to a reporter gene.
  • Such regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185. Academic Press, San Diego, Calif. (1990).
  • enhancer containing sequences are EPO 3' hypoxia enhancer, cytoplasmic actin promoter, VEGF hypoxia enhancer, LBP-32 enhancer, relA hypoxia enhancer, PROC hypoxia enhancer, DELTEX hypoxia enhancer, HMOX1 enhancer, GRAP enhancer, BTE ⁇ -4 hypoxia enhancer, CCRdelta5 lymphocyte promoter, and COL4A1.
  • This list is merely an exemplary list of the types of enhancers that can be used in the present invention.
  • Useful expression control modules can comprise for example, a viral LTR, such as the LTR of the Moloney murine leukemia virus, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the tip system, the TAC or TRC system, the T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters of the yeast alpha-mating factors, the polyhedron promoter of the baculo virus system, and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof.
  • a viral LTR such as the LTR of the Moloney murine leukemia virus, the early and late
  • Suitable eukaryotic promoters are the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus ("RSV”), and metallothionein promoters, such as the mouse metallothionein-I promoter.
  • RSV Rous sarcoma virus
  • metallothionein promoters such as the mouse metallothionein-I promoter.
  • DNA coding a member (or clone) of an expression library can be placed under the control of an inducible promoter, with the result that cells as produced or as introduced into an individual do not express the product but can be induced to do so.
  • a promoter can be a constitutively active promoter.
  • a control module can be located on the same vector as the regulatory module and/or on a different vector. For example, if needed the control sequence, i.e.
  • a reporter is used to report activated gene expression by providing an easily detectable protein product (e.g., an enzymatic activity such as chloramphenicol acetyl transferase, or CAT).
  • the reporter gene of the present invention can have additional nucleic acids at both ends or at one end of the reporter gene sequence.
  • the invention may also be practiced with various forms of GFP that exhibit colors other than green.
  • GFP isolated from sources other than the jellyfish Aequorea victoria such as the sea pansy Renilla reriformis, may be used.
  • the GFPs with GenBank accession numbers U47949 (AGP1); U43284; U36202; U36201; U19282; U19279; U19277; U19276; U19281; U19280; U19278; L29345 (Aequorea victoria); M62654 (Aequorea victoria); and M62653 (Aequorea victoria) may be used.
  • modified GFPs such as AF007834 (GFPuv); U73901 (Aequorea victoria mutant 3); U50963 (Synthetic); U70495 (soluble-modified green fluorescent protein (smGFP)); U57609 (enhanced green fluorescent protein gene); U57608 (enhanced green fluorescent protein gene); U57607 (enhanced green fluorescent protein gene); U57606 (enhanced green fluorescent protein gene); U55763 (enhanced green fluorescent protein (egfp); U55762 (enhanced green fluorescent protein (egfp); and U55761 (enhanced green fluorescent protein (egfp) may be used.
  • AF007834 GFPuv
  • U73901 Alquorea victoria mutant 3
  • U50963 Synthetic
  • U70495 soluble-modified green fluorescent protein
  • U57609 encoded green fluorescent protein gene
  • U57608 encoded green fluorescent protein gene
  • GFPs from microorganisms such as U89686 (Saccharomyces cerevisiae synthetic green fluorescent protein (cox3::GFPm-3) gene); and U89685 (Saccharomyces cerevisiae synthetic green fluorescent protein (cox3::GFPm) gene) may also be used in the present invention.
  • Synthetic GFPs such as U87974 (Synthetic construct modified green fluorescent protein GFP5-ER (mgfp5-ER)); U87973 (Synthetic construct modified green fluorescent protein GFP5 (mgfp5)); U87625 (Synthetic construct modified green fluorescent protein GFP-ER (mfgp4-ER)); U87624 (Synthetic construct green fluorescent protein (mgfp4) mRNA)); U54830 (Synthetic E. coli Tn3- derived transposon green fluorescent protein (GF); AAB47853 ((U87625) synthetic construct modified green fluorescent protein (GFP-ER)); and AAB47852 ((U87624) synthetic construct green fluorescent protein) may also be used.
  • GF Synthetic E. coli Tn3- derived transposon green fluorescent protein
  • AAB47853 ((U87625) synthetic construct modified green fluorescent protein (GFP-ER)
  • AAB47852 ((U87624) synthetic construct green fluorescent protein)
  • Nucleic acids encoding blue fluorescent proteins and identified by the following GenBank accession Nos. may be used: U70497 (soluble- modified blue fluorescent protein (smBFP); 1BFP (blue variant of green fluorescent protein); and AAB 16959 (soluble-modified blue fluorescent protein).
  • nucleic acids encoding red fluorescent proteins identified by the following GenBank accession Nos. may be used: U70496 (soluble-modified red-shifted green fluorescent protein (smRSGFP); and AAB16958 (U70496) soluble-modified red-shifted green fluorescent protein).
  • a fluorophore that changes color with time may be used in the present invention to provide the ability to follow expression over time or determine the approximate time point at which expression occurred. See Teiskikh et al.
  • indirect reporters can be used in the present invention.
  • a secondary protein or compound can be used that interacts with the reporter protein and is labeled with a fluorochrome, radioactivity, or any of the known labeling substances known to one skilled in the art.
  • the secondary protein could be a capture antibody that interacts with the reporter and is coupled to a label.
  • Excitation and emission maxima for various of the fluorescent proteins and fluorochromes listed above are known in the art.
  • Insulators mark the boundaries of chromatin domains by limiting the range of action of enhancers and silencers. Insulators, which flank many genes, may be responsible for providing a barrier against incursions from surrounding domains. Although the insulator elements vary greatly in their sequences and the specific proteins that bind to them, they have at least one of two properties related to barrier formation. First, insulators have the ability to act as a "positional enhancer Mocker.” If the insulator lies between a promoter and an enhancer, then enhancer mediated activation of the promoter is impaired, but if the insulator lies outside the region between enhancer and promoter, little or no effect is observed.
  • Insulators are neutral barriers to enhancer action; they do not inactivate either the enhancer or the promoter. [0058] Second, insulators have the ability to protect against position effects. When genes are removed from their native context, as in transgenic animals, the dominant effect of the new chromosomal environment becomes apparent. Expression levels at the new location often bear no resemblance to that of the gene in its native position. Flanking a transgene with insulators can suppress this variability. Having the ability to protect against position effects and/or to block distal enhancer activity has come to form the operational definition of an insulator. Insulators can act as a modulatable switch, allowing them to function as sophisticated regulatory elements (Bell, A.C., et al, Science, Vol.
  • Examples of insulators that can be used in the present invention are scs, scs', fab7, fab8, the gypsy Su(Hw) array, the cHS4 region from the chick globulin locus, VEGF-A basal promoter region, and the BEAD element. However, other sequences with insulator-like properties may also be used.
  • An insulator of the present invention can have additional nucleic acids at both ends or at one end of the insulator sequence.
  • Example 1 Using positive selection and a compound that inhibits a regulatory module from activating expression [0064]
  • the androgen receptor (AR) a nuclear hormone transcription factor that drives expression of the PSA gene, binds androgens as agonists but also binds bicalutamide, an antagonist. It is known that increased levels of AR expression can re-activate AR-dependent gene expression (a model of prostate cancer) in the presence of antagonists such as bicalutamide.
  • a marker/reporter construct containing a first nucleic acid molecule comprising a PSA regulatory module operably linked to a Simian Virus 40 (SV40) basal promoter operably linked to a luciferase coding sequence, an IRES, and a sequence encoding hygromycin resistance is used in combination with a prostate cDNA expression library (in expression constructs of the invention) in an androgen dependent prostate cell line to identify and quantify colonies rescued through AR transgene incorporation (via AR expression from an expression construct of the library).
  • SV40 Simian Virus 40
  • a cell containing the marker/reporter construct and an expression construct containing a member (or clone) of the cDNA library are contacted with both hygromycin and bicalutamide such that expression of hygromycin resistance is necessary for cell viability.
  • Bicalutamide acts as the inhibitor of the PSA regulatory module in the marker/reporter construct.
  • Cells able to express hygromycin resistance due to the presence of an expression construct expressing a cDNA that re-activates the PSA regulatory module in the presence of bicalutamide remain viable and selected to identify and/or isolate the cDNA.
  • cDNAs encoding other factors that reverse the inhibitor activity of bicalutamide are identified and/or isolated.
  • factors that are able to substitute for AR are identified via the cDNA encoding them.
  • This embodiment of the invention may also be practiced in the absence of hygromycin selection but in the presence of bicalutamide, which eventually kills the cells due to their androgen dependency. The cells need not express hygromycin resistance for viability, but only cells containing a cDNA expression construct that restores viability in the presence of bicalutamide will remain viable.
  • cDNAs encoding other factors that reverse the inhibitor activity of bicalutamide, such as, but not limited to, cDNAs that generally increase transcription from the basal SV40 promoter, are identified and/or isolated.
  • a comparison of the results from this and the above use of selection via the marker/reporter construct shows that the fraction of surviving cells that contain an AR transgene is higher when the marker/reporter construct is used.
  • Example 2 Using positive selection and a compound that inhibits a regulatory module from activating expression: inclusion of a second "control" construct
  • the assay system of Example 1 is modified such that the marker/reporter construct also contains a second nucleic acid molecule containing a basal S V40 promoter operably linked to a HSV thymidylate kinase (HSV tk) coding sequence.
  • This second nucleic acid molecule is operably linked to an insulator, which is also operably linked to the first nucleic acid molecule (described in Example 1) such that the PSA regulatory module does not affect expression of the HSV tk coding sequence.
  • Cells containing this construct and expression constructs of an expression library are grown in hygromycin, bicalutamide, and gancyclovir such that cells that specifically express hygromycin resistance without activation of gancyclovir by expression of HSV tk may be selected based upon cell viability.
  • Cells that express HSV tk, such that those containing a cDNA encoding a general activator of transcription from the basal SV40 promoter, will not be selected because they are not viable in the presence of gancyclovir.
  • Example 3 Using negative selection and a compound that activates a regulatory module controlling expression
  • a marker/reporter construct containing a first nucleic acid molecule comprising a regulatory module operably linked to a Simian Virus 40 (SV40) basal promoter operably linked to a luciferase coding sequence, an IRES, and a sequence encoding HSV tk is used in combination with an expression construct library in cells grown in the presence of gancyclovir.
  • SV40 Simian Virus 40
  • IRES luciferase coding sequence
  • HSV tk luciferase coding sequence
  • Introduction of a compound that activates the regulatory module to result in expression of HSV tk causes cell death unless expression from an expression construct results in a factor that suppresses the activity of the compound.
  • Use of a second nucleic acid molecule in the marker/reporter construct should include the use of a sequence encoding a positive selection marker to decrease identification of factors that do not act through the regulatory module to affect HSV tk expression.
  • the surviving cells are isolated, and the sequences responsible for suppressing the activity of the compound, and present in the expression construct, are isolated and/or identified by methods such as PCR or sequencing as described herein.
  • a marker/reporter construct comprising a silencer operably linked to a Simian Virus 40 (SV40) basal promoter operably linked to a luciferase coding sequence, an IRES, and a sequence encoding hygromycin resistance is used in combination with an expression construct library in cells grown in the presence of hygromycin.
  • the cells would not be viable in the presence of a compound that facilitates the silencer' s activity unless expression from an expression construct results in a factor that suppresses the activity of the compound such that hygromycin resistance is expressed.
  • the surviving cells are isolated, and the sequences responsible for suppressing the activity of the compound, and present in the expression construct, are isolated and/or identified by methods such as PCR or sequencing as described herein.
  • Example 5 Using negative selection and a compound that deactivates a silencer controlling its expression
  • a marker/reporter construct containing a first nucleic acid molecule comprising a silencer operably linked to a Simian Virus 40 (SV40) basal promoter operably linked to a luciferase coding sequence, an IRES, and a sequence encoding HSV tk is used in combination with an expression construct library in cells grown in the presence of gancyclovir.
  • SV40 Simian Virus 40
  • IRES luciferase coding sequence
  • HSV tk luciferase coding sequence
  • Introduction of a compound that deactivates the silencer to result in expression of HSV tk causes cell death unless expression from an expression construct results in a factor that suppresses the ability of the compound to deactivate the silencer.
  • Use of a second nucleic acid molecule in the marker/reporter construct should include the use of a sequence encoding a positive selection marker to decrease identification of factors that do not act through the silencer to affect HSV tk expression.
  • the surviving cells are isolated, and the sequences responsible for suppressing the activity of the compound, and present in the expression construct, are isolated and/or identified by methods such as PCR or sequencing as described herein.
  • a marker/reporter construct containing a first nucleic acid molecule comprising a regulatory module operably linked to a Simian Virus 40 (SV40) basal promoter operably linked to a luciferase coding sequence, an IRES, and a sequence encoding hygromycin resistance is used in combination with an expression construct library in cells.
  • the regulatory module is such that expression of hygromycin resistance is low or insignificant such that upon addition of hygromycin, the cells would die. Only cells wherein expression from an expression construct results in a factor that activates expression of hygromycin resistance will be viable.
  • the surviving cells are isolated, and the sequences responsible for activating expression of hygromycin resistance, and present in the expression construct, are isolated and/or identified by methods such as PCR or sequencing as described herein.
  • Example 7 Using positive selection with no compound to identify deactivators of a silencer
  • a marker/reporter construct containing a first nucleic acid molecule comprising a silencer operably linked to a Simian Virus 40 (SV40) basal promoter operably linked to a luciferase coding sequence, an IRES, and a sequence encoding hygromycin resistance is used in combination with an expression construct library in cells.
  • the silencer results in no or low expression of hygromycin resistance such that upon addition of hygromycin, the cells would die. Only cells wherein expression from an expression construct results in a factor that deactivates the silencer to result in expression of hygromycin resistance will be viable.

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Abstract

L'invention concerne des matériaux de construction d'acides nucléiques ainsi que leur utilisation dans l'identification de facteurs cellulaires qui interviennent dans divers processus cellulaires impliquant l'expression génique. Ces facteurs incluent ceux qui participent aux processus de signalisation pour réguler l'expression génique cellulaire. Ces facteurs peuvent être les cibles d'agents thérapeutiques connus, de nouvelles cibles pour un composé d'essai, ou soumis à une expression modifiée pour moduler les processus cellulaires.
PCT/US2003/014788 2003-05-09 2003-05-09 Procede d'identification de cibles cellulaires WO2005078069A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5767362A (en) * 1993-06-08 1998-06-16 Calgene, Inc. Methods and compositions for modulating lipid content of plant tissues
WO2002036760A2 (fr) * 2000-10-30 2002-05-10 Novozymes Biotech, Inc. Methodes d'expression des genes endogenes par integration mediee par enzymes de restriction

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5767362A (en) * 1993-06-08 1998-06-16 Calgene, Inc. Methods and compositions for modulating lipid content of plant tissues
WO2002036760A2 (fr) * 2000-10-30 2002-05-10 Novozymes Biotech, Inc. Methodes d'expression des genes endogenes par integration mediee par enzymes de restriction

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