US20030194727A1 - Phenotypic screen of chimeric proteins - Google Patents

Phenotypic screen of chimeric proteins Download PDF

Info

Publication number
US20030194727A1
US20030194727A1 US10/314,669 US31466902A US2003194727A1 US 20030194727 A1 US20030194727 A1 US 20030194727A1 US 31466902 A US31466902 A US 31466902A US 2003194727 A1 US2003194727 A1 US 2003194727A1
Authority
US
United States
Prior art keywords
cell
nucleic acid
protein
zinc finger
transcription factor
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US10/314,669
Other languages
English (en)
Inventor
Jin-soo Kim
Kyung-Soon Park
Dong-Ki Lee
Wongi Seol
Horim Lee
Seong-Il Lee
Hyo-Young Yang
Yangsoon Lee
Young-Soon Jang
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Toolgen Inc
Original Assignee
Toolgen Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Toolgen Inc filed Critical Toolgen Inc
Priority to US10/314,669 priority Critical patent/US20030194727A1/en
Assigned to TOOLGEN, INC. reassignment TOOLGEN, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: JANG, YOUNG-SOON, KIM, JIN-SOO, LEE, DONG-KI, LEE, HORIM, LEE, SEONG-IL, LEE, YANGSOON, PARK, KYUNG SOON, SEOL, WONGI, YANG, HYO-YOUNG
Priority to US10/669,861 priority patent/US7514257B2/en
Publication of US20030194727A1 publication Critical patent/US20030194727A1/en
Priority to US10/746,864 priority patent/US20040259258A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
    • 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
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/04—Plant cells or tissues
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • 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
    • C12N2510/00—Genetically modified cells
    • 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
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • genes are regulated at the transcriptional level by polypeptide transcription factors that bind to specific DNA sites within the gene, typically in promoter or enhancer regions. These proteins activate or repress transcriptional initiation by RNA polymerase at the promoter, thereby regulating expression of the target gene.
  • Many transcription factors, both activators and repressors include structurally distinct domains that have specific functions, such as DNA binding, dimerization, or interaction with the transcriptional machinery.
  • the DNA binding portion of the transcription factor itself can be composed of independent structural domains that contact DNA.
  • the three-dimensional structures of many DNA-binding domains, including zinc finger domains, homeodomains, and helix-turn-helix domains, have been determined from NMR and X-ray crystallographic data.
  • Effector domains such as activation domains or repression domains retain their function when transferred to DNA-binding domains of heterologous transcription factors (Brent and Ptashne, (1985) Cell 43:729-36; Dawson et al., (1995) Mol. Cell Biol. 15:6923-31).
  • Artificial transcription factors can be produced that are chimeras of zinc finger domains.
  • WO 01/60970 (Kim et al.) describes methods for determining the specificity of zinc finger domains and for constructing artificial transcription factors that recognize particular target sites.
  • One application for artificial transcription factors is to alter the expression of a particular target gene. Target sites are identified in the regulatory region of the target gene, and artificial transcription factors are engineered to recognize one or more of the target sites. When such artificial transcription factors are introduced into cells, they may bind to the corresponding target sites and modulate transcription.
  • This strategy for controlling the expression of a target gene is sometimes referred to as the “target-driven” approach for identifying transcription factors.
  • the invention features a method that includes: (1) providing a library of cells, the library comprising a plurality of cells that each have a heterologous nucleic acid that expresses an artificial, chimeric polypeptide comprising a first and a second binding domain, wherein the first and second binding domain are heterologous to each other, and the first and second binding domain of each member of the plurality differ from those of the other members of the plurality; and (2) identifying, from the library, a cell that has a trait that is altered relative to a reference cell.
  • the binding domains can be, for example, independently folded modules, e.g., zinc finger domains. In many embodiments, the binding domains are DNA binding domain.
  • the reference cell is a cell which does not include a library nucleic acid or which includes a control nucleic acid.
  • the reference cell may be a parental cell from which the library of cells was made, or a derivative thereof.
  • the trait can be any detectable phenotype, e.g., a phenotype that can be observed, selected, inferred, and/or quantitated.
  • a chimeric protein includes at least two binding domains that are heterologous to each other.
  • the two binding domains can be from different naturally occurring proteins.
  • the two regions can also be from the same naturally occurring protein, but are positioned in a different configuration in the chimeric protein relative to the corresponding naturally occurring protein.
  • a naturally occurring sequence is a sequence whose sequence was determined on or before Dec. 7, 2001; Apr. 26, 2002; Aug. 2, 2002; Aug. 5, 2002, or Dec. 9, 2002.
  • the cell does not include a reporter gene.
  • the cells can be screened without having, a priori, information about a target gene whose regulation is altered by expression of the chimeric polypeptide.
  • the cell may include a reporter gene as an additional indicator of a marker that is related or unrelated to the trait.
  • one or more target genes may be known prior to the screening.
  • the trait is production of a compound (e.g., a natural or artificial compound.
  • the compound can be an antibiotic, an anti-proliferative drug, an analgesic, a protein and so on.
  • the trait is resistance to an environmental condition, e.g., heavy metals, salinity, environmental toxins, biological toxins, pathogens, parasites, other environmental extremes (e.g., desiccation, heat, cold), and so forth.
  • the trait is stress resistance (e.g., to heat, cold, extreme pH, chemicals, such as ammonia, drugs, osmolarity, and ionizing radiation).
  • the trait is drug resistance. The change in the trait can be in either direction, e.g., towards sensitivity or further resistance.
  • the cell is a plant, animal (e.g., mammalian), fungal, or bacterial cell.
  • the trait can be cell proliferation, production of a cytokine, hormone, or signaling molecule, activation of a cell signaling pathway, activation of a physiological pathway (e.g., glucose homeostasis, metabolism, obesity).
  • the DNA binding domains can be, for example, zinc finger domains.
  • the first zinc finger domain varies among nucleic acids of the library, and the second zinc finger domain also varies among nucleic acids of the plurality.
  • the nucleic acid can also express at least a third DNA binding domain, e.g., a third zinc finger domain.
  • the zinc finger domains of each expressed polypeptide can be identical to zinc finger domains from different naturally occurring proteins, or can be variants of naturally-occurring proteins, e.g., mutants at the DNA contacting positions.
  • the naturally-occurring protein can be any eukaryotic zinc finger protein: for example, a fungal (e.g., yeast), plant, or animal protein (e.g., a mammalian protein, such as a human or murine protein).
  • Each polypeptide can further include a third, fourth, fifth, and/or sixth zinc finger domain.
  • Each zinc finger domain can be a mammalian, e.g., human zinc finger domain.
  • the nucleic acids of the plurality of cells encode a sufficient number of different zinc finger domains to recognize at least 10, 20, 30, 40, or 50 different 3-base pair DNA sites. In one embodiment, the nucleic acids the nucleic acids of the plurality encode a sufficient number of different zinc finger domains to recognize no more than 30, 20, 10, or 5 different 3-base pair DNA sites.
  • the polypeptide expressed from each nucleic acid of the library of cells can also include a functional transcriptional regulatory domain, e.g., a transcription activation, repression domain methylation domain, acetylation domain, or deacetylation domain. Also many chimeric polypeptides are functional without fusion to a particular transcriptional regulatory domain.
  • the nucleic acid encoding the polypeptide can be operably linked to a constitutive or inducible promoter.
  • the method can further include isolating the nucleic acid from the identified cell.
  • the nucleic acid can be sequenced.
  • the polypeptide encoded by the nucleic acid can be isolated.
  • the method can further include identifying a nucleic acid binding site specifically recognized by the polypeptide.
  • the binding site can be identified, e.g., by a computer string or profile search of a sequence database, particularly, a database of regulatory sequences or by selecting in vitro nucleic acids that bind to the polypeptide (e.g., SELEX).
  • a computer database of nucleic acid sequences can be analyzed to identify occurrences of the identified nucleic acid binding site or sites similar to the identified binding site.
  • the method can include analyzing the expression of one or more endogenous genes or the level/activity of one or more endogenously expressed polypeptides in the identified cell, e.g., using mRNA profiling (e.g., using microarray analysis), 2-D gel electrophoresis, an array of protein ligands (e.g., antibodies), and/or mass spectroscopy. Also, a single or small number of genes or proteins can also be profiled. In one embodiment, the profile is compared to a database of reference profiles. In another embodiment, regulatory regions of genes whose expression is altered by expression of the identified chimeric polypeptide are compared to identify candidate sites that determine coordinate regulation that results directly or indirectly from expression of the chimeric polypeptide.
  • the method can also include cultivating the cell to exploit the altered trait. For example, if the altered trait is increased production of a metabolite, the method can include cultivating the cell to produce the metabolite.
  • the cell can be the cell isolated from the library, or a cell into which the nucleic acid encoding the chimeric polypeptide has been re-introduced. Expression of the chimeric polypeptide can be tuned, e.g., using an inducible promoter, in order to finely vary the trait, or another conditional promoter (e.g., a cell type specific promoter).
  • a cell containing the nucleic acid encoding the chimeric polypeptide can be introduced into an organism (e.g., ex vivo treatment), or used to make a transgenic organism.
  • At least some library members encode proteins having different regulatory domains.
  • some library members can include an activation domain, and other members, an inhibitory domain.
  • a particular combination of DNA binding domains may be represented in the library in one instance as a fusion to an activation domain, and in another instance as a fusion to a repression domain.
  • some library members include an activation domain, whereas others do not have a regulatory domain.
  • stem cells populations e.g., hematopoietic stem cells, neuronal stem cells, epidermal stem cells, or cord blood stem cells
  • other cells having a limited ability to expand in vitro
  • inhibition of stem cell differentiation s not differentiate e.g., increased pluripotency of a cell (e.g., a differentiated cell or a stem cell); altered stress resistance (e.g., increased or decreased resistance to a condition such as heat, cold, extreme pH, chemicals such as ammonia which can be produced during cell culture, drugs, salt (osmality), ionizing irradiation, etc.); sensitivity to ionizing irradiation or a toxic agent (e.g., an anticancer drug cells), e.g., increased sensitivity in cancer cells; ability to support viral infection/replication (e.g., hepatitis C replication); ability to resist a pathogen, e.g., a virus, bacteria, or prot
  • Exemplary applications of these methods include: identifying essential genes in a pathogen (e.g., a pathogenic microbe), identifying genes (of the host or pathogen) required for pathogenesis of microbes, identifying targets of drug candidates, gene discovery in signal transduction pathways, microbial engineering and industrial biotechnology, increasing yield of metabolites of commercial interests, and modulating growth behavior (e.g. improving growth of a microorganism, or reducing growth of a cancer cell).
  • a pathogen e.g., a pathogenic microbe
  • identifying genes of the host or pathogen required for pathogenesis of microbes
  • identifying targets of drug candidates e.g., identifying targets of drug candidates
  • gene discovery in signal transduction pathways e.g., microbial engineering and industrial biotechnology
  • increasing yield of metabolites of commercial interests e.g. improving growth of a microorganism, or reducing growth of a cancer cell.
  • the invention features a cultured cell that includes (a) a gene (endogenous or exogenous) encoding a protein, and (b) an artificial transcription factor, wherein the cell produces the protein at a higher level than does an identical cell that comprises the gene but not the transcription factor, and wherein the transcription factor exerts its effect on production of the protein in a manner other than by binding to a regulatory region operably linked to the gene.
  • a gene endogenous or exogenous
  • an artificial transcription factor wherein the cell produces the protein at a higher level than does an identical cell that comprises the gene but not the transcription factor, and wherein the transcription factor exerts its effect on production of the protein in a manner other than by binding to a regulatory region operably linked to the gene.
  • artificial means not naturally-occurring.
  • gene means a “coding sequence”, either cDNA or genomic (i.e., with introns), and either endogenous or exogenous (transiently or stably transfected).
  • the artificial transcription factor can include a chimeric DNA binding domain that includes at least two, three, or four zinc finger domains.
  • the artificial transcription factor can further include a regulatory domain (list of activation and repression domain in summary).
  • At least one or two of the individual zinc finger domains may be naturally-occurring (e.g., mammalian, plant, or human). In one embodiment, all the zinc finger domains present are naturally occurring.
  • the artificial transcription factor can be encoded by a heterologous gene of the cell.
  • the heterologous gene encoding the heterologous transcription factor can be regulated by an inducible promoter.
  • the cell can further include at least a second artificial transcription factor.
  • the artificial transcription factor causes the cell or a culture cell lacking the first gene, but otherwise identical to produce a second protein encoded by a second gene operably linked to a regulatory region other than the regulatory region operably linked to the first gene at a higher level than does an identical cell including the second gene but not the transcription factor, and (ii) the transcription factor exerts its effect on production of the second protein in a manner other than by binding to a regulatory region operably linked to the second gene.
  • the cell further includes a second gene encoding a second protein
  • the cell produces the second protein at a higher level than does an identical cell including the second gene but not the transcription factor
  • the transcription factor exerts its effect on production of the second protein in a manner other than by binding to a regulatory region operably linked to the second gene.
  • the regulatory region operably linked to the first gene can be different from the regulatory region operably linked to the second gene.
  • the first and second transcription factors are selected from the group consisting of:
  • a polypeptide including the amino acid sequence of SEQ ID NO:21 (FECKDCGKAFIQKSNLIRHQRTHTGEKPYACPVESCDRRFSDSSNLTRHIRIHTGEKP YACPVESCDRRFSDSSNLTRHIRIH);
  • the invention features a method of producing a protein.
  • the method includes providing a cell described herein (e.g., above); culturing the cell under conditions that permit production of the protein at a level higher (e.g., at least two, three, five, ten, or a hundred fold) than the level produced by an identical cell that includes the gene but not the transcription factor; detecting the protein produced by the cell and/or purifying the expressed protein from the cell and/or from a medium that surrounds the cell.
  • the gene can be an endogenous or exogenous gene.
  • An endogenous gene can be a naturally occurring gene or a gene that is genetically altered relative to a naturally-occurring gene (e.g., by insertion or modification of regulatory sequences).
  • endogenous genes include genes encoding a hormone, a cell surface receptor, an antibody, a growth factor, an adhesion protein, a neurotransmitter, and an enzyme.
  • An exogenous gene can be operably linked to a viral promoter, e.g., a CMV or adenovirus promoter.
  • the cell can be a mammalian cell.
  • the method can further include introducing the cell into a subject.
  • the method can further include formulating the purified protein with a pharmaceutically acceptable carrier.
  • the method can include one or more of the following features: the transcription factor is present in the cell in an amount effective to increase production of a luciferase marker protein encoded by a gene operably linked to a CMV promoter at least 1.1, or 2 fold; the transcription factor is present in the cell in an amount effective to increase production of a secreted alkaline phosphatase marker protein encoded by a gene operably linked to an SV40 promoter at least 2, 5, 7, 10 fold; the transcription factor directly alters the expression of a plurality of endogenous genes; the transcription factor alters the rate of division of the cell; the transcription factor competes for binding to a naturally-occurring DNA binding site specifically recognized by PB08, K_F02, or K_D10, and has a dissociation constant for a DNA site of less than 50 nM; the transcription factor specifically recognizes a DNA site that partially overlaps the DNA binding site specifically recognized by PB08, K_F02, or K_D10; the transcription factor includes two consecutive zinc finger
  • the transcription factor can include an amino acid sequence that differs by 1 to 8 amino acid substitutions, insertions, or deletions of SEQ ID NO:21, 22, or 23.
  • the substitution may be at a position other than a DNA contacting residue, e.g., between a metal coordinating cysteine and position ⁇ 1.
  • the substitutions can be conservative substitutions.
  • the transcription factor includes an amino acid encoded by a nucleic acid sequence in FIGS. 17, 18, and 19 .
  • the transcription factor includes the following three zinc finger domains:
  • the transcription factor includes at least three of the following four zinc finger domains (in this order):
  • the transcription factor includes at least three of the following four zinc finger domains (in this order):
  • the invention features a method of producing a target protein that includes expressing a nucleic acid encoding the target protein in a cell (e.g., in vitro or in vivo), wherein the cell includes a heterologous, artificial transcription factor that increase the amount of protein produced relative to a cell that does not include the heterologous transcription factor.
  • the heterologous transcription factor causes the increase by a mechanism other than by directly regulating transcription of the gene encoding the target protein.
  • the heterologous transcription factor does not bind to a transcriptional regulatory region that directly regulates the nucleic acid encoding the target gene.
  • the transcription factor can be introduced into the cell as a protein or by introduction and transcription of a nucleic acid encoding it.
  • the method can include other features described herein.
  • the invention features a method of identifying a transcription factor.
  • the method includes: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including at least two zinc finger domains that are chimeric with respect to each other; introducing a member of the library into each of a plurality of cells; identifying a cell from the plurality that has enhanced production of a first target protein, wherein the first target protein is encoded by a gene operably linked to a first transcriptional regulatory sequence; and evaluating the ability of the member of the library to enhance production of a second target protein encoded by a gene operably linked to a second transcriptional regulatory sequence that is different from the first transcriptional regulatory sequence.
  • the method can include one or more of the following features: the first and second target proteins are the same; the cells into which the library members are introduced each includes the gene encoding the second target protein operably linked to the second transcriptional regulatory sequence; the cell is a eukaryotic cell; the first and/or second transcriptional regulatory sequences includes a viral regulatory sequence; the method further includes preparing a host cell that includes a gene encoding transcription factor encoded by a member of the library that enhances production of the first and the second target protein; the method further includes producing a third target protein, different from the first and second target protein from the host cell; and the evaluating includes evaluating the identified cell.
  • the second or third target protein can be, for example, a secreted protein, e.g., erythropoietin, thrombopoietin, a growth factor, an interleukin, or a chemokine.
  • the target protein is an enzyme, e.g., that catalyzes a reaction in a metabolite producing pathway. Other features described herein can also be included.
  • the invention features a method of identifying a protein chimera.
  • the method includes: providing a nucleic acid library that comprises a plurality of nucleic acids, each encoding a different artificial protein chimera that comprises at least two zinc finger domains; providing a test cell that produces a given level of a first target protein under a specific condition; wherein the first target protein is encoded by a gene operably linked to a first transcriptional regulatory sequence; introducing each member of the plurality into a replicate of the test cell to provide a plurality of transformed cells; identifying, from the plurality of transformed cells or progeny cells thereof, a cell that produces, under the specific condition, a level of the first target protein that differs from the given level; and evaluating the ability of the member of the library in the identified cell to enhance production of a second target protein, encoded by a gene operably linked to a second transcriptional regulatory sequence, different from the first transcriptional regulatory sequence.
  • Other features described herein can also be included.
  • the invention features a host cell, whose genetic material includes: a heterologous gene that encodes an artificial transcription factor that increases production of a target protein encoded by a target gene at least 30% relative to an otherwise identical control host cell lacking the heterologous gene.
  • the transcription factor does not directly regulate transcription of the target protein.
  • the transcription factor directly regulates transcription of the target protein.
  • the host cell can include a sequence or a reporter construct such as a sequence encoding lacZ, secreted alkaline phosphatase (SEAP), GFP, luciferase etc) operably linked to a promoter (e.g., a viral promoter).
  • SEAP secreted alkaline phosphatase
  • GFP secreted alkaline phosphatase
  • luciferase luciferase etc
  • the cell can include other features described herein.
  • the invention features a host cell, whose genetic material includes a heterologous gene that encodes a polypeptide that includes at least two, three, or four zinc finger domains, binds to an naturally-occurring DNA site, e.g., with an equilibrium dissociation constant of less than 50 nM, and competes with PB08, K_F02, or K_D10 for binding to the naturally-occurring DNA binding site.
  • the cell can include other features described herein.
  • the invention features a host cell, whose genetic material includes a first heterologous gene that encodes a target protein, and a second heterologous gene that encodes an artificial transcription factor that increases production of the target protein at least 30% relative to an otherwise identical control host cell lacking the second heterologous gene, wherein the transcription factor does not directly regulate transcription of the target protein.
  • the cell can include other features described herein.
  • the invention features an isolated polypeptide that includes: the sequence: X a -X-Cys-X 2-5 -Cys-X 3 -X a -X 5 -X b -X-Arg-His-X 3-5 -His-X 1-6 -X a -X-Cys-X 2-5 -Cys-X 3 -X a -X 5 -X b -X-Arg-His-X 3-5 -His-X 1-6 -X a -X-Cys-X 2-5 -Cys-X 3 -X a -X 5 -X b -X-Arg-His-X 3-5 -His (SEQ ID NO:35), and that has one or more of the following effects when present at an effective concentration in a human 293 cell: a) increases expression of a gene encoding a luciferase operably linked to a CMV promoter at
  • the invention features a method of altering the differentiated state of a metazoan cell, the method including: expressing an artificial transcription factor in the cell in an amount effective to alter the differentiated state of the cell.
  • the differentiated state can be characterized by a neuronal phenotype (e.g., neurite extension, synapse formation, or neuronal marker expression) or an osteoblasts phenotype (e.g., osteoblasts marker expression).
  • the differentiated state is altered so as to increase the pluripotency of the cell, e.g., to make it less differentiated, e.g., functional as a stem cell or a precursor cell.
  • the differentiated state is altered from one differentiated state to another (e.g., from a myogenic cell state to an osteoblast state, from a neuronal state to a glial state, and so forth).
  • the artificial transcription factor induces neurite extension.
  • the cell can be, for example, a stem cell, a neuronal cell, a neural crest cell, or a neuronal progenitor cell.
  • the artificial transcription factor can be a transcription factor described herein, e.g., Neuro1-p65 or a Neuro1 related molecule (see below).
  • the artificial transcription factor can compete for binding to a natural DNA binding site with Neuro1-p65.
  • the artificial transcription factor may bind to the same site as Neuro1-p65 or a site that overlaps a DNA site bound by Neuro1-p65.
  • the artificial transcription factor induces an osteoblasts-specific marker, e.g., in an initially non-osteoblast cell, e.g., a myoblast.
  • the invention features a method of identifying a transcription factor.
  • the method includes: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including at least two zinc finger domains; introducing a member of the library into each of a plurality of cells; and identifying a cell from the plurality that has an altered differentiated state.
  • the method can include one or more of the following features: the cell is a stem cell; the cell is a neuronal cell, a neural crest cell, or a neuronal progenitor cell; and the differentiated state includes neuronal outgrowth or neurite formation. Other features can also be included.
  • the invention features a method of identifying a plurality of transcription factors.
  • the method includes: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including at least two zinc finger domains; identifying a first member of the library which alters a given trait of a cell; and screening cells to identify a cell in which the given trait is further altered, wherein each screened cell expresses the transcription factor encoded by the first member of the library and a transcription factor encoded by second member of the same nucleic acid library or another nucleic acid library of artificial transcription factors.
  • the method can include additional rounds of screening in the presence of the first and second transcription factors.
  • the method includes identifying a second member of the library that reverses the change in phenotypic state effected by the first member.
  • the method can include other features described herein.
  • the invention features a method of preparing a modified cell, the method including providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including at least two zinc finger domains; identifying a first and a second member of the library which alters a given trait of a cell; and preparing a cell that can express first and second polypeptides, the first and second polypeptides being encoded respectively by the first and second identified library members.
  • the method can also be extended to additional member, e.g., a third member.
  • the method can further include evaluating the given trait for the prepared cell.
  • the method can further include one or more of the following features: the preparing includes introducing a first gene encoding the first polypeptide and a second gene encoding the second polypeptide into the cell; the first and second gene are components of the same nucleic acid; wherein the preparing includes fusing a first cell that includes a first gene encoding the first polypeptide to a second cell that includes a second gene encoding the second polypeptide; the given trait is production of a metabolite; the given trait is production of a target polypeptide; the production includes secretion.
  • the preparing includes introducing a first gene encoding the first polypeptide and a second gene encoding the second polypeptide into the cell; the first and second gene are components of the same nucleic acid; wherein the preparing includes fusing a first cell that includes a first gene encoding the first polypeptide to a second cell that includes a second gene encoding the second polypeptide; the given trait is production of a metabolite
  • a method of identifying a transcription factor can include: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including at least two zinc finger domains; packaging each member of the library into a virus or viral particle that infects mammalian cells to form a plurality of viruses or viral particles; introducing the plurality of viruses or viral particles into a plurality of non-human mammalian subjects; and identifying a subject from the plurality that has an altered phenotype.
  • each subject has a detectable disorder; the plurality of viruses or viral particles is divided into pools, and each pool is introduced into one of the subjects from the plurality of subjects; the plurality of viruses or viral particles includes separate samples, each sample having packaged within a single nucleic acid of the nucleic acid library, and a single sample is introduced into each subject.
  • the invention features a modified cell that includes a heterologous nucleic acid encoding an artificial transcription factor that confers stress resistance to the modified cell relative to a reference cell that is substantially identical to the modified cell and that lacks the heterologous nucleic acid and the artificial transcription factor.
  • the artificial transcription factor includes at least two zinc finger domains.
  • One or more of the zinc finger domains can be naturally occurring, e.g., a naturally occurring domain in Table 1.
  • one or more of the zinc finger domains can include at least 17, 18, 19, 20, or 21 amino acids that are identical to a zinc finger domain sequence in Table 1.
  • the DNA contacting residues can be among the identical amino acids.
  • Exemplary artificial transcription factors include transcription factors that have one or more consecutive motifs as described herein, e.g., a thermotolerant or solvent tolerant protein described herein.
  • the modified cell can be a prokaryotic or eukaryotic cell.
  • the stress resistance can include one or more the following traits: heat resistance, solvent resistance, heavy metal resistance, osmolarity resistance, resistance to extreme pH, chemical resistance, cold resistance, and resistance to a genotoxic agent, resistance to radioactivity. Stress resistance enables a resistant cell to survive or grow in a condition in which a non-resistant cell would die or fail to grow.
  • the modified cell can express the artificial transcription factor and resist stress to a greater extent than a substantially identical culture cell that lacks the artificial transcription factor.
  • the invention also provides such artificial proteins, and proteins that alter sensitivity of the cell to a toxic agent relative to an identical cell that does not contain the nucleic acid.
  • the method includes a method of producing a cellular product.
  • the method includes providing a modified cell that includes a heterologous nucleic acid encoding an artificial transcription factor; maintaining the modified cell under conditions in which the artificial transcription factor is produced; and recovering a product produced by the cultured cell, wherein the product is other than the artificial transcription factor.
  • the artificial transcription factor can confer stress resistance, or another property described herein, e.g., altered protein production, altered metabolite production, and so forth.
  • the artificial transcription factor includes at least two zinc finger domains. One or more of the zinc finger domains can be naturally occurring, e.g., a naturally occurring domain in Table 1.
  • one or more of the zinc finger domains can include at least 17, 18, 19, 20, or 21 amino acids that are identical to a zinc finger domain sequence in Table 1.
  • Exemplary artificial transcription factors include transcription factors that have one or more consecutive motifs (e.g., at least two, three or four consecutive motifs, or at least three motifs in the same pattern, including non-consecutive patterns) as described herein.
  • Exemplary products include a metabolite or a protein (e.g., an endogenous or heterologous protein.
  • the modified cell includes a nucleic acid encoding a heterologous protein, other than the artificial transcription factor, and the product is the heterologous protein.
  • the modified cell further includes a second nucleic acid encoding a heterologous protein, and the heterologous protein participates in production of the metabolite.
  • the modified cell can be maintained at a temperature between 20° C. and 40° C. or greater than 37° C. In one embodiment, the modified cell is maintained under conditions which would inhibit the growth of a substantially identical cell that lacks the artificial transcription factor.
  • the zinc finger domain includes a set of DNA contacting residues that correspond to DNA contacting residues of a zinc finger domain listed in Table 15.
  • the artificial transcription factor includes an array of at least three zinc finger domains, wherein the DNA contacting residues of each of the zinc finger domains of the array correspond respectively to DNA contacting residues of any three consecutive zinc finger domains listed in a row of Table 15.
  • the artificial transcription factor competes with a protein that includes the array of zinc finger domains listed in a row of Table 15.
  • the invention features a cell that contains a gene (e.g., an endogenous or heterologous gene) encoding a target protein and a heterologous nucleic acid that includes a sequence encoding an artificial protein chimera that (1) increases the amount of the protein produced by the cell relative to a cell that does not include the heterologous nucleic acid, and (2) does not bind to a transcriptional regulatory region that directly regulates transcription of the gene encoding the target protein.
  • the artificial transcription factor includes at least two zinc finger domains, and, e.g., binds DNA.
  • One or more of the zinc finger domains can be naturally occurring, e.g., a naturally occurring domain in Table 1.
  • one or more of the zinc finger domains can include at least 17, 18, 19, 20, or 21 amino acids that are identical to a zinc finger domain sequence in Table 1.
  • Exemplary artificial transcription factors include transcription factors that have one or more consecutive motifs as described herein,
  • the cell is a eukaryotic cell, and the artificial protein chimera competes with PB08, K_F02, or K_D10 for binding to a genomic DNA site.
  • the artificial protein chimera alters (e.g., increases or decrease) the rate of cell cycle progression by the cell.
  • the invention also provides such artificial transcription factors.
  • the cell can be used in a method of producing a protein, the method including: providing the cell, and maintaining the cell under conditions in which the artificial protein chimera increases the amount of the target protein produced by the cell relative to a cell that does not include the heterologous nucleic acid.
  • the protein is a secreted protein, a cytoplasmic protein, or a nuclear protein.
  • the invention features a cell that contains an endogenous gene encoding a secreted protein and a heterologous nucleic acid that includes a sequence encoding an artificial transcription factor that increases the amount of the secreted protein produced by the cell relative to a cell that does not include the heterologous nucleic acid.
  • the cell is a eukaryotic cell, and the secreted protein is insulin.
  • the artificial transcription factor specifically binds to an endogenous DNA site that is also specifically bound by 08_D04_p65.
  • the artificial transcription factor specifically binds to an endogenous DNA site and the artificial transcription factor competes with 08_D04_p65 for binding to the endogenous DNA site.
  • the artificial transcription factor includes an amino acid sequence as follows:
  • B is phenylalanine or tyrosine
  • J is a hydrophobic amino acid
  • the cell can be used in a method of producing a secreted protein (e.g., insulin).
  • a secreted protein e.g., insulin
  • the cell is maintained under conditions in which the artificial transcription factor increases the amount of insulin produced by the cell relative to a cell that does not include the heterologous nucleic acid.
  • the invention provides an artificial transcription factor including at least two zinc finger domains, wherein the artificial transcription factor induces expression of an endogenous insulin gene in a mammalian cell that does not express an endogenous insulin gene in the absence of the artificial transcription factor.
  • the invention features an artificial transcription factor that alters sensitivity of the cell to a toxic agent (e.g., a drug, e.g., an anti-fungal drug, e.g., ketoconazole) relative to an identical cell that does not contain the nucleic acid.
  • a toxic agent e.g., a drug, e.g., an anti-fungal drug, e.g., ketoconazole
  • the cell is a fungal cell.
  • the artificial transcription factor includes at least two zinc finger domains, e.g., at least three.
  • One or more of the zinc finger domains can be naturally occurring, e.g., a naturally occurring domain in Table 1.
  • Exemplary artificial transcription factors include transcription factors that have one or more consecutive motifs as described herein.
  • the artificial transcription factor binds to an endogenous DNA site and the artificial transcription factor competes with a zinc finger protein listed in Table 5 for binding to the endogenous DNA site.
  • the invention features a method of altering the drug resistance of a fungal cell.
  • the method includes altering the expression or activity of a protein that is at least 70% identical to AQY1, YJR147W, YLL052C, YLL053C, or YPL091W in the cell.
  • the expression can be altered, e.g., using a transcription factor.
  • the invention features a method of identifying an artificial chimeric protein that alters the sensitivity of a cell to a toxic agent, the method including: providing a nucleic acid library that includes a plurality of nucleic acids, each nucleic acid of the plurality encoding a chimeric protein that includes an array of at least three zinc finger domains wherein at least two adjacent zinc finger domains do not occur adjacent to each other in a naturally occurring protein; introducing members of the library into replicates of a test cell to yield transformed cells; cultivating the transformed cells in the presence of a toxic agent; and identifying, from the transformed cells, a cell whose sensitivity to the toxic agent is altered relative to the test cell.
  • test cell is a fungal cell, and the toxic agent is an anti-fungal agent.
  • test cell is a cancer cell, and the toxic agent is an anti-mitotic agent.
  • the chimeric protein encoded by each nucleic acid of the plurality can include a transcriptional regulatory domain.
  • the method can further include constructing a nucleic acid that encodes a second chimeric protein that includes the array of zinc finger domains of the chimeric protein encoded by the library member in the selected cell, but does not include the transcriptional regulatory domain of the chimeric protein encoded by the library member in the identified cell.
  • the method can further include constructing a nucleic acid that encodes a second chimeric protein that includes (i) the array of zinc finger domains of the chimeric protein encoded by the library member in the selected cell, and (ii) a transcriptional regulatory domain, other than the transcriptional regulatory domain of the chimeric protein encoded by the library member in the identified cell.
  • the invention features a nucleic acid that includes a sequence encoding an artificial transcription factor including three zinc finger domains, wherein expression of the artificial transcription factor induces a neuronal phenotype in at least one vertebrate cell.
  • at least one of the zinc finger domains has the sequence:
  • the artificial transcription factor includes the sequence: Cys-X 2-5 -Cys-X 3 -X a -X-Gln-X b -X-Ser-Asn-His-X 3-5 -His-X 1-6 -Cys-X 2-5 -Cys-X 3 -X a -X-Gln-X b -X-Ser-Asn-His-X 3-5 -His-X 1-6 -Cys-X 2-5 -Cys-X3-X a -X-Cys-X b -X-Ser-Asn-His-X 3-5 -His (SEQ ID NO:253), wherein X a is phenylalanine or tyrosine; and X b is a hydrophobic amino acid.
  • One method includes providing a vertebrate cell (e.g., a mammalian cell, a human) that contains the nucleic acid; and maintaining the vertebrate cell under conditions in which the artificial transcription factor is produced and neurite formation is induced.
  • the vertebrate cell is a stem cell prior to production of the artificial transcription factor.
  • the invention features a nucleic acid that includes a sequence encoding an artificial transcription factor included of three zinc finger domains, wherein expression of the artificial transcription factor induces osteogenesis in at least one vertebrate cell.
  • at least one of the zinc finger domains has the sequence:
  • One method includes providing a vertebrate cell that contains the nucleic acid; and maintaining the vertebrate cell under conditions in which the artificial transcription factor is produced and osteogenesis is induced.
  • the invention features a method of altering the differentiative capacity of a stem cell.
  • the method includes: providing a stem cell and a nucleic acid that includes a sequence encoding an artificial transcription factor includes of a plurality of zinc finger domains, wherein the artificial transcription factor alters the differentiative capacity of the stem cell; introducing the nucleic into the stem cell; and maintaining the stem cell under conditions in which the artificial transcription factor is produced thereby altering the differentiative capacity of the stem cell.
  • the artificial transcription factor induces differentiation of the stem cell.
  • the artificial transcription factor enhances self-renewal potential of the stem cell.
  • the stem cell can be an embryonic stem cell. a vertebrate stem cell, a plant stem cell, a hematopoietic stem cell, a neuronal progenitor cell or muscular progenitor cell.
  • the invention features a method that includes: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each artificial transcription including an array of at least two zinc finger domains and a regulatory domain that activates or represses transcription; providing cell that have a given trait; introducing members of the nucleic acid library into the cells; identifying a member of the library that alters the given trait; and preparing a coding nucleic acid that includes a sequence encoding a DNA binding polypeptide that includes the array of zinc finger domains from the identified member, but does not include a regulatory domain identical to the regulatory domain of the identified member.
  • the DNA binding polypeptide is lacks the regulatory domain of the identified member.
  • the DNA binding polypeptide includes a regulatory domain that is mutated relative to the regulatory domain of the identified member.
  • the DNA binding polypeptide includes a regulatory domain of opposite functionality relative to the regulatory domain of the identified member.
  • the method can further include introducing the coding nucleic acid into a cell and assessing the given trait of the cell.
  • the step of identifying can include identifying cells having a property selected from the group consisting of: resistance to a given environmental condition; differentiation; dedifferentiation; proliferation; apoptosis; serum-independence; pathogen resistance; and pathogen sensitivity.
  • the invention features an isolated nucleic acid encoding an artificial transcription factor that improves solubility of a heterologous, over-expressed protein in a cell that produces the artificial transcription factor.
  • the artificial transcription factor includes a plurality of zinc finger domains.
  • the cell can be a bacterial cell or a eukaryotic cell.
  • the protein is a mammalian protein, e.g., AKT protein.
  • the plurality of zinc finger domains include domains: QSTR-DSAR-RDHT-WSNR or VSTR-DGNV-QSNR-QSNK.
  • the invention also features a modified cell that includes the heterologous nucleic acid of any of claims 99 to 104.
  • the invention also provides a method of producing a heterologous target protein.
  • the method includes providing the modified cell wherein the modified cell includes a second nucleic acid that includes a sequence encoding a heterologous target protein; and maintaining the modified cell under conditions wherein the artificial transcription factor and the heterologous target protein are produced.
  • the modified cell can be a cultured cell or an in vivo cell, e.g., in a subject.
  • the invention features a method of transferring an altered trait from a first cell to a second cell.
  • the method includes: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial protein chimera that includes at least two zinc finger domains; introducing members of the nucleic acid library into first cells that have a given trait to provide transformed cells; identifying an altered cell from the transformed cells in which a member of the library alters the given trait; recovering a nucleic acid library member from the identified, altered cell; introducing the nucleic acid library member into second cells, wherein the second cells differ from the first cells by a phenotypic trait other than the given trait; and evaluating a second cell that includes the nucleic acid library member and expresses the artificial protein chimera that the nucleic acid library member encodes.
  • the first and second cells are eukaryotic, e.g., yeast cells or mammalian cells.
  • the first and second cells can differ by the proliferative properties or differentiative properties.
  • the first cells are cancer cells, and the second cells are non-cancerous, or vice versa.
  • the evaluated second cell can be evaluated for an alteration to the given trait.
  • the invention features a nucleic acid including a sequence encoding an artificial transcription factor included of three zinc finger domains.
  • expression of the artificial transcription factor alters a property of at least one eukaryotic cell, wherein the property is selected from the group consisting of viral replication, virus production, and viral infectivity; expression of the artificial transcription factor alters an ability of a eukaryotic cell to regulate a stem cell that is co-cultured with the eukaryotic cell or that is cultured in media conditioned by the eukaryotic cell; expression of the artificial transcription factor alters an ability of a mammalian culture cell (e.g., a CHO cell) to glycosylate a secreted protein (e.g., an antibody); and expression of the artificial transcription factor alters the ability of a cell to take up exogenous nucleic acid.
  • a mammalian culture cell e.g., a CHO cell
  • glycosylate a secreted protein e.g., an antibody
  • the invention features a method of identifying a transcription factor.
  • the method includes: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including at least two zinc finger domains; introducing members of the nucleic acid library into cells that have a given trait; identifying a member of the library which alters the given trait; and preparing a second library that includes a plurality of nucleic acids, each encoding (1) a variant that differs from the artificial protein chimera corresponding to the identified member by between one and six amino acid substitutions, insertions, or deletions, (2) a protein chimera that comprises the zinc finger domains of the artificial protein chimera corresponding to the identified member and an additional zinc finger domain, wherein the additional zinc finger domain varies among the members of the second library, (3) a variant of the artificial protein chimera corresponding to the identified member, wherein the variant has a subset of the zinc finger domain positions replaced with other zinc finger domain
  • the invention features a method of identifying a transcription factor, the method includes: providing a nucleic acid library that includes a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor including a DNA binding component having at least two zinc finger domains and a first regulatory domain that activates or represses transcription; introducing members of the nucleic acid library into cells that have a given trait; identifying a member of the library which alters the given trait; and preparing a nucleic acid that encodes the DNA binding component of the identified member and a second regulatory domain that differs from the first regulatory domain.
  • a related method includes identifying a transcription factor that lacks a regulatory domain, and adding one; or removing a regulatory domain from a transcription factor that includes one. Some transcription factors can function without a transcriptional regulatory domain.
  • the invention also features an artificial transcription factor that comprises a plurality of zinc finger domains and a first regulatory domain. The artificial transcription factor produces a first trait when produced in a cell, but produces a second trait if the first regulatory domain is inactivated and a second regulatory domain, of opposite functionality from the first, is included.
  • the first regulatory domain activates transcription and the second regulatory domain represses transcription. In another example, the first regulatory domain represses transcription and the second regulatory domain activates transcription. In still another example, the first regulatory domain activates transcription to a first extent and the second regulatory domain activates transcription to an extent at least 50% less than the first extent.
  • the method can further include introducing the prepared nucleic acid into a test cell and assessing the test cell, e.g., the given trait of the test cell.
  • the identified member increases the rate of cell division, and the prepared nucleic acid encodes a transcription factor that decreases the rate of cell division. In another embodiment, the identified member causes resistance to a compound, and the prepared nucleic acid encodes a transcription factor that causes sensitivity to the compound.
  • the invention features a method of identifying a transcription factors, the method including: providing a nucleic acid library that includes first and second pluralities of nucleic acids, wherein each nucleic acid of the first plurality encodes a different artificial transcription factor that includes at least two zinc finger domains and a regulatory domain that activates transcription, and each nucleic acid of the second plurality encodes a different artificial transcription factor that includes at least two zinc finger domains and a regulatory domain that represses transcription; introducing members of the nucleic acid library into cells that have a given trait; and identifying a member of the library which alters the given trait.
  • the invention also features a method that includes evaluating the phenotype of a chimeric artificial zinc finger protein in a first cell, expressing the protein in a second cell, and evaluating a phenotype of the second cell.
  • the method can include identifying a member of a transcription factor library that alters a given phenotype in a first cell (a yeast strain or a human cell line such as 293 cells, for example) and then expressing the member in a different cell (a different yeast strain or HeLa cells, for example).
  • the first cell may be more amenable for screening than other strains or cell lines.
  • the invention features a method of altering the sensitivity of a fungal strain to an anti-fungal agent.
  • the method includes: introducing a nucleic acid encoding an artificial chimeric protein that comprises at least three zinc finger domains into a fungal cell; and maintaining the fungal cell under conditions that allow for expression of the introduced nucleic acid in the cell.
  • the fungal cell is a yeast cell, e.g., Candida, Pichia, Hanseula, Histoplasma, or Cryptococcus.
  • the artificial chimeric protein comprises the amino acid sequence of the zinc finger array of a protein selected from K1 through K11 (see Example 3).
  • the method can include one or more of the following features: the artificial chimeric protein comprises a transcriptional regulatory domain (e.g., an activation or repression domain); expression of the artificial chimeric protein in the cell alters the transcript level of a water transporter; expression of the artificial chimeric protein in a S. cerevisiae cell alters the transcript level of the YLL053 gene or the PDR5 gene; the artificial chimeric protein competes a polypeptide selected from K1 through K11 for binding to a specific DNA site.
  • a transcriptional regulatory domain e.g., an activation or repression domain
  • expression of the artificial chimeric protein in the cell alters the transcript level of a water transporter
  • expression of the artificial chimeric protein in a S. cerevisiae cell alters the transcript level of the YLL053 gene or the PDR5 gene
  • the artificial chimeric protein competes a polypeptide selected from K1 through K11 for binding to a specific DNA site.
  • the invention features a method of altering the sensitivity of a fungal strain to an anti-fungal agent.
  • the method includes altering the activity or expression of a protein that comprises an amino acid sequence of at least 50 amino acids that is at least 30, 50, 60, 70, 80, 90, or 95% identical to YLL053, AQY1, YJR147W, YLL052C, or YPL091W.
  • the fungal cell is a yeast cell, e.g., Candida, Pichia, Hanseula, Histoplasma, or Cryptococcus.
  • the activity or expression is increased to reduce sensitivity, i.e., increase resistance.
  • the activity or expression is reduced to increase sensitivity.
  • Altering the activity or expression can include expressing an artificial transcription factor, contacting the cell with a double-stranded RNA (dsRNA) that includes a sequence of at least 20 nucleotides complementary to YLL053, AQY1, YJR147W, YLL052C, or YPL091W gene, or contacting the cell with a chemical compound.
  • dsRNA double-stranded RNA
  • a related method includes screening a test compound (e.g., a small organic compound) for interaction with such a protein, e.g., to identify an inhibitor of a YLL053/AQY1-related protein or screening a test compound for ability to alter the activity or expression of AQY1, YJR147W, YLL052C, YLL053C or YPL091W.
  • a test compound e.g., a small organic compound
  • the invention features a method of identifying an artificial chimeric protein that alters the sensitivity of a fungal strain to an anti-fungal agent.
  • the method includes: providing a nucleic acid library that comprises a plurality of nucleic acids, each nucleic acid of the plurality comprising a coding sequence that encodes an artificial, chimeric protein that comprises an array of at least three zinc finger domains wherein at least two adjacent zinc finger domains do not occur adjacent to each other in a naturally occurring protein; introducing each nucleic acid of the plurality into a fungal cell to yield transformed fungal cells; maintaining (e.g., culturing) the transformed fungal cells in the presence of an anti-fungal agent; and selecting a cell from the transformed fungal cells whose sensitivity to the anti-fungal agent is altered relative to a control fungal cell.
  • the method can include one or more of the following features: the control fungal cell is not transformed or is transformed with a reference nucleic acid; the chimeric protein encoded by each nucleic acid of the plurality comprises a transcriptional regulatory domain; the fungal cell is a pathogenic fungal cell, e.g., a Candida, Histoplasma, or Cryptococcus.
  • the method can further include, for example, constructing a nucleic acid that encodes a second chimeric protein that comprises (i) the array of zinc finger domains of the chimeric protein encoded by the library member in the selected cell, and (ii) a transcriptional regulatory domain, other than the transcriptional regulatory domain of the chimeric protein encoded by the library member in the selected cell.
  • the method can include other features described herein.
  • the invention features a method of identifying an agent that counters resistance of a fungal cell to an anti-fungal agent.
  • the method includes: contacting a test compound to a polypeptide that includes YJR147W, YLL052C, YLL053C or YPL091 W; and evaluating interaction between the test compound and the polypeptide, wherein an interaction indicates that the test compound may be useful as an agent to counter resistance to an anti-fungal agent.
  • the method can further include contacting a fungal cell with the test compound and the anti-fungal agent, e.g., and evaluating viability or growth of the cell.
  • the cell can be a cell that is resistant to the anti-fungal agent (e.g., ketoconazole).
  • the invention features a method of identifying a target gene that is regulated by artificial transcription factors.
  • the method includes: providing a nucleic acid library that comprises a plurality of nucleic acids, each encoding a different artificial transcription factor, each transcription factor comprising at least two zinc finger domains; introducing each member of the plurality into a replicate of a test cell to provide a plurality of transformed cells; identifying a plurality of phenotypically altered cells from the plurality of transformed cells, wherein each phenotypically altered cell has an altered phenotype relative to the test cell; and identifying one or more transcripts or proteins whose abundance is similarly altered in at least two phenotypically altered cells of the plurality relative to the test cell.
  • the method further includes, in a test cell, altering the activity of a transcript or protein whose abundance is similarly altered in at least two phenotypically altered cells, e.g., by genetic alteration (e.g., mutation or overexpression) or otherwise (e.g., RNA interference, anti-sense, or antibody binding). In some cases, the activity of a plurality of transcripts or proteins is altered.
  • the similarly altered transcripts or proteins are identified by profiling transcripts or protein abundance in each phenotypically altered cell of the plurality to provide a profile for each phenotypically altered cell; and comparing the profiles to each other.
  • Profiles can be obtained using, for example, a nucleic acid or protein array, SAGE tags, differential display, or subtractive hybridization.
  • the cell is a cancer cell, e.g., a human cancer cell.
  • One or more of the identified transcripts can be a transcript that is absent from the cell in the absence of any artificial transcription factors.
  • a polypeptide encoded by one of the identified transcripts can be used as a target polypeptide for screening to find test compounds that interact with the target.
  • the test compound can be evaluated to determine if they enhance or inhibit activity of the target polypeptide.
  • the test compound is a small molecule having a molecular weight of less than 10, 5, or 2 kDa.
  • a library of nucleic acids that encode chimeric zinc finger proteins can be used.
  • the term “library” refers to a physical collection of similar, but non-identical biomolecules. The collection can be, for example, together in one vessel or physically separated (into groups or individually) in separate vessels or on separate locations on a solid support. Duplicates of individual members of the library may be present in the collection.
  • a library can include at least 10, 10 2 , 10 3 , 10 5 , 10 7 , or 10 9 different members, or fewer than 10 13 , 10 12 , 10 10 , 10 9 , 10 7 , 10 5 , or 10 3 different members.
  • a first exemplary library includes a plurality of nucleic acids, each nucleic acid encoding a polypeptide comprising at least a first, second, and third zinc finger domains.
  • first, second and third denotes three separate domains that can occur in any order in the polypeptide: e.g., each domain can occur N-terminal or C-terminal to either or both of the others.
  • the first zinc finger domain varies among nucleic acids of the plurality.
  • the second zinc finger domain varies among nucleic acids of the plurality. At least 10 different first zinc finger domains are represented in the library.
  • At least 0.5, 1, 2, 5%, 10%, or 25% of the members of the library have one or both of the following properties: (1) each represses transcription of at least one pIG reporter plasmid at least 1.25 fold in vivo; and (2) each binds at least one target site with a dissociation constant of no more than 7, 5, 3, 2, 1, 0.5, or 0.05 nM.
  • the first and second zinc finger domains can be from different naturally-occurring proteins or are positioned in a configuration that differs from their relative positions in a naturally-occurring protein.
  • the first and second zinc finger domains may be adjacent in the polypeptide, but may be separated by one or more intervening zinc finger domains in a naturally occurring protein.
  • a second exemplary library includes a plurality of nucleic acids, each nucleic acid encoding a polypeptide that includes at least first and second zinc finger domains.
  • the first and second zinc finger domains of each polypeptide (1) are identical to zinc finger domains of different naturally occurring proteins (and generally do not occur in the same naturally occurring protein or are positioned in a configuration that differs from their relative positions in a naturally-occurring protein), (2) differ by no more than four, three, two, or one amino acid residues from domains of naturally occurring proteins, or (3) are non-adjacent zinc finger domains from a naturally occurring protein.
  • Identical zinc finger domains refer to zinc finger domains that are identical at each amino acid from the first metal coordinating residue (typically cysteine) to the last metal coordinating residue (typically histidine).
  • the first zinc finger domain varies among nucleic acids of the plurality
  • the second zinc finger domain varies among nucleic acids of the plurality.
  • the naturally occurring protein can be any eukaryotic zinc finger protein: for example, a fungal (e.g., yeast), plant, or animal protein (e.g., a mammalian protein, such as a human or murine protein).
  • Each polypeptide can further include a third, fourth, fifth, and/or sixth zinc finger domain.
  • Each zinc finger domain can be a mammalian, e.g., human, zinc finger domain.
  • a library of nucleic acids encoding zinc finger proteins or a library of such proteins themselves can include members with different regulatory domains.
  • the library can include at least 10% of members with an activation domain, and at least another 10% of members with a repression domain.
  • at least 10% have an activation domain or repression domain; another at least 10% has no regulatory domain.
  • some include an activation domain; others, a repression domain; still others, no regulatory domain at all.
  • Other percentages e.g., at least 20, 25, 30, 40, 50, 60% can also be used.
  • the invention features an artificial polypeptide that includes: the sequence:
  • B is any amino acid, or optionally phenylalanine or tyrosine
  • J is any amino acid, or optionally, a hydrophobic amino acid.
  • QSNR-QSNK-CSNR is also abbreviated as:
  • the polypeptide can, for example, induce neurites when present at an effective concentration in a mouse Neuro2a cell.
  • the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:2 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:2. The substitutions can be conservative substitutions.
  • the isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:2. In one embodiment, the polypeptide can specifically bind to a target DNA site.
  • the polypeptide can compete with the Neuro1p chimeric ZFP (SEQ ID NO:2) for binding to a target DNA site, e.g., a site bound with a K d of less than 10 nM.
  • the polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain.
  • the polypeptide can include one, two, or three or more additional zinc finger domains.
  • nucleic acids encoding the above polypeptides.
  • the isolated polypeptide that includes the amino acid of SEQ ID NO:2 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:1.
  • Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth.
  • the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter.
  • the nucleic acid can be included in a vector or integrated into a chromosome.
  • the invention features host cells (e.g., mammalian host cells) that include a polypeptide described above.
  • the host cell can include a nucleic acid described above and express the nucleic acid.
  • the host cell can be a neuronal cell that extends neurites (e.g., at least in part as a consequence of the polypeptide).
  • the invention features an artificial polypeptide that includes: the sequence:
  • B is any amino acid, or optionally phenylalanine or tyrosine
  • J is any amino acid, or optionally, a hydrophobic amino acid.
  • This array is also abbreviated as: RDKR-QTHR-VSTR-RDKR.
  • Other exemplary artificial polypeptides include:
  • the polypeptide can, for example, induce alkaline phosphatase or other indicators of osteoblasts differentiation, when present at an effective concentration in a C2C12 myoblast cell line cell.
  • the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:4 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:4. The substitutions can be conservative substitutions.
  • the isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:4. In one embodiment, the polypeptide can specifically bind to a target DNA site.
  • the polypeptide can compete with the Osteo1p chimeric ZFP (SEQ ID NO:4) for binding to a target DNA site, e.g., a site bound with a K d of less than 10 nM.
  • the polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain.
  • the polypeptide can include one, two, or three or more additional zinc finger domains.
  • nucleic acids encoding the above polypeptides.
  • the isolated polypeptide that includes the amino acid of SEQ ID NO:4 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:3.
  • Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth.
  • the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter.
  • the nucleic acid can be included in a vector or integrated into a chromosome.
  • the invention features host cells (e.g., mammalian host cells) that include a polypeptide described above.
  • the host cell can include a nucleic acid described above and express the nucleic acid.
  • the host cell can be a stem cell that has the phenotype of an osteoblasts, e.g., at least partially as a consequence of the artificial chimeric polypeptide.
  • the invention features an artificial polypeptide that includes: the sequence:
  • B is any amino acid, or optionally phenylalanine or tyrosine
  • J is any amino acid, or optionally, a hydrophobic amino acid.
  • This array is also abbreviated as: RSHR-RDHT-VSSR.
  • Other exemplary artificial polypeptides include:
  • the polypeptide can, for example, induce insulin gene expression, e.g., when present at an effective concentration in a human 293 cell.
  • the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:6 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:6. The substitutions can be conservative substitutions.
  • the isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:6. In one embodiment, the polypeptide can specifically bind to a target DNA site.
  • the polypeptide can compete with the 08_D04 — 6 chimeric ZFP (SEQ ID NO:6) for binding to a target DNA site, e.g., a site bound with a K d of less than 10 nM.
  • the polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain.
  • the polypeptide can include one, two, or three or more additional zinc finger domains.
  • nucleic acids encoding the above polypeptides.
  • the isolated polypeptide that includes the amino acid of SEQ ID NO:6 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:5.
  • Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth.
  • the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter.
  • the nucleic acid can be included in a vector or integrated into a chromosome.
  • the invention features host cells (e.g., mammalian host cells) that include a polypeptide described above.
  • the host cell can include a nucleic acid described above and express the nucleic acid.
  • the host cell can be a human cell that expresses the insulin gene, e.g., at least partially as a consequence of the artificial chimeric polypeptide.
  • the invention also features a method of producing insulin that includes culturing a cell described herein in vitro, or of producing insulin in a subject by introducing a polypeptide described above or a nucleic acid encoding the polypeptide into a cell of the subject.
  • the invention features an artificial polypeptide that includes: the sequence:
  • B is any amino acid, or optionally phenylalanine or tyrosine
  • J is any amino acid, or optionally, a hydrophobic amino acid.
  • QSNR-DSNR-DSNR is also abbreviated as: QSNR-DSNR-DSNR.
  • Other exemplary artificial polypeptides include:
  • the polypeptide can, for example, increase expression of a heterologous polypeptide, e.g., a reporter polypeptide encoded by a SV40-SEAP reporter construct in a 293 cell when present at an effective concentration.
  • the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:8 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:8. The substitutions can be conservative substitutions.
  • the isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:8. In one embodiment, the polypeptide can specifically bind to a target DNA site.
  • the polypeptide can compete with the P_B08 chimeric ZFP (SEQ ID NO:8) for binding to a target DNA site, e.g., a site bound with a K d of less than 10 nM.
  • the polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain.
  • the polypeptide can include one, two, or three or more additional zinc finger domains.
  • nucleic acids encoding the above polypeptides.
  • the isolated polypeptide that includes the amino acid of SEQ ID NO:8 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:7.
  • Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth.
  • the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter.
  • the nucleic acid can be included in a vector or integrated into a chromosome.
  • the invention features host cells (e.g., mammalian host cells) that include a polypeptide described above.
  • the host cell can include a nucleic acid described above and express the nucleic acid.
  • the host cell can be a mammalian cell that has increased heterologous polypeptide production.
  • the invention features an artificial polypeptide that includes: the sequence:
  • B is any amino acid, or optionally phenylalanine or tyrosine
  • J is any amino acid, or optionally, a hydrophobic amino acid.
  • QSHV-WSNR-WSNR-RDNQ is also abbreviated as:
  • the polypeptide can decrease the cell proliferation when present at an effective concentration in a mammalian cell, e.g., by at least 30, 40, 50, or 60%. Cell proliferation may be assessed by cell number after a predefined incubation time.
  • the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:10 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:10. The substitutions can be conservative substitutions.
  • the isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:10. In one embodiment, the polypeptide can specifically bind to a target DNA site.
  • the polypeptide can compete with the K_D10 chimeric ZFP (SEQ ID NO:10) for binding to a target DNA site, e.g., a site bound with a K d of less than 10 nM.
  • the polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain.
  • the polypeptide can include one, two, or three or more additional zinc finger domains.
  • nucleic acids encoding the above polypeptides.
  • the isolated polypeptide that includes the amino acid of SEQ ID NO:10 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:9.
  • Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth.
  • the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter.
  • the nucleic acid can be included in a vector or integrated into a chromosome.
  • the invention features host cells (e.g., eukaryotic, e.g., mammalian host cells) that include a nucleic acid described herein, e.g., a nucleic acid encoding polypeptide described herein.
  • the host cell can also express the nucleic acid.
  • the host cell can be a mammalian cell that has a reduced proliferation rate relative to an otherwise identical cell that does not include the polypeptide.
  • the invention features an artificial polypeptide that includes: the sequence:
  • B is any amino acid, or optionally phenylalanine or tyrosine
  • J is any amino acid, or optionally, a hydrophobic amino acid.
  • This array is also abbreviated as: DSAR-RDKR-RDER-QTHR.
  • Other exemplary artificial polypeptides include:
  • the polypeptide can increase cell proliferation, e.g., by at least 50, 100, or 120%, in a mammalian cell, e.g., a 293 cell.
  • the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:12 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:12. The substitutions can be conservative substitutions.
  • the isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:12. In one embodiment, the polypeptide can specifically bind to a target DNA site.
  • the polypeptide can compete with the K_F02 chimeric ZFP (SEQ ID NO:12) for binding to a target DNA site, e.g., a site bound with a K d of less than 10 nM.
  • the polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain.
  • the polypeptide can include one, two, or three or more additional zinc finger domains.
  • nucleic acids encoding the above polypeptides.
  • the isolated polypeptide that includes the amino acid of SEQ ID NO:12 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:11.
  • Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth.
  • the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter.
  • the nucleic acid can be included in a vector or integrated into a chromosome.
  • the invention features host cells (e.g., mammalian host cells) that include a nucleic acid described above.
  • the host cell can include a nucleic acid described above and express the nucleic acid.
  • the host cell can be a mammalian cell that has an increased rate of cell proliferation relative to an otherwise identical cell that does not include the polypeptide.
  • the invention features an artificial polypeptide that includes: the sequence:
  • B is any amino acid, or optionally phenylalanine or tyrosine
  • J is any amino acid, or optionally, a hydrophobic amino acid.
  • This array is also abbreviated as: RDHT-QSNV-QTHR-QFNR.
  • Other exemplary polypeptides include:
  • the polypeptide can increase heterologous gene expression, e.g., a gene operable linked to a strong promoter such as a viral promoter, in a mammalian cell.
  • the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:260 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:260. The substitutions can be conservative substitutions.
  • the isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:260. In one embodiment, the polypeptide can specifically bind to a target DNA site.
  • the polypeptide can compete with the K12_A — 11 chimeric ZFP (SEQ ID NO:260) for binding to a target DNA site, e.g., a site bound with a K d of less than 10 nM.
  • the polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain.
  • the polypeptide can include one, two, or three or more additional zinc finger domains. Since the K12_A — 11 chimeric ZFP includes a repression domain it is unlikely to increase heterologous gene expression by directly binding to the promoter of the heterologous gene.
  • nucleic acids encoding the above polypeptides.
  • the isolated polypeptide that includes the amino acid of SEQ ID NO:260 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:259.
  • Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth.
  • the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter.
  • the nucleic acid can be included in a vector or integrated into a chromosome.
  • the invention features host cells (e.g., mammalian host cells) that include a nucleic acid described above.
  • the host cell can include a nucleic acid described above and express the nucleic acid.
  • the invention features an artificial polypeptide that includes: the sequence:
  • B is any amino acid, or optionally phenylalanine or tyrosine
  • J is any amino acid, or optionally, a hydrophobic amino acid.
  • QSHV-QSSR-QTHR is also abbreviated as:
  • the polypeptide can increase heterologous gene expression, e.g., a gene operable linked to a strong promoter such as a viral promoter, in a mammalian cell.
  • the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:262 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:262. The substitutions can be conservative substitutions.
  • the isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:262. In one embodiment, the polypeptide can specifically bind to a target DNA site.
  • the polypeptide can compete with the K44-16-E12 chimeric ZFP (SEQ ID NO:262) for binding to a target DNA site, e.g., a site bound with a K d of less than 10 nM.
  • the polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain.
  • the polypeptide can include one, two, or three or more additional zinc finger domains. Since the K44-16-E12 chimeric ZFP includes a repression domain it is unlikely to increase heterologous gene expression by directly binding to the promoter of the heterologous gene.
  • nucleic acids encoding the above polypeptides.
  • the isolated polypeptide that includes the amino acid of SEQ ID NO:262 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:261.
  • Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth.
  • the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter.
  • the nucleic acid can be included in a vector or integrated into a chromosome.
  • the invention features host cells (e.g., mammalian host cells) that include a nucleic acid described above.
  • the host cell can include a nucleic acid described above and express the nucleic acid.
  • the host cell can be a mammalian cell (e.g., a 293 cell that has the phenotype of increased heterologous protein production (e.g., both a secreted and intracellular reporter protein encoded by genes operably linked to the CMV promoter).
  • the invention features an artificial polypeptide that includes: the sequence:
  • B is any amino acid, or optionally phenylalanine or tyrosine
  • J is any amino acid, or optionally, a hydrophobic amino acid.
  • This array is also abbreviated as: QSNR-QSSR-QTHR-RDKR.
  • Other exemplary artificial polypeptides include:
  • the polypeptide can cause increased SEAP production for an SEAP protein encoded by a heterologous gene encoded operably linked to the CMV promoter.
  • the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:264 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:264. The substitutions can be conservative substitutions.
  • the isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:264. In one embodiment, the polypeptide can specifically bind to a target DNA site.
  • the polypeptide can compete with the K13_B08 chimeric ZFP (SEQ ID NO:264) for binding to a target DNA site, e.g., a site bound with a K d of less than 10 nM.
  • the polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain.
  • the polypeptide can include one, two, or three or more additional zinc finger domains.
  • nucleic acids encoding the above polypeptides.
  • the isolated polypeptide that includes the amino acid of SEQ ID NO:264 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:263.
  • Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth.
  • the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter.
  • the nucleic acid can be included in a vector or integrated into a chromosome.
  • the invention features host cells (e.g., mammalian host cells) that include a nucleic acid described above.
  • the host cell can include a nucleic acid described above and express the nucleic acid.
  • the invention features an artificial polypeptide that includes: the sequence:
  • B is any amino acid, or optionally phenylalanine or tyrosine
  • J is any amino acid, or optionally, a hydrophobic amino acid.
  • This array is also abbreviated as: RDHT-RSHR-QSHR.
  • Other exemplary artificial polypeptides include:
  • the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:18 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:18.
  • the substitutions can be conservative substitutions.
  • the isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:18.
  • the polypeptide can specifically bind to a target DNA site.
  • the polypeptide can compete with the F104_p65 chimeric ZFP (SEQ ID NO:18) for binding to a target DNA site, e.g., a site bound with a K d of less than 10 nM.
  • the polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain.
  • the polypeptide can include one, two, or three or more additional zinc finger domains.
  • nucleic acids encoding the above polypeptides.
  • the isolated polypeptide that includes the amino acid of SEQ ID NO:18 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:17.
  • Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth.
  • the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter.
  • the nucleic acid can be included in a vector or integrated into a chromosome.
  • the invention features host cells (e.g., mammalian host cells) that include a nucleic acid described above.
  • the host cell can include a nucleic acid described above and express the nucleic acid.
  • the invention features an artificial polypeptide that includes: the sequence:
  • B is any amino acid, or optionally phenylalanine or tyrosine
  • J is any amino acid, or optionally, a hydrophobic amino acid.
  • This array is also abbreviated as: QSHT-RSHR-RDHT.
  • Other exemplary polypeptides include:
  • the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:20 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:20.
  • the substitutions can be conservative substitutions.
  • the isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:20.
  • the polypeptide can specifically bind to a target DNA site.
  • the polypeptide can compete with the F121_p65 chimeric ZFP (SEQ ID NO:20) for binding to a target DNA site, e.g., a site bound with a K d of less than 10 nM.
  • the polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain.
  • the polypeptide can include one, two, or three or more additional zinc finger domains.
  • the polypeptide can be used, for example, to regulate expression of insulin-like growth factor 2.
  • nucleic acids encoding the above polypeptides.
  • the isolated polypeptide that includes the amino acid of SEQ ID NO:20 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:19.
  • Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth.
  • the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter.
  • the nucleic acid can be included in a vector or integrated into a chromosome.
  • the invention features host cells (e.g., mammalian host cells) that include a nucleic acid described above.
  • the host cell can include a nucleic acid described above and express the nucleic acid.
  • the invention features an artificial polypeptide that includes: the sequence:
  • B is any amino acid, or optionally phenylalanine or tyrosine
  • J is any amino acid, or optionally, a hydrophobic amino acid.
  • the isolated polypeptide can include an amino acid sequence that is identical to the zinc finger array within SEQ ID NO:14 or 16 or that differs by no more than 8, 6, 4, 3, or 2 substitutions within the zinc finger domains present in SEQ ID NO:20.
  • the substitutions can be conservative substitutions.
  • the isolated polypeptide can have a sequence that is at least 80, 85, 90, 95, or 97% identical to the zinc finger array within SEQ ID NO:14 or 16.
  • the polypeptide can specifically bind to a target DNA site.
  • the polypeptide can compete with the F121_p65 chimeric ZFP (SEQ ID NO:14 or 16) for binding to a target DNA site, e.g., a site bound with a K d of less than 10 nM.
  • the polypeptide can further include a transcriptional regulatory domain, e.g., an activation or repression domain.
  • the polypeptide can include one, two, or three or more additional zinc finger domains.
  • the polypeptide can be used, for example, to regulate protein production.
  • nucleic acids encoding the above polypeptides.
  • the isolated polypeptide that includes the amino acid of SEQ ID NO:14 or 16 can be encoded by a nucleic acid sequence that includes the sequence of SEQ ID NO:13 or 15.
  • Featured nucleic acids can include operably linked regulatory sequences, e.g., a promoter sequence, an enhancer sequence, an insulator sequence, untranslated regulatory regions, a polyA addition site, and so forth.
  • the coding nucleic acid is operably linked to a conditional promoter such as an inducible promoter or a cell-type specific promoter.
  • the nucleic acid can be included in a vector or integrated into a chromosome.
  • the invention features host cells (e.g., mammalian host cells) that include a nucleic acid described above.
  • the host cell can include a nucleic acid described above and express the nucleic acid.
  • the “dissociation constant” refers to the equilibrium dissociation constant of a polypeptide for binding to a 28-basepair double-stranded DNA that includes one 9-basepair target site.
  • the dissociation constant is determined by gel shift analysis using a purified protein that is bound in 20 mM Tris pH 7.7, 120 mM NaCl, 5 mM MgCl 2 , 20 ⁇ M ZnSO 4 , 10% glycerol, 0.1% Nonidet P-40, 5 mM DTT, and 0.10 mg/mL BSA (bovine serum albumin) at room temperature. Additional details are provided in Example 10 and Rebar and Pabo (1994) Science 263:671-673.
  • the term “screen” refers to a process for evaluating members of a library to find one or more particular members that have a given property.
  • each member of the library is evaluated. For example, each cell is evaluated to determine if it is extending neurites.
  • a selection each member is not directly evaluated. Rather the evaluation is made by subjecting the members of the library to conditions in which only members having a particular property are retained. Selections may be mediated by survival (e.g., drug resistance) or binding to a surface (e.g., adhesion to a substrate). Such selective processes are encompassed by the term “screening.”
  • base contacting positions refers to the four amino acid positions of zinc finger domains that structurally correspond to the positions of amino acids arginine 73, aspartic acid 75, glutamic acid 76, and arginine 79 of ZIF268.
  • the query sequence is aligned to the zinc finger domain of interest such that the cysteine and histidine residues of the query sequence are aligned with those of finger 3 of Zif268.
  • the ClustalW WWW Service at the European Bioinformatics Institute provides one convenient method of aligning sequences.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; a group of amino acids having acidic side chains is aspartic acid and glutamic acid; and a group of amino acids having sulfur-containing side chains is cysteine and methionine.
  • amino acids within the same group may be interchangeable.
  • Some additional conservative amino acids substitution groups are: valine-leucine-isoleucine; phenylalanine-tyrosine; lysine-arginine; alanine-valine; aspartic acid-glutamic acid; and asparagine-glutamine.
  • heterologous polypeptide refers either to a polypeptide with a non-naturally occurring sequence (e.g., a hybrid polypeptide) or a polypeptide with a sequence identical to a naturally occurring polypeptide but present in a milieu in which it does not naturally occur.
  • hybrid and chimera refer to a non-naturally occurring polypeptide that comprises amino acid sequences derived from either (i) at least two different naturally occurring sequences, or non-contiguous regions of the same naturally occurring sequence, wherein the non-contiguous regions are made contiguous in the hybrid; (ii) at least one artificial sequence (i.e., a sequence that does not occur naturally) and at least one naturally occurring sequence; or (iii) at least two artificial sequences (same or different). Examples of artificial sequences include mutants of a naturally occurring sequence and de novo designed sequences. An “artificial sequence” is not present among naturally occurring sequences.
  • the invention also refers to a sequence with the same elements, but which is not presenting each of the following organisms whose genomes are sequenced: Homo sapiens, Mus musculus, Arabidopsis thaliana, Drosophila melanogaster, Escherichia coli, Saccharomyces cerevisiae, and Oryza sativa.
  • a molecule with such a sequence can be expressed as a heterologous molecule in a cell of one of the afore-mentioned organisms.
  • the invention also includes sequences (not necessarily termed “artificial”) which are made by a method described herein, e.g., a method of joining nucleic acid sequences encoding different zinc finger domains or a method of phenotypic screening.
  • sequences not necessarily termed “artificial” which are made by a method described herein, e.g., a method of joining nucleic acid sequences encoding different zinc finger domains or a method of phenotypic screening.
  • the invention also features a cell that includes such a sequence.
  • hybridizes under stringent conditions refers to conditions for hybridization in 6 ⁇ sodium chloride/sodium citrate (SSC) at 45° C., followed by two washes in 0.2 ⁇ SSC, 0.1% SDS at 65° C.
  • SSC sodium chloride/sodium citrate
  • binding preference refers to the discriminative property of a polypeptide for selecting one nucleic acid binding site relative to another. For example, when the polypeptide is limiting in quantity relative to two different nucleic acid binding sites, a greater amount of the polypeptide will bind the preferred site relative to the other site in an in vivo or in vitro assay described herein.
  • a “reference cell” refers to any standard cell.
  • the reference cell is a parental cell for a cell that expresses a zinc finger protein, e.g., a cell that is substantially identical to the zinc finger protein expressing cell, but which does not produce the zinc finger protein.
  • a “transformed” or “transfected” cell refers to a cell that includes a heterologous nucleic acid. The cell can be made by introducing (e.g., transforming, transfecting, or infecting, e.g., using a viral particle) a nucleic acid into the cell or the cell can be a progeny or derivative of a cell thus made.
  • chimeric transcription factors relate to the identification and use of new and useful chimeric proteins, e.g., chimeric transcription factors.
  • Some embodiments may include one or more of the following advantages: i) Endogenous genes can be either up- or down-regulated. Any given artificial chimeric transcription factor can be converted to transcriptional activator by fusing to appropriate transcriptional activation domains or to transcriptional repressor by fusing to repression domains.
  • chimeric transcription factors can be potent repressors, e.g., when they bind to sites near the TATA box and the initiator element.
  • Gene expression can be finely regulated. Depending on the DNA-binding affinity, chimeric transcription factors can cause a range of effects, e.g., moderate to strong activation and repression. This may lead to diverse phenotypes that are not necessarily obtained by completely inactivation or high level over-expressed of a particular target gene. For example, in some cases, broad specificity may be an advantage where the coordinated action of several genes is required to drive a desired phenotype. In other cases, narrow specificity is desired.
  • One method for flexibly controlling specificity is by adding or removing zinc finger domains.
  • 3-finger ZFPs theoretically regulate more genes than 6-finger ZFPs.
  • ZFP library approach is universally applicable. Since transcriptional regulatory mechanisms are highly conserved and because all known organism use DNA and transcription for life, chimeric proteins that bind to DNA can be used to regulate any desired cell. Further, as described herein, many methods do not require a priori information (e.g., genome sequence) of the cell in order to identify useful chimeric proteins.
  • Artificial chimeric proteins can be used as a tool to dissect pathways within a cell. For example, target genes responsible for the phenotypic changes in selected clones can be identified, e.g., as described herein.
  • a ZFP-TF may mimic the function of a master regulatory protein, such as a master regulatory transcription factor.
  • the ZFP-TF may bind to the same site as the master regulatory, or to an overlapping site.
  • the level of gene expression change thus the extent of the phenotype generated by ZFP-TF, can be precisely controlled by altering the expression level of ZFP-TF in cells.
  • FIG. 1 is a diagram of a region of the yeast plasmid pYTC-Lib.
  • FIG. 2 is a schematic of an exemplary target-driven approach.
  • FIG. 3A is a schematic of a method for preparing an exemplary zinc finger protein library.
  • FIG. 3B is a schematic of an exemplary phenotype-driven approach.
  • FIG. 4 depicts an expression profile of transiently transfected ZFPs.
  • a cell expressing p65 activation domain alone is compared to a control.
  • a cell expressing the zinc finger protein F121_p65 is compared to a control.
  • Each dot displaced from the diagonal represents a gene whose expression is substantially altered.
  • FIG. 5 depicts expression profiles of stably transfected ZFPs.
  • FIG. 6A depicts the effects of DNA-binding domain mutants and effector domain alterations to the K5 ZFP on drug resistance.
  • FIG. 6B depicts the effects of K5 and YLL053C overexpression.
  • FIG. 7 depicts one method of constructing a diverse three finger library.
  • FIG. 8 is a photograph of control and ZFP-expressing cells in which neurite formation is induced.
  • FIG. 8A pcDNA3(empty vector)-transfected Neuro2A cells without retinoic acid (RA) treatment.
  • FIG. 8B Neuro1-p65 (also called 08_D01-65) expression without RA treatment.
  • FIG. 8C pcDNA3(empty vector)-transfected Neuro2A cells with 10 ⁇ M RA.
  • FIG. 8D Neuro1-p65 expression with 10 ⁇ M RA.
  • FIG. 9 depicts comparative microarray data for cells in which the insulin gene is over-expressed.
  • the chimeric ZFP is 08_D04-p65. The sequence of which is listed in FIG. 16.
  • FIG. 10A is a truncated table listing ZFP DNA binding specificities.
  • FIG. 10B is a schematic illustrating the correspondence between zinc finger domains and the recognition site sequence.
  • FIG. 11 is a flowchart for a method of identifying targets of artificial ZFPs.
  • FIG. 12 is a schematic of a method of identifying ZFPs that induce differentiation.
  • FIG. 13 depicts comparative microarray data at various time points for cells in which the F104-p65 ZFP is present.
  • FIG. 14 lists the coding nucleic acid (SEQ ID NO:1) and amino acid (SEQ ID NO:2) sequences for Neuro1-p65, a protein that can induce neurites.
  • Neuro1-p65 includes the zinc finger domains QSNR1-QSNK-CSNR1 and the p65 activation domain.
  • Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in QSNR1-QSNK-CSNR1-p65 may also induce neurites.
  • FIG. 15 lists the coding nucleic acid (SEQ ID NO:3) and amino acid (SEQ ID NO:4) sequences for Osteo1-p65, a protein that can induce osteocytes.
  • Osteo1-p65 includes the zinc finger domains RDKR-QTHR1-VSTR-RDKR and the p65 activation domain.
  • Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in RDKR-QTHR1-VSTR-RDKR-p65 may also induce osteocytes.
  • FIG. 16 lists the coding nucleic acid (SEQ ID NO:5) and amino acid (SEQ ID NO:6) sequences for 08_D04_p65, a protein that can enhance insulin production (See FIG. 9).
  • 08 —D 04_p65 includes the zinc finger domains RSHR-RDHT-VSSR and the p65 activation domain.
  • Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in RSHR-RDHT-VSSR may also enhance insulin production.
  • FIG. 17 lists the coding nucleic acid (SEQ ID NO:7) and amino acid (SEQ ID NO:8) sequences for P_B08, a protein that can enhances SV40-SEAP.
  • P_B08 includes the zinc finger domains QSNR1-DSNR-DSNR and the p65 activation domain.
  • Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in QSNR1-DSNR-DSNR may also enhances SV40-SEAP.
  • FIG. 18 lists the coding nucleic acid (SEQ ID NO:9) and amino acid (SEQ ID NO:10) sequences for K_D10, a protein that can decrease cell proliferation.
  • K_D10 includes the zinc finger domains QSHV-WSNR-WSNR-RDNQ and the kid repression domain.
  • Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in QSHV-WSNR-WSNR-RDNQ may also decrease cell proliferation.
  • FIG. 19 lists the coding nucleic acid (SEQ ID NO:11) and amino acid (SEQ ID NO:12) sequences for K_F02, a protein that can enhance cell proliferation.
  • K_F02 includes the zinc finger domains DSAR2-RDKR-RDER1-QHR1 and the kid repression domain.
  • Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in DSAR2-RDKR-RDER1-QHR1-kid may also enhance cell proliferation.
  • FIG. 20 lists the coding nucleic acid (SEQ ID NO:13) and amino acid (SEQ ID NO:14) sequences for K44 — 11_D01, a protein that can enhance protein expression.
  • K44 — 11D01 includes the zinc finger domains QSHV-QSNI-QTHR1-CSNR1 and the kid repression domain.
  • Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in QSHV-QSNI-QTHR1-CSNR1 may also enhance protein expression.
  • FIG. 21 lists the coding nucleic acid (SEQ ID NO:15) and amino acid (SEQ ID NO:16) sequences for K44-11-G12, a protein that can enhance protein expression.
  • K44-11-G12 includes the zinc finger domains QSHV-VSTR-RDNQ-QTHR1 and the kid repression domain.
  • Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in QSHV-VSTR-RDNQ-QTHR1 may also enhance protein expression.
  • FIG. 22 lists the coding nucleic acid (SEQ ID NO:17) and amino acid (SEQ ID NO:18) sequences for F104_p65.
  • F104_p65 includes the zinc finger domains RDHT-RSHR-QSHR2-p65.
  • FIG. 23 lists the coding nucleic acid (SEQ ID NO:19) and amino acid (SEQ ID NO:20) sequences for F121_p65.
  • F121_p65 includes the zinc finger domains QSHT-RSHR-RDHT-p65.
  • Other artificial proteins with at least two or three consecutive zinc finger domains that have the same pattern of DNA contacting residues as domains in QSHT-RSHR-RDHT may also increase transcription of insulin-like growth factor 2
  • FIG. 24 lists the coding nucleic acid (SEQ ID NO:259) and amino acid (SEQ ID NO:260) sequences for K12_A11, which includes the zinc finger domains RDHT-QSNV2-QTHR1-QFNR-kid.
  • FIG. 25 lists the coding nucleic acid (SEQ ID NO:261) and amino acid (SEQ ID NO:262) sequences for K44-16-E12, which includes the zinc finger domains QSHV-QSSR1-QTHR1-kid.
  • FIG. 26 lists the coding nucleic acid (SEQ ID NO:263) and amino acid (SEQ ID NO:264) sequences for K13_B08, which includes the zinc finger domains QSNR1-QSSR1-QTHR1-RDKR-KID.
  • DNA sequences in the FIGS. 14 to 26 can include a sequence encoding a HA tag (gray underlined), and a sequence encoding a nuclear localization signal (boxed).
  • the initiation and stop codons are underlined.
  • the shaded letters indicate sequences encoding a regulatory domain, e.g., a p65 or kid domain.
  • the small letters indicated the linker sequences.
  • italicized and shaded letters indicate zinc finger domains and shaded letters, not italicized indicate a regulatory domain, e.g., a Kid or p65 domain.
  • DNA sequences in the FIGS. 14 to 24 include a sequence encoding a HA tag (gray underlined), and a sequence encoding a nuclear localization signal (boxed). The initiation and stop codons are underlined. The shaded letters indicate sequences encoding a regulatory domain, e.g., a p65 or kid domain. The small letters indicated the linker sequences. With respect to the amino acid sequences, italicized and shaded letters indicate zinc finger domains and shaded letters, not italicized indicate a regulatory domain, e.g., a Kid or p65 domain.
  • a library of nucleic acids that encode different artificial, chimeric proteins is screened to identify a chimeric protein that alters a phenotypic trait of a cell or organism.
  • the chimeric protein can be identified without a priori knowledge of a particular target gene or pathway.
  • each nucleic acid of the library encodes an artificial polypeptide that includes a plurality of zinc finger domains.
  • a nucleic acid library that encodes different chimeric proteins can be prepared, for example, as described in the sections below. Members of the library are introduced into cells in culture or cells in an organism. After an interval to allow for expression of the encoded polypeptides, cells or organisms which have an altered phenotypic trait are identified. The library nucleic acid in at least one of these phenotypically altered cells is recovered, thus identifying an artificial chimeric polypeptide that produces the phenotypic effect.
  • the nucleic acid library is constructed so that it includes nucleic acids that each encode and can express an artificial protein that is a chimera of one or more structural domains.
  • the structural domains are nucleic acid binding domains that vary in specificity such that the library encodes a population of proteins with different binding specificities.
  • nucleic acid binding domains are known to bind nucleic acids with high affinity and high specificity.
  • nucleic acid binding domains include:
  • Zinc fingers are small polypeptide domains of approximately 30 amino acid residues in which there are four amino acids, either cysteine or histidine, appropriately spaced such that they can coordinate a zinc ion (For reviews, see, e.g., Klug and Rhodes, (1987) Trends Biochem. Sci. 12:464-469(1987); Evans and Hollenberg, (1988) Cell 52:1-3; Payre and Vincent, (1988) FEBS Lett. 234:245-250; Miller et al., (1985) EMBO J. 4:1609-1614; Berg, (1988) Proc. Natl. Acad. Sci. U.S.A. 85:99-102; Rosenfeld and Margalit, (1993) J.
  • zinc finger domains can be categorized according to the identity of the residues that coordinate the zinc ion, e.g., as the Cys 2 -His 2 class, the Cys 2 -Cys 2 class, the Cys 2 -CysHis class, and so forth.
  • the zinc coordinating residues of Cys 2 -His 2 zinc fingers are typically spaced as follows: X a -X-C-X 2-5 -C-X 3 -X a -X 5 - ⁇ -X 2 -H-X 3-5 -H (SEQ ID NO:72), where ⁇ (psi) is a hydrophobic residue (Wolfe et al., (1999) Annu. Rev. Biophys. Biomol. Struct. 3:183-212), wherein “X” represents any amino acid, wherein X a is phenylalanine or tyrosine, the subscript indicates the number of amino acids, and a subscript with two hyphenated numbers indicates a typical range of intervening amino acids.
  • the intervening amino acids fold to form an anti-parallel ⁇ -sheet that packs against an ⁇ -helix, although the anti-parallel ⁇ -sheets can be short, non-ideal, or non-existent.
  • the fold positions the zinc-coordinating side chains so they are in a tetrahedral conformation appropriate for coordinating the zinc ion.
  • the base contacting residues are at the N-terminus of the finger and in the preceding loop region.
  • X a is typically phenylalanine or tyrosine
  • X b is typically a hydrophobic residue.
  • the DNA contacting residues are Cys (C), Ser (S), Asn (N), and Arg (R).
  • CSNR As used herein, such abbreviation refers to a class of sequences which include a domain corresponding to the motif.
  • the class also includes a species whose sequence includes a particular polypeptide sequence, typically a sequence listed in Table 1 or 2 which corresponds to the motif. Where two sequences have the same motif, a number may be used to indicate the sequence.
  • a zinc finger protein typically consists of a tandem array of three or more zinc finger domains.
  • zinc finger domains whose motifs are listed consecutively are not interspersed with other folded domains, but may include a linker, e.g, a flexible linker described herein between domains.
  • the zinc finger domain (or “ZFD”) is one of the most common eukaryotic DNA-binding motifs, found in species from yeast to higher plants and to humans. By one estimate, there are at least several thousand zinc finger domains in the human genome alone, possibly at least 4,500. Zinc finger domains can be isolated from zinc finger proteins.
  • Non-limiting examples of zinc finger proteins include CF2-II, Kruppel, WT1, basonuclin, BCL-6/LAZ-3, erythroid Kruppel-like transcription factor, Sp1, Sp2, Sp3, Sp4, transcriptional repressor YY1, EGR1/Krox24, EGR2/Krox20, EGR3/Pilot, EGR4/AT133, Evi-1, GLI1, GLI2, GLI3, HIV-EP1/ZNF40, HIV-EP2, KR1, ZfX, ZfY, and ZNF7.
  • zinc finger domains bind to ligands other than DNA, e.g., RNA or protein.
  • a chimera of zinc finger domains or of a zinc finger domain and another type of domain can be used to recognize a variety of target compounds, not just DNA.
  • WO 01/60970 U.S. Serial No. 60/374,355, filed Apr. 22, 2002, and U.S. Ser. No. 10/223,765, filed Aug. 19, 2002, describe exemplary zinc finger domains which can be used to construct an artificial zinc finger protein. See also the Table 1, below.
  • Homeodomains are eukaryotic domains that consist of a N-terminal arm that contacts the DNA minor groove, followed by three ⁇ -helices that contact the major groove (for a review, see, e.g., Laughon, (1991) Biochemistry 30:11357-67). The third ⁇ -helix is positioned in the major groove and contains critical DNA-contacting side chains.
  • Homeodomains have a characteristic highly-conserved motif present at the turn leading into the third ⁇ -helix. The motif includes an invariant tryptophan that packs into the hydrophobic core of the domain.
  • This motif is represented in the Prosite database as PDOC00027 ([L/I/V/M/F/Y/G]-[A/S/L/V/R]-X(2)-[L/I/V/M/S/T/A/C/N]-X-[L/I/V/M]-X(4)-[L/I/V]-[R/K/N/Q/E/S/T/A/I/Y]-[L/I/V/F/S/T/N/K/H]-W-[F/Y/V/C]-X-[N/D/Q/T/A/H]-X(5)-[R/K/N/A/I/M/W].
  • Homeodomains are commonly found in transcription factors that determine cell identity and provide positional information during organismal development. Such classical homeodomains can be found in the genome in clusters such that the order of the homeodomains in the cluster approximately corresponds to their expression pattern along a body axis. Homeodomains can be identified by alignment with a homeodomain, e.g., Hox-1, or by alignment with a homeodomain profile or a homeodomain hidden Markov Model (HMM; see below), e.g., PF00046 of the Pfam database or “HOX” of the SMART database, or by the Prosite motif PDOC00027 as mentioned above.
  • HMM homeodomain hidden Markov Model
  • Helix-turn-helix proteins This DNA binding motif is common among many prokaryotic transcription factors. There are many subfamilies, e.g., the LacI family, the AraC family, to name but a few. The two helices in the name refer to a first ⁇ -helix that packs against and positions a second ⁇ -helix in the major groove of DNA.
  • HMM e.g., HTH_ARAC, HTH_ARSR, HTH_ASNC, HTH_CRP, HTH_DEOR, HTH_DTXR, HTH_GNTR, HTH_ICLR, HTH_LACI, HTH_LUXR, HTH_MARR, HTH_MERR, and HTH_XRE profiles available in the SMART database.
  • HMM e.g., HTH_ARAC, HTH_ARSR, HTH_ASNC, HTH_CRP, HTH_DEOR, HTH_DTXR, HTH_GNTR, HTH_ICLR, HTH_LACI, HTH_LUXR, HTH_MARR, HTH_MERR, and HTH_XRE profiles available in the SMART database.
  • nucleic acids encoding identified domains are used to construct the nucleic acid library. Further, nucleic acid encoding these domains can also be varied (e.g., mutated) to provide additional domains that are encoded by the library.
  • the amino acid sequence of a known structural domain can be compared to a database of known sequences, e.g., an annotated database of protein or nucleic acid sequences.
  • databases of uncharacterized sequences e.g., unannotated genomic, EST or full-length cDNA sequence; of characterized sequences, e.g., SwissProt or PDB; and of domains, e.g., Pfam, ProDom (Corpet et al. (2000) Nucleic Acids Res. 28:267-269), and SMART (Simple Modular Architecture Research Tool, Letunic et al.
  • Nucleic Acids Res 30, 242-244 can provide a source of structural domain sequences.
  • Nucleic acid sequence databases can be translated in all six reading frames for the purpose of comparison to a query amino acid sequence.
  • Nucleic acid sequences that are flagged as encoding candidate nucleic acid binding domains can be amplified from an appropriate nucleic acid source, e.g., genomic DNA or cellular RNA. Such nucleic acid sequences can be cloned into an expression vector.
  • the procedures for computer-based domain identification can be interfaced with an oligonucleotide synthesizer and robotic systems to produce nucleic acids encoding the domains in a high-throughput platform.
  • Cloned nucleic acids encoding the candidate domains can also be stored in a host expression vector and shuttled easily into an expression vector, e.g., into a translational fusion vector with other domains (of a similar or different type), either by restriction enzyme mediated subcloning or by site-specific, recombinase mediated subcloning (see U.S. Pat. No. 5,888,732).
  • the high-throughput platform can be used to generate multiple microtitre plates containing nucleic acids encoding different candidate chimeras.
  • Domains similar to a query domain can be identified from a public database, e.g., using the XBLAST programs (version 2.0) of Altschul et al., (1990) J. Mol. Biol. 215:403-10.
  • Gaps can be introduced into the query or searched sequence as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. Default parameters for XBLAST and Gapped BLAST programs are available at National Center for Biotechnology Information (NCBI), National Institutes of Health, Bethesda Md.
  • NCBI National Center for Biotechnology Information
  • the Prosite profiles PS00028 and PS50157 can be used to identify zinc finger domains. In a SWISSPROT release of 80,000 protein sequences, these profiles detected 3189 and 2316 zinc finger domains, respectively. Profiles can be constructed from a multiple sequence alignment of related proteins by a variety of different techniques. Gribskov and co-workers (Gribskov et al., (1990) Meth. Enzymol. 183:146-159) utilized a symbol comparison table to convert a multiple sequence alignment supplied with residue frequency distributions into weights for each position. See, for example, the PROSITE database and the work of Luethy et al., (1994) Protein Sci. 3:139-1465.
  • HMM's representing a DNA binding domain of interest
  • a database can be searched, e.g., using the default parameters, with the HMM in order to find additional domains (see, e.g., Bateman et al. (2002) Nucleic Acids Research 30:276-280).
  • the user can optimize the parameters.
  • a threshold score can be selected to filter the database of sequences such that sequences that score above the threshold are displayed as candidate domains.
  • Acids Res 28:231) provides a catalog of zinc finger domains (ZnF_C2H2; ZnF_C2C2; ZnF_C2HC; ZnF_C3H1; ZnF_C4; ZnF_CHCC; ZnF_GATA; and ZNF_NFX) identified by profiling with the hidden Markov models of the HMMer2 search program (Durbin et al., (1998) Biological sequence analysis: probabilistic models of proteins and nucleic acids. Cambridge University Press).
  • Hybridization-based Methods A collection of nucleic acids encoding various forms of a structural domain can be analyzed to profile sequences encoding conserved amino- and carboxy-terminal boundary sequences. Degenerate oligonucleotides can be designed to hybridize to sequences encoding such conserved boundary sequences. Moreover, the efficacy of such degenerate oligonucleotides can be estimated by comparing their composition to the frequency of possible annealing sites in known genomic sequences. If desired, multiple rounds of design can be used to optimize the degenerate oligonucleotides.
  • Nucleic acids that are used to assemble the library can be obtained by a variety of methods. Some component nucleic acids of the library can encode naturally occurring domains. In addition, some component nucleic acids are variants that are obtained by mutation or other randomization methods. The component nucleic acids, typically encoding just a single domain, can be joined to each other to produce nucleic acids encoding a fusion of the different domains.
  • a library of domains can be constructed by isolation of nucleic acid sequences encoding domains from genomic DNA or cDNA of eukaryotic organisms such as humans. Multiple methods are available for doing this. For example, a computer search of available amino acid sequences can be used to identify the domains, as described above.
  • a nucleic acid encoding each domain can be isolated and inserted into a vector appropriate for the expression in cells, e.g., a vector containing a promoter, an activation domain, and a selectable marker.
  • degenerate oligonucleotides that hybridize to a conserved motif are used to amplify, e.g., by PCR, a large number of related domains containing the motif.
  • Kruppel-like Cys 2 His 2 zinc fingers can be amplified by the method of Agata et al., (1998) Gene 213:55-64. This method also maintains the naturally occurring zinc finger domain linker peptide sequences, e.g., sequences with the pattern: Thr-Gly-(Glu/Gln)-(Lys/Arg)-Pro-(Tyr/Phe) (SEQ ID NO:74).
  • screening a collection limited to domains of interest unlike screening a library of unselected genomic or cDNA sequences, significantly decreases library complexity and reduces the likelihood of missing a desirable sequence due to the inherent difficulty of completely screening large libraries.
  • the human genome contains numerous zinc finger domains, many of which are uncharacterized and unidentified. It is estimated that there are thousands of genes encoding proteins with zinc finger domains (Pellegrino and Berg, (1991) Proc. Natl. Acad. Sci. USA 88:671-675). These human zinc finger domains represent an extensive collection of diverse domains from which novel DNA-binding proteins can be constructed. Many exemplary human zinc finger domains are described in WO 01/60970, U.S. Serial No. 60/374,355, filed Apr. 22, 2002, and U.S. Ser. No. 10/223,765, filed Aug. 19, 2002. See also Table 1 below.
  • a nucleic acid library can include nucleic acids encoding proteins that include naturally occurring zinc finger domains, artificial mutants of such domains, and combinations thereof.
  • the library includes nucleic acids encoding at least one structural domain that is an artificial variant of a naturally-occurring sequence.
  • such variant domains are assembled from a degenerate patterned library.
  • positions in close proximity to the nucleic acid binding interface or adjacent to a position so located can be targeted for mutagenesis.
  • a mutated test zinc finger domain for example, can be constrained at any mutated position to a subset of possible amino acids by using a patterned degenerate library. Degenerate codon sets can be used to encode the profile at each position.
  • codon sets are available that encode only hydrophobic residues, aliphatic residues, or hydrophilic residues.
  • the library can be selected for full-length clones that encode folded polypeptides. Cho et al. ((2000) J. Mol. Biol. 297(2):309-19) provides a method for producing such degenerate libraries using degenerate oligonucleotides, and also provides a method of selecting library nucleic acids that encode full-length polypeptides. Such nucleic acids can be easily inserted into an expression plasmid, e.g., using convenient restriction enzyme cleavage sites.
  • Selection of the appropriate codons and the relative proportions of each nucleotide at a given position can be determined by simple examination of a table representing the genetic code, or by computational algorithms. For example, Cho et al., supra, describe a computer program that accepts a desired profile of protein sequence and outputs a preferred oligonucleotide design that encodes the sequence.
  • a chimeric protein can include one or more of the zinc finger domains that have at least 17, 18, 19, 20, or 21 amino acids that are identical to a zinc finger domain sequence in Table 1.
  • the DNA contacting residues can be identical.
  • a library of nucleic acids encoding diverse chimeric zinc finger proteins can be formed by serial ligation, e.g., as described in Example 1.
  • the library can be constructed such that each nucleic acid encodes a protein that has at least three, four, or five zinc finger domains.
  • each zinc finger coding segment can be designed to randomly encode any one of a set of zinc finger domains.
  • the set of zinc finger domains can be selected to represent domains with a range of specificities, e.g., covering 30, 40, 50 or more of the 64 possible 3-basepair subsites.
  • the set can include at least about 12, 15, 20, 25, 30, 40 or 50 different zinc finger domains. Some or all of these domains can be domains isolated from naturally occurring proteins.
  • One exemplary library includes nucleic acids that encode a chimeric zinc finger protein having three fingers and 30 possible domains at each finger position. In its fully represented form, this library includes 27,000 sequences (i.e., the result of 30 3 ).
  • the library can be constructed by serial ligation in which a nucleic acid from a pool of nucleic acids encoding all 30 possible domains is added at each step.
  • the library can be stored as a random collection.
  • individual members can be isolated, stored at an addressable location (e.g., arrayed), and sequenced. After high throughput sequencing of 40 to 50 thousand constructed library members, missing chimeric combinations can be individually assembled in order to obtain complete coverage.
  • arrayed e.g., in microtitre plates
  • each individual member can be recovered later for further analysis, e.g., for a phenotypic screen. For example, equal amounts of each arrayed member can be pooled and then transformed into a cell. Cells with a desired phenotype are selected and characterized.
  • each member is individually transformed into a cell, and the cell is characterized, e.g., using a nucleic acid microarray to determine if the transcription of endogenous genes is altered (see “Profiling Regulatory Properties of a Chimeric Zinc Finger Protein,” below).
  • Library nucleic acids can be introduced into cells by a variety of methods.
  • the library is stored as a random pool including multiple replicates of each library nucleic acid. An aliquot of the pool is transformed into cells.
  • individual library members are stored separately (e.g., in separate wells of a microtitre plate or at separate addresses of an array) and are individually introduced into cells.
  • the library members are stored in pools that have a reduced complexity relative to the library as a whole.
  • each pool can include 10 3 different library members from a library of 10 5 or 10 6 different members.
  • the pool is deconvolved to identify the individual library member that mediates the phenotypic effect. This approach is useful when recovery of the altered cell is difficult, e.g., in a screen for chimeric proteins that cause apoptosis.
  • Library nucleic acids can be introduced into cells by a variety of methods. Exemplary methods include electroporation (see, e.g., U.S. Pat. No. 5,384,253); microprojectile bombardment techniques (see, e.g., U.S. Pat. Nos.
  • liposome-mediated transfection e.g., using LIPOFECTAMINETM (Invitrogen) or SUPERFECTTM (QIAGEN GmbH); see, e.g., Nicolau et al., Methods Enzymol., 149:157-176, 1987.
  • calcium phosphate or DEAE-Dextran mediated transformation see, e.g., Rippe et al., (1990) Mol.
  • transformation encompasses any method that introduces an exogenous nucleic acid into a cell.
  • viral particle it is also possible to use a viral particle to deliver a library nucleic acid into a cell in vitro or in vivo.
  • viral packaging is used to deliver the library nucleic acids to cells within an organism.
  • the library nucleic acids are introduced into cells in vitro, after which the cells are transferred into an organism.
  • the library nucleic acids are expressed so that the chimeric proteins encoded by the library are produced by the cells.
  • Constant regions of the library nucleic acid can provide necessary regulatory and supporting sequences to enable expression.
  • sequences can include transcriptional promoters, transcription terminators, splice site donors and acceptors, untranslated regulatory regions (such as polyA addition sites), bacterial origins of replication, markers for indicating the presence of the library nucleic acid or for selection of the library nucleic acid.
  • a screen the cells or organisms are evaluated to identify ones that have an altered phenotype. This process can be adapted to the phenotype of interest. As the number of possible phenotypes is vast, so too are the possibilities for screening. Numerous genetic screens and selections have been conducted to identify mutants or overexpressed naturally occurring genes that result in particular phenotypes. Any of these methods can be adapted to identify useful members of a nucleic acid library encoding chimeric proteins.
  • a screen can include evaluating each cell or organism that includes a library nucleic acid or a selection, e.g., evaluating cells or organisms that survive or otherwise withstand a particular treatment.
  • Exemplary methods for evaluating cells include microscopy (e.g., light, confocal, fluorescence, scanning electron, and transmission electron), fluorescence based cell sorting, differential centrifugation, differential binding, immunoassays, enzymatic assays, growth assays, and in vivo assays.
  • microscopy e.g., light, confocal, fluorescence, scanning electron, and transmission electron
  • fluorescence based cell sorting e.g., fluorescence based cell sorting
  • differential centrifugation e.g., differential centrifugation
  • differential binding e.g., immunoassays, enzymatic assays, growth assays, and in vivo assays.
  • Some screens involve particular environmental conditions. Cells that are sensitive or resistant to the condition are identified.
  • Some screens require detection of a particular behavior of a cell (e.g., chemotaxis, morphological changes, or apoptosis), or a particular behavior of an organism (e.g., phototaxis by a plant, mating behavior by a Drosophila, and so forth).
  • the cells or organisms can be evaluated directly, e.g., by visual inspection, e.g., using a microscope and optionally computer software to automatically detect altered cells.
  • the cells or organisms can be evaluated using an assay or other indicator associated with the desired phenotype.
  • Some screens relate to cell proliferation.
  • Cells that proliferate at a different rate relative to a reference cell e.g., a normal cell
  • a proliferative signal e.g., a growth factors or other mitogen
  • the cells may be more or less sensitive to the signal.
  • Screens that relate to cell differentiation can also be used.
  • the screening and use of chimeric zinc finger proteins can be used to modulate the differentiative and proliferative capacity of a variety of cells, including stem cells, such as ES cells and somatic stem cells, both human and otherwise.
  • Zinc finger proteins can be found that direct ES cells to differentiate into a restricted lineage, such as neuronal progenitor cells or hematopoietic stem cells. It should be also possible to screen for zinc finger proteins that can direct differentiation of stem cells toward a defined post-mitotic cell subtype, for example, directing differentiation of ES cells and/or neural stem cells to dopaminergic or cholinergic neurons.
  • marker genes and marker proteins include:
  • CD4, CD8, and CD45 for white blood cells (Ody et al., (2000) Blood 96:3988-90, Martin et al., (2000) Blood 96:2511-9)
  • ABP adipocyte lipid-binding protein
  • fatty acid transporter for adipocytes
  • CD133 for neural stem cells (Uchida N et al., (2000) Proc. Natl. Acad. Sci. USA 97:14720-5)
  • microtubule-associated protein-2 for neurons (Roy et al., (2000) Nat Med 6:271-7)
  • mammalian cells for other properties, such as anti-tumorigenesis, altered apoptosis, and anti-viral phenotypes. For example, by selecting for cells that are resistant to viral infection or virus production, it is possible to identify artificial chimeric proteins that can be used as anti-viral agents.
  • Some screens relate to production of a compound of interest, e.g., a metabolite, a secreted protein, and a post-translationally modified protein.
  • a compound of interest e.g., a metabolite, a secreted protein, and a post-translationally modified protein.
  • cells can be identified that produce an increased amount of a compound.
  • cells can be identified that produce a reduced amount of a compound, e.g., an undesired byproduct.
  • Cells of interest can be identified by a variety of means, including the use of a responder cell, microarrays, chemical detection assays, and immunoassays.
  • More examples of particular embodiments include:
  • Protein solubility In E. coli, many heterologous proteins are expressed as inclusion bodies. We identified chimeric zinc finger proteins that increase the soluble fraction of a human protein expressed in E. coli. See Example 12. Accordingly, the invention features an artificial transcription factor or a chimeric zinc finger protein that alters (e.g., increases) the solubility of a heterologous protein expressed (e.g., overexpressed in a cell).
  • a library encoding chimeric proteins is screened to identify a CHO cell that is modified so that a secreted protein, e.g., an antibody, includes one or more (e.g., all) glycosylations that characterize an antibody produced by a B cell.
  • the invention features an artificial transcription factor or a chimeric zinc finger protein that alters glycosylation of a secreted protein, e.g., a protein secreted by a CHO cell, e.g., an antibody secreted by a CHO cell.
  • a library encoding chimeric proteins is screened to identify a chimeric protein that increases or decreases viral titer in a cell culture.
  • Viruses can be used as a delivery vehicle, e.g., a gene delivery vehicle.
  • therapeutic viruses are now being developed to cure certain type of cancer (e.g. an adenovirus).
  • Increasing the viral titer is useful for preparing viruses for therapy.
  • suppressing viral production in a cell culture and in vivo is useful for treating viral disease.
  • the invention features an artificial transcription factor or a chimeric zinc finger protein that alters (e.g., increases or decreases) virus production in a cell, e.g., a eukaryotic or mammalian cell.
  • Transformation efficiency Genetic engineering in many eukaryotic cell lines or prokaryotic organisms is limited by low transfection or transformation efficiency. Artificial transcription factors can be selected that modify cells so that transfection or transformation efficiency is improved. A selection for such a factor can be performed by a transformation with a reporter or marker at limiting concentrations, followed by selection of those cells that take up the reporter. Accordingly, the invention features an artificial transcription factor or a chimeric zinc finger protein that alters (e.g., increases) the DNA uptake efficiency or tolerance of DNA uptake procedures of a cell.
  • Feeder cells It is possible to identify artificial transcription factors and other chimeras that modify the properties of a culture cell so that the culture cell can support proliferation or differentiation of a stem cells, e.g., thereby producing a feeder cell.
  • the culture cell can be a human or mammalian cell.
  • the cells can be screened (e.g., by pooling library members) to identify a cell which causes a stem cell cultivated in the same milieu (e.g, the same well) to proliferate or differentiation.
  • the artificial transcription factor may activate key cytokines and growth factors, which are secreted to the media.
  • the media can be used to induce differentiation or proliferate (e.g., by supporting self-renewal) the stem cell.
  • the invention features an artificial transcription factor or a chimeric zinc finger protein that alters (e.g., increases) the ability of a mammalian cell to condition media or otherwise alter the behavior of a stem cell, e.g., regulate proliferation or differentiation of a stem cell.
  • Stem cells are cells with the capacity self-renewal and also the potential for differentiation. The self-renewal can be prolonged, and even indefinite. Stem cells can produce highly differentiated descendants (Watt and Hogan (2000) Science 287:1427-1430). Recent success in culturing human embryonic stem cells has provided a potential source of cells for cell-based therapies. However, maintaining, replicating, and differentiating stem cells can, at least in some cases, be difficult. ES cells, for example, have a tendency to differentiate randomly in vitro.
  • a library of chimeric transcription factors can be used to identify proteins that can control cell differentiation, e.g., stem cell differentiation. For example, one may identify chimeric transcription factors that direct the differentiation of stem cells towards a defined post-mitotic cell subtype (for example, a dopaminergic or cholinergic neuron).
  • a defined post-mitotic cell subtype for example, a dopaminergic or cholinergic neuron.
  • Proteins can be identified that increase the potential for self-renewal, that prevent differentiation, or that direct the extent and character of differentiation. These proteins are generally identified by introducing nucleic acids encoding artificial zinc finger proteins into stem cells or stem cell progenitors, and evaluating the phenotype of the cell.
  • a means to control the differentiative and proliferative potential of stem cells would enable, among other things, the provision of a large supply of undifferentiated cells, and the regulated differentiation toward a specific cell types. Such control can be tailored to a therapeutic use or other applications (e.g., development of transgenic animals, in vitro cell culture, and so forth).
  • ES embryonic stem
  • chimeric proteins e.g., chimeric ZFPs, that cause 1) a differentiated cell to adopt a different differentiated state or 2) a differentiated cell to a adopt a non-differentiated state, e.g., thereby generating a stem cell or a pluripotent progenitor cell.
  • the identification method does not require information about a particular target gene.
  • Target genes can be identified after screening, e.g., by transcript or protein profiling to identify genes whose expression or activity is altered by an identified chimeric transcription factor.
  • the identification of genes regulated by ZFP-TFs will advance understanding of cell differentiation.
  • the invention features artificial transcription factors (e.g., chimeric zinc finger proteins) that alter the ability of a cell to produce a cellular product, e.g., a protein or metabolite.
  • a cellular product can be an endogenous or heterologous molecule.
  • an artificial transcription factor that increases the ability of a cell to produce proteins, e.g., particular proteins (e.g., particular endogenous proteins), overexpressed proteins, heterologous proteins, or mis-folded proteins.
  • cells are screened for their ability to produce a reporter protein, e.g., a protein that can be enzymatically or fluorescently detected.
  • the reporter protein is insoluble when overexpressed in a reference cell.
  • bacterial cells can be screened for artificial transcription factors that reduce inclusion bodies.
  • the reporter protein is secreted, e.g., by a prokaryotic or eukaryotic cell.
  • Cells can be screened for higher secretory through-put, or improved post-translational modification, e.g., glycosylation, phosphorylation, or proteolytic processing.
  • cells are screened for their ability to alter (e.g., increase or decrease) the activity of two different reporter proteins.
  • the reporter proteins may differ, e.g., by activity, localization (e.g., secreted/cytoplasmic/nuclear), size, solubility, isoelectric point, oligomeric state, post-translational regulation, translational regulation, and transcriptional regulation (e.g., the gene encoding them may be regulated by different regulatory sequences).
  • the invention includes artificial transcription factors (e.g., zinc finger proteins) that alter at least two different reporter genes that differ by these properties, and zinc finger proteins that selectively regulate a reporter gene, or a class of reporter genes defined by one of these properties.
  • the phenotypic screening method can be used to isolate the artificial transcription factor, it is not necessary to know a priori how the zinc finger protein mediates increased protein production. Possible mechanisms, which can be verified, include alteration of one or more of the following: translation machinery, transcript processing, transcription, secretion, protein degradation, stress resistance, catalytic activity, e.g., metabolite production.
  • an artificial transcription factor may modulate expression of one or more enzymes in a metabolic pathway and thereby enhance production of a cellular product such as a metabolite or a protein.
  • a chimeric DNA binding protein Once a chimeric DNA binding protein is identified, its ability to alter a phenotypic trait of a cell can be further improved by a variety of strategies. Small libraries, e.g., having about 6 to 200 or 50 to 2000 members, or large libraries can be used to optimize the properties of a particular identified chimeric protein.
  • mutagenesis techniques are used to alter the original chimeric DNA binding protein.
  • the techniques are applied to construct a second library whose members include members that are variants of an original protein, for example, a protein identified from a first library.
  • Examples of these techniques include: error-prone PCR (Leung et al. (1989) Technique 1:11-15), recombination, DNA shuffling using random cleavage (Stemmer (1994) Nature 389-391), Coco et al. (2001) Nature Biotech. 19:354, site-directed mutagenesis (Zollner et al.
  • a library is constructed that mutates a set of amino acid positions.
  • the set of amino acid positions may be positions in the vicinity of the DNA contacting residues, but not the DNA contacting residues themselves.
  • the library varies each encoded domain in a chimeric protein, but to a more limited extent than the initial library from which the chimeric DNA binding protein was identified.
  • the nucleic acids that encode a particular domain can be varied among other zinc finger domains whose recognition specificity is known to be similar to that of the domain present in the original chimeric protein.
  • Some techniques include generating new chimeric DNA binding proteins from nucleic acids encoding domains of at least two chimeric DNA binding proteins that are known to have a particular functional property. These techniques, which include DNA shuffling and standard domain swapping, create new combinations of domains. See, e.g., U.S. Pat. No. 6,291,242. DNA shuffling can also introduce point mutations in addition to merely exchanging domains. The shuffling reaction is seeded with nucleic acid sequences encoding chimeric proteins that induces a desired phenotype. The nucleic acids are shuffled.
  • a secondary library is produced from the shuffling products and screened for members that induce the desired phenotype, e.g., under similar or more stringent conditions. If the initial library is comprehensive such that chimeras of all possible domain combinations are screened, DNA shuffling of domains isolated from the same initial library may be of no avail. DNA shuffling may be useful in instances where coverage is comprehensive and also in instances where comprehensive screening may not be practical.
  • a chimeric DNA binding protein that produces a desired phenotype is altered by varying each domain. Domains can be varied sequentially, e.g., one-by-one, or greater than one at a time.
  • the following example refers to an original chimeric protein that includes three zinc finger domains: fingers I, II, and III and that produces a desired phenotype.
  • a second library is constructed such that each nucleic acid member of the second library encodes the same finger II and finger III as the initially identified protein. However, the library includes nucleic acid members whose finger I differs from finger I of the original protein. The difference may be a single nucleotide that alters the amino acid sequence of the encoded chimeric protein or may be more substantial.
  • the second library can be constructed, e.g., such that the base-contacting residues of finger I are varied, or that the base-contacting residues of finger I are maintained but that adjacent residues are varied.
  • the second library can also to include a large enough set of zinc finger domains to recognize at least 20, 30, 40, or 60 different trinucleotide sites.
  • the second library is screened to identify members that alter a phenotype of a cell or organism.
  • the extent of alteration can be similar to that produced by the original protein or greater than that produced by the original protein.
  • a third library can be constructed that varies finger II, and a fourth library can be constructed that varies finger III. It may not be necessary to further improve a chimeric protein by varying all domains, if the chimeric protein or already identified variants are sufficient. In other cases, it is desirable to re-optimize each domain.
  • the method includes adding, substituting, or deleting a domain, e.g., a zinc finger domain or a regulatory domain.
  • a domain e.g., a zinc finger domain or a regulatory domain.
  • An additional zinc finger domain may increase the specificity of a chimeric protein and may increase its binding affinity. In some cases, increased binding affinity may enhance the phenotype that the chimeric protein produces.
  • An additional regulatory domain e.g., a second activation domain or a domain that recruits an accessory factor, may also enhance the phenotype that the chimeric protein produces.
  • a deletion may improve or broaden the specificity of the activity of the chimeric protein, depending on the contribution of the domain that is deleted, and so forth.
  • the method includes co-expressing the original chimeric protein and a second chimeric DNA binding protein in a cell.
  • the second chimeric protein can be also identified by screening a nucleic acid library that encodes different chimeras.
  • the second chimeric protein is identified by screening the library in a cell that expresses the original chimeric protein.
  • the second chimeric protein is identified independently.
  • a chimeric transcription factor that alters a phenotype of a cell can be further characterized to identify the endogenous genes that it directly or indirectly regulates. Typically, the chimeric transcription factor is produced within the cell. At an appropriate time, e.g., before, during, or after the phenotypic change occurs, the cell is analyzed to determine the levels of transcripts or proteins present in the cell or in the medium surrounding the cell. For example, mRNA can be harvested from the cell and analyzed using a nucleic acid microarray.
  • Nucleic acid microarrays can be fabricated by a variety of methods, e.g., photolithographic methods (see, e.g., U.S. Pat. No. 5,510,270), mechanical methods (e.g., directed-flow methods as described in U.S. Pat. No. 5,384,261), and pin based methods (e.g., as described in U.S. Pat. No. 5,288,514).
  • the array is synthesized with a unique capture probe at each address, each capture probe being appropriate to detect a nucleic acid for a particular expressed gene.
  • the mRNA can be isolated by routine methods, e.g., including DNase treatment to remove genomic DNA and hybridization to an oligo-dT coupled solid substrate (e.g., as described in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y). The substrate is washed, and the mRNA is eluted. The isolated mRNA is then reversed transcribed and optionally amplified, e.g., by rtPCR, e.g., as described in (U.S. Pat. No. 4,683,202).
  • the nucleic acid can be labeled during amplification or reverse transcription, e.g., by the incorporation of a labeled nucleotide.
  • nucleic acid can be labeled with biotin, and detected after hybridization with labeled streptavidin, e.g., streptavidin-phycoerythrin (Molecular Probes).
  • streptavidin e.g., streptavidin-phycoerythrin (Molecular Probes).
  • the labeled nucleic acid is then contacted to the array.
  • a control nucleic acid or a reference nucleic acid can be contacted to the same array.
  • the control nucleic acid or reference nucleic acid can be labeled with a label other than the sample nucleic acid, e.g., one with a different emission maximum.
  • Labeled nucleic acids are contacted to an array under hybridization conditions. The array is washed, and then imaged to detect fluorescence at each address of the array.
  • a general scheme for producing and evaluating profiles includes detecting hybridization at each address of the array.
  • the extent of hybridization at an address is represented by a numerical value and stored, e.g., in a vector, a one-dimensional matrix, or one-dimensional array.
  • the vector x has a value for each address of the array.
  • a numerical value for the extent of hybridization at a particular address is stored in variable x a .
  • the numerical value can be adjusted, e.g., for local background levels, sample amount, and other variations.
  • Nucleic acid is also prepared from a reference sample and hybridized to the same or a different array.
  • the vector y is construct identically to vector x.
  • the sample expression profile and the reference profile can be compared, e.g., using a mathematical equation that is a function of the two vectors.
  • the comparison can be evaluated as a scalar value, e.g., a score representing similarity of the two profiles.
  • Either or both vectors can be transformed by a matrix in order to add weighting values to different genes detected by the array.
  • the expression data can be stored in a database, e.g., a relational database such as a SQL database (e.g., Oracle or Sybase database environments).
  • the database can have multiple tables.
  • raw expression data can be stored in one table, wherein each column corresponds to a gene being assayed, e.g., an address or an array, and each row corresponds to a sample.
  • a separate table can store identifiers and sample information, e.g., the batch number of the array used, date, and other quality control information.
  • Genes that are similarly regulated can be identified by clustering expression data to identify coregulated genes. Such cluster may be indicative of a set of genes coordinately regulated by the chimeric zinc finger protein. Genes can be clustered using hierarchical clustering (see, e.g., Sokal and Michener (1958) Univ. Kans. Sci. Bull. 38:1409), Bayesian clustering, k-means clustering, and self-organizing maps (see, Tamayo et al. (1999) Proc. Natl. Acad. Sci. USA 96:2907).
  • the similarity of a sample expression profile to a reference expression profile can also be determined, e.g., by comparing the log of the expression level of the sample to the log of the predictor or reference expression value and adjusting the comparison by the weighting factor for all genes of predictive value in the profile.
  • Proteins can also be profiled in a cell that has an active chimeric protein with in it.
  • One exemplary method for profiling proteins includes 2-D gel electrophoresis and mass spectroscopy to characterize individual protein species. Individual “spots” on the 2-D gel are proteolyzed and then analyzed on the mass spectrometer. This method can identify both the protein component and, in many cases, translational modifications.
  • the protein and nucleic acid profiling methods can not only provide information about the properties of the chimeric protein, but also information about natural mechanisms operating within the cell.
  • the proteins or nucleic acids upregulated by expression of the chimeric protein may be the natural effectors of the phenotypic change caused by expression of the chimeric protein.
  • alterations that compensate (e.g., suppress) the phenotypic effect of the artificial chimeric protein are characterized. These alterations include genetic alterations such as mutations in chromosomal genes and overexpression of a particular gene, as well as other alterations, such as RNA interference (e.g., by double-stranded RNA).
  • a chimeric ZFP is isolated that causes a growth defect or lethality when conditionally expressed in a cell, e.g., a pathogenic bacteria or fungi.
  • a ZFP can be identified by transforming the cell with the ZFP libraries that include nucleic acids encoding ZFPs, expression of the nucleic acids being controlled by an inducible promoter. Transformants are cultured on non-inducible media and then replica-plated on both inducible and non-inducible plates. Colonies that grow normally on non-inducible plate, but show defective growth on inducible plate are identified as “conditional lethal” or “conditional growth defective” colonies.
  • a cDNA expression library is then transformed into the “conditional lethal” or “conditional growth defective” strains described above. Transformants are plated on inducible plates. Colonies that survive, despite the presence and expression of the ZFP that causes the defect, are isolated. The nucleic acid sequences of cDNAs that complement the defect are characterized. These cDNA can be transcripts of direct or indirect target genes that are regulated by chimeric ZFP that mediates the defect.
  • a second chimeric protein that suppresses the effect of the first chimeric protein is identified.
  • the targets of the second chimeric protein in the presence or absence of the first chimeric protein are identified.
  • a ZFP library is transformed into “conditional lethal” or “conditional growth defective” colonies (which include a first chimeric ZFP that causes the defect).
  • Transformants are plated on inducible plates. Colonies that can survive by the expression of introduced ZFP are identified as “suppressed strains”.
  • Target genes of the second ZFPs can be characterized by DNA microarray analysis. The comparative analysis can be done between four strains: 1) no ZFP; 2) the first ZFP alone; 3) the second ZFP alone; and 4) the first and second ZFP.
  • genes that are regulated in opposing directions by the first and second chimeric ZFPs are candidates for targets that mediate the growth-defective phenotype. This method can be applied to any phenotype, not just a growth defect.
  • a candidate target of a chimeric ZFP can be identified by expression profiling. Subsequently, to determine if the candidate target mediates the phenotype of the chimeric ZFP, the candidate target can be independently over-expressed or inhibited (e.g., by genetic deletion or RNA interference). In addition, it may be possible to apply this analysis to multiple candidate targets since in at least some cases more than one candidate may need to be perturbed to cause the phenotype. An example of this approach is provided in Example 3 (Ketoconazole resistance).
  • the targets of a chimeric ZFP can be identified by a characterizing changes in gene expression with respect to time after a cell is exposed to the chimeric ZFP.
  • a gene encoding the chimeric ZFP can be attached to an inducible promoter.
  • An exemplary inducible promoter is regulated by a small molecule such as doxycycline.
  • the gene encoding the chimeric ZFP is introduced into cells. mRNA samples are obtained from cells at various times after induction of the inducible promoter. See, e.g., FIG. 13, which depicts genes that are activated and repressed in the course of induction of the ZFP F104-p65.
  • a chimeric protein into a cell by transduction.
  • the protein is provided to the extracellular milieu and the cell transduces the protein into itself.
  • the cell does not have to include a gene encoding the chimeric protein.
  • This approach may obviate concerns about exogenous nucleic acid integration, propagation, and so forth. Levels of the protein can be precisely controlled.
  • the chimeric ZFP is fused to protein transduction domain of Tat or VP22.
  • the chimeric protein is added to the culture media, e.g., as a fusion to a protein transduction domain.
  • Detection of regulated target genes can be enhanced by addition of an inhibitor of protein synthesis such as cycloheximide.
  • an inhibitor of protein synthesis such as cycloheximide.
  • translation of the primary target genes is blocked, and genes that would be regulated by proteins encoded by the primary target genes would be detected.
  • the identity of primary target genes can be found by DNA microarray analysis.
  • cyclohexamide to identify primary target genes can also be used when the chimeric protein is encoded by a heterologous nucleic acid in the cell.
  • expression of the heterologous nucleic acid can be induced for less than 30, 20, 15, 10, or 5 minutes, and then cyclohexamide can be added.
  • RNA interference with double-stranded RNAs (dsRNA), anti-sense technology, ribozymes, or targeted genetic mutation.
  • dsRNA double-stranded RNAs
  • a cell or organism in which activity of the target gene is reduced can be evaluated and compared to a control cell or organism that is not treated with RNAi.
  • a cell or organism that expresses the artificial zinc finger protein believed to regulate the target gene is treated with RNAi.
  • the ability of the artificial zinc finger protein to induce a phenotype is evaluated in the presence and absence of RNAi.
  • the RNAi treatment inactivating the potential target may attenuate the phenotype induced by the artificial zinc finger protein.
  • dsRNA can be produced by transcribing a cassette in both directions, for example, by including a T7 promoter on either side of the cassette.
  • the insert in the cassette is selected so that it includes a sequence complementary to the potential target gene.
  • HiScribeTM RNAi Transcription Kit New England Biolabs, MA
  • Fire A. (1999) Trends Genet. 15, 358-363.
  • dsRNA can be digested into smaller fragments. See, e.g., U.S. patent application Ser. No. 2002-0086356.
  • dsRNAs can be used to silence gene expression in mammalian cells. See, e.g., Clemens, et al. (2000) Proc. Natl. Sci.
  • chimeric DNA binding proteins With respect to chimeric DNA binding proteins, a variety of methods can be used to determine the target site of a chimeric DNA binding protein that produces a phenotype of interest. Such methods can be used, alone or in combination, to find such a target site.
  • information from expression profile is used to identify the target site recognized by a chimeric zinc finger protein.
  • the regulatory regions of genes that are co-regulated by the chimeric zinc finger protein are compared to identify a motif that is common to all or many of the regulatory regions.
  • biochemical means are used to determine what DNA site is bound by the chimeric zinc finger protein.
  • chromatin immuno-precipitation experiments can be used to isolate nucleic acid to which the chimeric zinc finger protein is bound. The isolated nucleic acid is PCR amplified and sequence. See, e.g., Gogus et al. (1996) Proc. Natl. Acad. Sci. USA. 93:2159-2164.
  • the SELEX method is another exemplary method that can be used.
  • information about the binding specificity of individual zinc finger domains in the chimeric zinc finger protein can be used to predict the target site. The prediction can be validated or can be used to guide interpretation of other results (e.g., from chromatin immunoprecipitation, in silico analysis of co-regulated genes, and SELEX).
  • a potential target site is inferred based on information about the binding specificity of each component zinc finger.
  • a chimera that includes the zinc finger domains, from N- to C-terminus,: CSNR, RSNR, and QSNR is expected to recognize the target site 5′-GAAGAGGACC-3′ (SEQ ID NO:130).
  • the domains CSNR, RSNR, and QSNR have the following respective DNA binding specificities GAC, GAG, and GAA.
  • the expected target site is formed by considering the domains in C terminal to N-terminal order and concatenating their recognition specificities to obtain one strand of the target site in 5′ to 3′ order.
  • chimeric zinc finger proteins are likely to function as transcriptional regulators, it is possible that in some cases the chimeric zinc finger proteins mediate their phenotypic effect by binding to an RNA or protein target. Some naturally-occurring zinc finger proteins in fact bind to these macromolecules.
  • the encoded polypeptides can also include one or more of the following features. These features may be constant among all members of the library or may also vary. In one example, some nucleic acids encode polypeptides that include an activation domain, whereas others include a repression domain, or no transcriptional regulatory domain.
  • Transcriptional activation domains that may be used in the present invention include but are not limited to the Gal4 activation domain from yeast and the VP16 domain from herpes simplex virus.
  • the ability of a domain to activate transcription can be validated by fusing the domain to a known DNA binding domain and then determining if a reporter gene operably linked to sites recognized by the known DNA-binding domain is activated by the fusion protein.
  • An exemplary activation domain is the following domain from p65: (SEQ ID NO:131) YLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRS SASVPKPAPQPYPFTSSLSTINYDEFPTMVFPSGQISQASALAPAPPQVL PQAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLS EALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPH TTEPMLMEYPEAITRLVTAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIA DMDFSALLSQ
  • Gal4 activation domain The sequence of an exemplary Gal4 activation domain is as follows: (SEQ ID NO:132) NFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSNVHDNFM NNEITASKIDDGNNSKPLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYL FDDEDTPPNPKKEISMAYPYDVPDYAS
  • activation domain function can be emulated by a domain that recruits a wild-type RNA polymerase alpha subunit C-terminal domain or a mutant alpha subunit C-terminal domain, e.g., a C-terminal domain fused to a protein interaction domain.
  • Repression domains If desired, a repression domain instead of an activation domain can be fused to the DNA binding domain.
  • eukaryotic repression domains include repression domains from Kid, UME6, ORANGE, groucho, and WRPW (see, e.g., Dawson et al., (1995) Mol. Cell Biol. 15:6923-31).
  • the ability of a domain to repress transcription can be validated by fusing the domain to a known DNA binding domain and then determining if a reporter gene operably linked to sites recognized by the known DNA-binding domain is repressed by the fusion protein.
  • An exemplary repression domain is the following domain from UME6 protein: (SEQ ID NO:133) NSASSSTKLDDDLGTAAAVLSNMRSSPYRTHDKPISNVNDMNNTNALGVP ASRPHSSSFPSKGVLRPILLRIHNSEQQPIFESNNSTACI
  • Kid protein (SEQ ID NO:134) VSVTFEDVAVLFTRDEWKKLDLSQRSLYREVMLENYSNLASMAGFLFTKP KVISLLQQGEDPW
  • Still other chimeric transcription factors include neither an activation or repression domain. Rather, such transcription factors may alter transcription by displacing or otherwise competing with a bound endogenous transcription factor (e.g., an activator or repressor).
  • a bound endogenous transcription factor e.g., an activator or repressor
  • Peptide Linkers DNA binding domains can be connected by a variety of linkers.
  • the utility and design of linkers are well known in the art.
  • a particularly useful linker is a peptide linker that is encoded by nucleic acid.
  • PCT WO 99/45132 and Kim and Pabo ((1998) Proc. Natl. Acad. Sci. USA 95:2812-7) describe the design of peptide linkers suitable for joining zinc finger domains.
  • peptide linkers are available that form random coil, ⁇ -helical or ⁇ -pleated tertiary structures.
  • Polypeptides that form suitable flexible linkers are well known in the art (see, e.g., Robinson and Sauer (1998) Proc Natl Acad Sci USA. 95:5929-34).
  • Flexible linkers typically include glycine, because this amino acid, which lacks a side chain, is unique in its rotational freedom. Serine or threonine can be interspersed in the linker to increase hydrophilicity.
  • amino acids capable of interacting with the phosphate backbone of DNA can be utilized in order to increase binding affinity. Judicious use of such amino acids allows for balancing increases in affinity with loss of sequence specificity.
  • ⁇ -helical linkers such as the helical linker described in Pantoliano et al. (1991) Biochem. 30:10117-10125, can be used.
  • Linkers can also be designed by computer modeling (see, e.g., U.S. Pat. No. 4,946,778). Software for molecular modeling is commercially available (e.g., from Molecular Simulations, Inc., San Diego, Calif.).
  • the linker is optionally optimized, e.g., to reduce antigenicity and/or to increase stability, using standard mutagenesis techniques and appropriate biophysical tests as practiced in the art of protein engineering, and functional assays as described herein.
  • the peptide that occurs naturally between zinc fingers can be used as a linker to join zinc fingers together.
  • An example of a naturally occurring linker is: Thr-Gly-(Glu or Gln)-(Lys or Arg)-Pro-(Tyr or Phe) (SEQ ID NO:74) (Agata et al., supra).
  • linkers can be selected or based on the sequences that join zinc fingers in naturally occurring proteins.
  • Dimerization Domains An alternative method of linking DNA binding domains is the use of dimerization domains, especially heterodimerization domains (see, e.g., Pomerantz et al (1998) Biochemistry 37:965-970).
  • DNA binding domains are present in separate polypeptide chains. For example, a first polypeptide encodes DNA binding domain A, linker, and domain B, while a second polypeptide encodes domain C, linker, and domain D.
  • An artisan can select a dimerization domain from the many well-characterized dimerization domains. Domains that favor heterodimerization can be used if homodimers are not desired.
  • a particularly adaptable dimerization domain is the coiled-coil motif, e.g., a dimeric parallel or anti-parallel coiled-coil. Coiled-coil sequences that preferentially form heterodimers are also available (Lumb and Kim, (1995) Biochemistry 34:8642-8648).
  • Another species of dimerization domain is one in which dimerization is triggered by a small molecule or by a signaling event.
  • a dimeric form of FK506 can be used to dimerize two FK506 binding protein (FKBP) domains.
  • FKBP FK506 binding protein
  • chimeric proteins that include different combinations of non-DNA binding domains, e.g., intracellular signal transduction domains (e.g., SH2, SH3, PDZ, Che domains, or kinase domains).
  • the chimeric proteins encoded by the library can be expressed in cells, and cells having an altered phenotypic trait are identified. For example, chimeric proteins formed of different combinations of signaling domains can be identified that decrease or increase the rate of cell proliferation.
  • Method described herein can include use of routine techniques in the field of molecular biology, biochemistry, classical genetics, and recombinant genetics.
  • Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (2nd ed. 1989); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).
  • nucleic acids encoding zinc proteins can be constructed using synthetic oligonucleotides as linkers to construct a synthetic gene.
  • synthetic oligonucleotides are used and/or primers to amplify sequences encoding one or more zinc finger domains, e.g., from an RNA or DNA template, artificial or synthetic. See U.S. Pat. Nos. 4,683,195 and 4,683,202; PCR Protocols: A Guide to Methods and Applications (Innis et al., eds, 1990)).
  • PCR polymerase chain reaction
  • Gene expression of zinc finger proteins can also be analyzed by techniques known in the art, e.g., reverse transcription and amplification of mRNA, isolation of total RNA or polyA + RNA, northern blotting, dot blotting, in situ hybridization, RNase protection, nucleic acid array technology, e.g., and the like.
  • the polynucleotide encoding an artificial zinc finger protein can be cloned into vectors before transformation into prokaryotic or eukaryotic cells for replication and/or expression.
  • vectors are typically prokaryote vectors, e.g., plasmids, phage or shuttle vectors, or eukaryotic vectors.
  • Protein Expression To obtain recombinant expression (e.g., high level) expression of a polynucleotide encoding an artificial zinc finger protein, one can subclone the relevant coding nucleic acids into an expression vector that contains a strong promoter to direct transcription, a transcription/translation terminator, and a ribosome binding site for translational initiation. Suitable bacterial promoters are well known in the art and described, e.g., in Sambrook et al., and Ausubel et al, supra. Bacterial expression systems for expression are available in, e.g., E.
  • Kits for such expression systems are commercially available.
  • Eukaryotic expression systems for mammalian cells, yeast (e.g., S. cerevisiae, S. pombe, Pichia, and Hanseula), and insect cells are well known in the art and are also commercially available.
  • the promoter used to direct expression of a heterologous nucleic acid depends on the particular application.
  • the promoter is preferably positioned about the same distance from the heterologous transcription start site as it is from the transcription start site in its natural setting. As is known in the art, however, some variation in this distance can be accommodated without loss of promoter function.
  • the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for expression in host cells.
  • a typical expression cassette thus contains a promoter operably linked to the coding nucleic acid sequence and signals required for efficient polyadenylation of the transcript, ribosome binding sites, and translation termination. Additional elements of the cassette may include enhancers and, if genomic DNA is used as the structural gene, introns with functional splice donor and acceptor sites.
  • the expression cassette should also contain a transcription termination region downstream of the structural gene to provide for efficient termination.
  • the termination region may be obtained from the same gene as the promoter sequence or may be obtained from different genes.
  • the particular expression vector used to transport the genetic information into the cell is not particularly critical. Any of the conventional vectors used for expression in eukaryotic or prokaryotic cells may be used. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and fusion expression systems such as MBP, GST, and LacZ. Epitope tags can also be added to recombinant proteins to provide convenient methods of isolation, e.g., c-myc-, or a hexa-histidine tag.
  • Expression vectors can contain regulatory elements from eukaryotic viruses, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus.
  • eukaryotic vectors include pMSG, pAV009/A + , pMTO10/A + , pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the CMV promoter, SV40 early promoter, SV40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
  • Expression of proteins from eukaryotic vectors can be also be regulated using inducible promoters.
  • inducible promoters expression levels are tied to the concentration of inducing agents, such as tetracycline or ecdysone, by the incorporation of response elements for these agents into the promoter. Generally, high level expression is obtained from inducible promoters only in the presence of the inducing agent; basal expression levels are minimal.
  • Inducible expression vectors are often chosen if expression of the protein of interest is detrimental to eukaryotic cells.
  • Some expression systems have markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase.
  • markers that provide gene amplification such as thymidine kinase and dihydrofolate reductase.
  • high yield expression systems not involving gene amplification are also suitable, such as using a baculovirus vector in insect cells, with mitochondrial respiratory chain protein encoding sequences and glycolysis protein encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters
  • the elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of eukaryotic sequences.
  • the prokaryotic sequences can be chosen such that they do not interfere with the replication of the DNA in eukaryotic cells.
  • Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of zinc finger proteins, which are then purified using standard techniques (see, e.g., Colley et al., J. Biol. Chem. 264:17619-17622 (1989); Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, J. Bact. 132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).
  • Any of the well-known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, protoplast fusion, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell.
  • the transfected cells are cultured under conditions favoring expression or activating expression.
  • the protein can then be isolated from a cell extract, cell membrane component or vesicle, or media.
  • Expression vectors with appropriate regulatory sequences can also be used to express a heterologous gene encoding an artificial zinc finger in a model organism, e.g., a Drosophila, nematode, zebrafish, Xenopus, or mouse. See, e.g., Riddle et al., eds., C. elegans II. Plainview (N.Y.): Cold Spring Harbor Laboratory Press; 1997.
  • Zinc finger protein can be purified from materials generated by any suitable expression system, e.g., those described above.
  • Zinc finger proteins may be purified to substantial purity by standard techniques, including selective precipitation with such substances as ammonium sulfate; column chromatography, affinity purification, immunopurification methods, and others (see, e.g., Scopes, Protein Purification. Principles and Practice (1982); U.S. Pat. No. 4,673,641; Ausubel et al., supra; and Sambrook et al., supra).
  • zinc finger proteins can include an affinity tag that can be used for purification, e.g., in combination with other steps.
  • Recombinant proteins are expressed by transformed bacteria in large amounts, typically after promoter induction; but expression can be constitutive.
  • Promoter induction with IPTG is one example of an inducible promoter system.
  • Bacteria are grown according to standard procedures in the art. Fresh or frozen bacteria cells are used for isolation of protein. Proteins expressed in bacteria may form insoluble aggregates (“inclusion bodies”). Several protocols are suitable for purifying proteins from inclusion bodies. See, e.g., Sambrook et al., supra; Ausubel et al., supra). If the proteins are soluble or exported to the periplasm, they can be obtained from cell lysates or periplasmic preparations.
  • Salting-in or out can be used to selectively precipitate a zinc finger protein or a contaminating protein.
  • An exemplary salt is ammonium sulfate.
  • Ammonium sulfate precipitates proteins on the basis of their solubility. The more hydrophobic a protein is, the more likely it is to precipitate at lower ammonium sulfate concentrations.
  • a typical protocol includes adding saturated ammonium sulfate to a protein solution so that the resultant ammonium sulfate concentration is between 20-30%. This concentration precipitates many of the more hydrophobic proteins.
  • the precipitate is analyzed to determine if the protein of interest is precipitated or in the supernatant. Ammonium sulfate is added to the supernatant to a concentration known to precipitate the protein of interest. The precipitate is then solubilized in buffer and the excess salt removed if necessary, either through dialysis or diafiltration.
  • a zinc finger protein can be separated from other proteins on the basis of its size, net surface charge, hydrophobicity, and affinity for ligands.
  • antibodies raised against proteins can be conjugated to column matrices and the proteins immunopurified. All of these methods are well known in the art. Chromatographic techniques can be performed at any scale and using equipment from many different manufacturers (e.g., Pharmacia Biotech). See, generally, Scopes, Protein Purification: Principles and Practice (1982).
  • a “vector” is a nucleic acid molecule competent to transport another nucleic acid molecule to which it has been covalently linked.
  • Vectors include plasmids, cosmids, artificial chromosomes, and viral elements.
  • the vector can be competent to replicate in a host cell or to integrate into a host DNA.
  • Viral vectors include, e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses.
  • a gene therapy vector is a vector designed for administration to a subject, e.g., a mammal, such that a cell of the subject is able to express a therapeutic gene contained in the vector.
  • the gene therapy vector can contain regulatory elements, e.g., a 5′ regulatory element, an enhancer, a promoter, a 5′ untranslated region, a signal sequence, a 3′ untranslated region, a polyadenylation site, and a 3′ regulatory region.
  • regulatory elements e.g., a 5′ regulatory element, an enhancer, a promoter, a 5′ untranslated region, a signal sequence, a 3′ untranslated region, a polyadenylation site, and a 3′ regulatory region.
  • the 5′ regulatory element, enhancer or promoter can regulate transcription of the DNA encoding the therapeutic polypeptide.
  • the regulation can be tissue specific.
  • the regulation can restrict transcription of the desired gene to brain cells, e.g., cortical neurons or glial cells; hematopoietic cells; or endothelial cells.
  • regulatory elements can be included that respond to an exogenous drug, e.g., a steroid, tetracycline, or the like.
  • an exogenous drug e.g., a steroid, tetracycline, or the like.
  • the level and timing of expression of the therapeutic zinc finger polypeptide e.g., a polypeptide that regulates VEGF
  • the level and timing of expression of the therapeutic zinc finger polypeptide e.g., a polypeptide that regulates VEGF
  • Gene therapy vectors can be prepared for delivery as naked nucleic acid, as a component of a virus, or of an inactivated virus, or as the contents of a liposome or other delivery vehicle.
  • the gene delivery agent e.g., a viral vector
  • the gene delivery agent can be produced from recombinant cells which produce the gene delivery system.
  • Appropriate viral vectors include retroviruses, e.g., Moloney retrovirus, adenoviruses, adeno-associated viruses, and lentiviruses, e.g., Herpes simplex viruses (HSV). HSV is potentially useful for infecting nervous system cells.
  • a gene therapy vector can be administered to a subject, for example, by intravenous injection, by local administration (see U.S. Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057).
  • the gene therapy agent can be further formulated, for example, to delay or prolong the release of the agent by means of a slow release matrix.
  • One method of providing a recombinant therapeutic tri-domain polypeptide is by inserting a gene therapy vector into bone marrow cells harvested from a subject. The cells are infected, for example, with a retroviral gene therapy vector, and grown in culture. Meanwhile, the subject is irradiated to deplete the subject of bone marrow cells. The bone marrow of the subject is then replenished with the infected culture cells. The subject is monitored for recovery and for production of the therapeutic polypeptide.
  • Cell based-therapeutic methods include introducing a nucleic acid that encoding the artificial zinc finger protein operably linked to a promoter into a cell in culture.
  • the artificial zinc finger protein can be selected to regulate an endogenous gene in the culture cell or to produce a desired phenotype in the cultured cell.
  • cells e.g., stem cells
  • nucleic acid recombination e.g., to insert a transgene, e.g., a transgene encoding an artificial zinc finger protein that regulates an endogenous gene.
  • the modified stem cell can be administered to a subject. Methods for cultivating stem cells in vitro are described, e.g., in U.S. application Ser. No.
  • the stem cells can be induced to differentiate in the subject and express the transgene.
  • the stem cells can be differentiated into liver, adipose, or skeletal muscle cells.
  • the stem cells can be derived from a lineage that produces cells of the desired tissue type, e.g., liver, adipose, or skeletal muscle cells.
  • recombinant cells that express or can express an artificial zinc finger protein can be used for replacement therapy in a subject.
  • a nucleic acid encoding the artificial zinc finger protein operably linked to a promoter e.g., an inducible promoter, e.g., a steroid hormone receptor-regulated promoter
  • a promoter e.g., an inducible promoter, e.g., a steroid hormone receptor-regulated promoter
  • the cell is cultivated and encapsulated in a biocompatible material, such as poly-lysine alginate, and subsequently implanted into the subject. See, e.g., Lanza (1996) Nat.
  • production of the secreted polypeptide can be regulated in the subject by administering an agent (e.g., a steroid hormone) to the subject.
  • an agent e.g., a steroid hormone
  • the recombinant cells that express or can express an artificial zinc finger protein are cultivated in vitro.
  • a protein produced by the recombinant cells can be recovered (e.g., purified) from the cells or from media surrounding the cells.
  • a nucleic acid library is screened to identify an artificial zinc finger protein that alters production, synthesis or activity of one or more particular target proteins.
  • the alteration can increase or decrease activity or abundance of the target protein.
  • the phenotype screened for can be associated with altered production or activity of one or more target proteins or can be the level of production or activity itself.
  • Some exemplary target proteins include: cell surface proteins (e.g., glycosylated surface proteins), cancer-associated proteins, cytokines, chemokines, peptide hormones, neurotransmitters, cell surface receptors (e.g., cell surface receptor kinases, seven transmembrane receptors, virus receptors and co-receptors, extracellular matrix binding proteins, cell-binding proteins, antigens of pathogens (e.g., bacterial antigens, malarial antigens, and so forth).
  • Additional protein targets include enzymes such as enolases, cytochrome P450s, acyltransferases, methylases, TIM barrel enzymes, isomerases, acyl transferases, and so forth.
  • More specific examples include: integrins, cell attachment molecules or “CAMs” such as cadherins, selections, N-CAM, E-CAM, U-CAM, I-CAM and so forth); proteases (e.g., subtilisin, trypsin, chymotrypsin; a plasminogen activator, such as urokinase or human tissue-type plasminogen activator); bombesin; factor IX, thrombin; CD-4; platelet-derived growth factor; insulin-like growth factor-I and -II; nerve growth factor; fibroblast growth factor (e.g., aFGF and bFGF); epidermal growth factor (EGF); VEGFa; transforming growth factor (TGF, e.g., TGF- ⁇ and TGF- ⁇ ; insulin-like growth factor binding proteins; erythropoietin; thrombopoietin; mucins; human serum albumin; growth hormone (e.g., human
  • various phenotypes of Saccharomyces cerevisiae are altered by regulating gene expression using zinc finger protein (ZFP) expression libraries.
  • the zinc finger proteins in these exemplary libraries consist of three or four zinc finger domains (ZFDs) and recognize 9- to 12-bp DNA sequences respectively.
  • the chimeric zinc finger protein is identified without a priori knowledge of the target genes.
  • Three different class of transcription factors are produced from the ZFPs in the libraries: isolated ZFPs themselves function as efficient transcriptional repressors when they bind to a site near the promoter region; ZFPs are also expressed as fusion proteins to a transcriptional activation domain or to a repression domain to yield transcriptional activators or repressors, respectively.
  • the S. cerevisiae strain used for this experiment was YPH499a (MATa, ade2-101, ura3-52, lys2-801, trp1- ⁇ 63, his3- ⁇ 200, leu2- ⁇ 1, GAL+). Transformation of yeast cells was carried out by using the lithium acetate transformation method (see, e.g., Gietz et al., (1992) Nucl. Acids Res 20:1245).
  • the parental vector that we used to construct libraries of zinc finger proteins is the plasmid p3.
  • p3 was constructed by modifying the pcDNA3 vector (Invitrogen, San Diego Calif.) as follows.
  • the pcDNA3 vector was digested with HindIII and XhoI.
  • a synthetic oligonucleotide duplex with compatible overhangs was ligated into the digested pcDNA3.
  • the duplex contains nucleic acid that encodes the hemagglutinin (HA) tag and a nuclear localization signal.
  • the duplex also includes: restriction sites for BamHI, EcoRI, NotI, and BglII; and a stop codon.
  • the XmaI site in SV40 origin of the vector was destroyed by digestion with XmaI, filling in the overhanging ends of the digested XmaI restriction site, and religation of the ends.
  • pYCT-Lib is a yeast shuttle vector that includes the inducible GAL1 promoter (FIG. 1).
  • Other features can include (i) a sequence encoding a nuclear localization signal (NLS) and a hemagglutinin tag (HA) and (ii) a sequence including the TRP1 gene for selection of plasmid containing cells on synthetic minimal media lacking tryptophan.
  • the polylinker from the T7 primer site to the SphI site region can include: (SEQ ID NO:135) TAATACGACTCACTATAGGGAATATTAAGCTAAGCTCACCATGGGTAAGC CTATCCCTAACCCTCTCCTCGGTCTCGATTCTACACAAGCTATGGGTGCT CCTCCAAAAAAGAAGAGAAAGGTAGCTGGATCCACTAGTAACGGCCGCCA GTGTGCTGGAATTCTGCAGATATCCATCACACTGGCGGCGCTCGAGGCAT GCATCTA
  • p-YCT-Lib was constructed as follows.
  • the yeast expression plasmid pYESTrp2 (Invitrogen, San Diego Calif.) was digested with NgoM4 and then was partially digested with PstI to remove the 2 ⁇ ori fragment from the vector.
  • the 5.0 kb DNA fragment from the NgoM4-PstI digested vector was purified after gel electrophoresis and ligated with a CEN-ARS fragment that was amplified from pRS313 (forward primer:5′-CGATCTGCAGGG TCCTTTTCATCACGTGCT-3′ (SEQ ID NO:136), reverse primer:5′-CGATCGATGCCG GCGGACGGATCGCTTGCCT (SEQ ID NO:137)).
  • the DNA segment that encodes the B42 activation domain was removed by digestion with NcoI and BamHI and replaced with a DNA fragment encoding the V5 epitope tag and the nuclear localization signal.
  • the latter DNA fragment was PCR-amplified from pYESTrp2 (forward primer: 5′-AATTCCATGGGTAAGCCTATCCCTAACC-3′ (SEQ ID NO:138), reverse primer:5′-AATTGGATCCAGCTACCTTTCTCTTCTT-3′(SEQ ID NO:139)) and ligated into the NcoI and BamHI sites.
  • the resulting plasmid was named as pYTC-Lib (FIG. 1).
  • the Gal4 activation domain was PCR-amplified from yeast genomic DNA (forward primer: 5′-AAGGAAGGAAGGAAGCGGC CGCAGCCAATTTTAATCAAAGTGG-3′ (SEQ ID NO:140), reverse primer: 5′-ACATACATGCATGCGCCGTTACTAGTGGATCC-3′ sequence (SEQ ID NO:141)) and inserted between the NotI and SphI recognition sites of pYTC-Lib to generate pYTC-Lib-Gal4.
  • An exemplary sequence encoding the Gal4 activation domain and linking sequences includes: (SEQ ID NO:142) GGCCGCCAGTGTGCTGGAATTCTGCAGATATCCATCACACTGGCGGCCGC AGCCAATTTTAATCAAAGTGGGAATATTGCTGATAGCTCATTGTCCTTCA CTTTCACTAACAGTAGCAACGGTCCGAACCTCATAACAACTCAAACAAAT TCTCAAGCGCTTTCACAACCAATTGCCTCCTCTAACGTTCATGATAACTT CATGAATAATGAAATCACGGCTAGTAAAATTGATGATGGTAATAATTCAA AACCACTGTCACCTGGTTGGACGGACCAAACTGCGTATAACGCGTTTGGA ATCACTACAGGGATGTTTAATACCACTACAATGGATGATGTATATAACTA TCTATTCGATGATGAAGATACCCCACCAAACCCAAAAAAAGAGATCTCTA TGGCTTACCCATACGTATGGATGGATGATGTATATAACTA TCTATTCGATGATGAAGATACCCCACCAA
  • pYCT-Lib-Ume6 To generate pYCT-Lib-Ume6, a DNA fragment encoding amino acids 508 to 594 of S. cerevisiae Ume6 was amplified from yeast genomic DNA (forward primer: 5′-AAGGAAGGAAGGAAGCGGCCGCAAATTCTGCATCTTCATCTACC-3′ (SEQ ID NO:143), reverse primer:5′-ACATACATGCATGCTGTAGAATTGTTGCTTTCG-3′ (SEQ ID NO:144)) and inserted between the NotI and SphI recognition sites of pYTC-Lib. This 87-amino acid region functions as a transcriptional repression domain (Kadosh and Struhl (1997) Cell 89:365-371).
  • An exemplary sequence encoding the Ume6 repression domain and linking sequences includes: (SEQ ID NO:145) GGCCGCCAGTGTGCTGGAATTCTGCAGATATCCATCACACTGGCGGCCGC AAATTCTGCATCTTCATCTACCAAACTAGACGACGACTTGGGTACAGCAG CAGCAGTGCTATCAAACATGAGATCATCCCCATATAGAACTCATGATAAA CCCATTTCCAATGTCAATGACATGAATAACACAAATGCGCTCGGTGTGCC GGCTAGTAGGCCTCATTCGTCATCTTTTCCATCAAAGGGTGTCTTAAGAC CAATTCTGTTACGTATCCATAATTCCGAACAACAACCCATTTTCGAAAGC AACAATTCTACAGCATGCATCTAGGGCC
  • a three-fingered protein library (the “3-F library”), encoding zinc finger proteins that have an array of three ZFDs, was constructed from nucleic acids encoding 40 different ZFDs or “fingers.”
  • a four-fingered protein library (the “4-F library”) was constructed from nucleic acids encoding 27 different ZFDs (Table 2, below). TABLE 2 Zinc finger domains for construction of 3-finger or 4-finger ZFP libraries.
  • FIG. 7 depicts a method of constructing a diverse three finger library. Nucleic acid fragments encoding each ZFD were individually cloned into the p3 vector to form “single fingered” vectors. Equal amounts of each “single fingered” vector were combined to form a pool. One aliquot of the pool was digested with AgeI and XhoI to obtain digested vector fragments. These vector fragments were treated with phosphatase for 30 minutes. Another aliquot of the pool was digested with XmaI and XhoI to obtain segments encoding single fingers.
  • the digested vector nucleic acids from the AgeI and XhoI digested pool were ligated to the nucleic acid segments released from the vector by the XmaI and XhoI digestion.
  • the ligation generated vectors that each encodes two zinc finger domains.
  • approximately 1.4 ⁇ 10 4 independent transformants were obtained, thereby forming a two-fingered library.
  • the size of the insert region of the two-fingered library was verified by PCR analysis of 40 colonies. The correct size insert was present in 95% of the library members.
  • the 3-F libraries were screened to identify chimeric zinc finger proteins that impair the growth of yeast cells.
  • This screen uses the GAL promoter to conditionally express the chimeric zinc finger proteins.
  • Previous studies have used the GAL promoter to yeast cDNA and genomic DNA sequences to identify genes whose overexpression is lethal (Liu et al., (1992) Genetics 132:665-673; Ramer et al. (1992) Proc. Natl. Acad. Sci. USA 89:11589-11593; Espinet et al., (1995) Yeast 11:25-32; Akada et al., (1997) Mol. Gen. Genet 254:267-274; Stevenson et al., (2001) Proc. Natl. Acad. Sci. USA. 98:3946-3951).
  • Yeast strain YPH499a was transformed with nucleic acid from the 3-F libraries. Transformants were grown for two days at 30° C. on plates containing synthetic minimal medium that lack tryptophan and that include glucose. Each of these glucose plates was replica-plated onto a galactose plate and a second glucose plate. The replica plates were grown overnight at 30° C. Then, each galactose plate was compared to its corresponding glucose replica plate. Colonies were identified that did not grow on galactose, but grew on glucose. These colonies were recovered from the glucose plate and retested by streaking selected colonies on galactose media. Library plasmids were rescued from each colony that retested.
  • the plasmids were retransformed into YPH499a to confirm that the growth defect is caused by expression of the zinc finger protein.
  • Plasmids were recovered from ten colonies that were growth-defective on galactose.
  • the plasmids (L1 to L10) were retransformed into yeast cells. All ten plasmids retested. Cells transformed with the recovered plasmids were unable to grow on galactose media but were able to grow on glucose media.
  • the zinc finger proteins encoded by these ten plasmids were characterized by DNA sequencing (Table 4). The potential target DNA binding sites for these proteins were inferred from information about the binding specificities of the component zinc finger domains.
  • ZFPs encoded in plasmids isolated from growth-defective transformants Name of ZFD (N to C) Potential target No 1 2 3 sequence L1 RSHR CSNR1 RDHT 5′-NGG GAV GGG-3′ (SEQ ID NO:200) L2 RSNR RDHT TDKR 5′-GGK NGG GAG-3′ (SEQ ID NO:201) L3 RDHT QSHR3 RDHT 5′-NGG GRA NTT-3′ (SEQ ID NO:202) L4 QSDR RDHT RSNR 5′-GAANGG GCT-3′ (SEQ ID NO:203) L5 CSNR1 RDTN VSSR 5′-GTD AAG GAV-3′ (SEQ ID NO:204) L6 RDHT RDHT RDER1 5′-GHG NGG KGG-3′ (SEQ ID NO:205) L7 QSNR1 RDTN QTHQ 5′-HGA AAG GAA-3′ (SEQ ID NO:206) L8
  • Ketoconazole is an orally absorbed antimycotic imidazole drug. It can be administered for the treatment of certain mucoses. Ketoconazole blocks the biosynthesis of ergosterol in yeasts and other fungi (Burden et al., (1989) Phytochemistry 28:1791-1804) and has additional effects on cellular metabolism (Kelly et al., (1992) In Fernandes, P. B. (Ed.) New Approaches for Antifungal Drug, Birkhäuser, Boston, pp.155-187).
  • the resistant phenotype was verified by plasmid rescue.
  • the plasmids were isolated, transformed into E. coli for amplification, and retransformed into yeast strain YPH499a (Ausubel, et al. (Eds) (1995) Current Protocols in Molecular Biology John Wiley and Sons Ltd, New York). Equal numbers of retransformants were spotted onto synthetic galactose agar plates with or without 35 ⁇ M ketoconazole. The retransformants were also spotted onto synthetic glucose agar plates with or without ketoconazole to verify that the drug resistance was induced by galactose-inducible expression of zinc finger protein.
  • K1:QSHV-QFNR-RSHR-Ume6 includes the following amino acid sequence: MGKPIPPLLGLNSTQAMGAPPKKKRKVGIRIPGEKPECDHCGKSFSQSSHLNVHKRTHTGEKP YKC (SEQ ID NO:221) HQCGKAFIQSFNLRRHERTHT GEKP YKCMECGKAFNRRSHLTRHQRIH AAAA NSASSSTKLDDDLGTA AAVLSNMRSSPYRTHDKPISNVNDMNNTNALGVPASRPHSSSFPSKGVLRPILLRIHNSEQQPIFESN NSTACI
  • K2 RSNR-RSNR-QSSR1-QSHT-Ume6 includes the following amino acid sequence: MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKP YICRKCGRGFSRKSNLIRHQRTH TGEKPYIC (SEQ ID NO:222) RKCGRGFSRKSNLIRHQRTHTGEKP YKCPDCGKSFSQSSSLIRHQRTH TGEKP YKCEECGKAFRQSSH LTTHKIIH AAAA NSASSSTKLDDDLGTAAAVLSNMRSSPYRTHDKPISNVNDMNNTNALGVPASRPHS SSFPSKGVLRPILLRIHNSEQQPIFESNNSTACI
  • K3 RSNR-RSNR-QGTR-QSHR5-Ume6 includes the following amino acid sequence: MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKP YICRKCGRGFSRKSNLIRHQRTH TGEKP YIC (SEQ ID NO:223) RKCGRGFSRKSNLIRHQR THTGEKP FQCRICMRNFSQRGTLTRHIRTH TGEKP YVCRECGRGFRQHSH LVRHKRTH AAAA NSASSSTKLDDDLGTAAAVLSNMRSSPYRTHDKPISNVNDMNNTNALGVPASRPHS SSFPSKGVLRPILLRIHNSEQQPIFESNNSTACI
  • K4 RSNR-RSNR-QGTR-QTHQ-Ume6 includes the following amino acid sequence: MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKP YICRKCGRGFSRKSNLIRHQRTH TGEKP YIC (SEQ ID NO:224) RKCGRGFSRKSNLIRHQRTH TGEKP FQCRICMRNFSQRGTLTRHIRTH TGEKP YECHDCGKSFRQSTH LTQHRRIH AAAA NSASSSTKLDDDLGTAAAVLSNMRSSPYRTHDKPISNVNDMNNTNALGVPASRPHS SSFPSKGVLRPILLRIHNSEQQPIFESNNSTACI
  • K5 VSSR-DGNV-VSSR-VDYK-Gal4 includes the following amino acid sequence: MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKP YTCKQCGKAFSVSSSLRRHETTH TGEKP FQC (SEQ ID NO:225) RICMRNFSDSGNLRVHIRTH TGEKP YTCKQCGKAFSVSSSLRRHETTH GEKP FHCGYCEKSFSVKDY LTKIRTH AAAA NFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSNVHDNEMNNEITTASKIDDGNNSKP LSPGWTDQTAYNAFGITTGMFNTTIMDDVYNYLFDDEDTPPNPKKEISMAYPYDVPDYAS
  • K6 MHHE-QSNR1-VSSR-QGDR-Gal4 includes the following amino acid sequence: MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKP YACPVESCDRRFSMSHHLKEHIRTH GEKP F (SEQ ID NO:226) ECKDCGKAFIQKSNLIRHQRTH TGEKP YTCKQCGKAFSVSSSLRRHETTH TGEKP FQCRICMRNFSQS GDLRRHIRTH AAAA NFNQSGNIADSSLSFIFTNSSNGPNLITTQTNSQALSQPIASSNVHDNFMNNEITASKIDDGN NSKPLSPGWTDQTAYNAFGITTGMFNTTIMDDVYNYLFDDEDTPPNPKKEISMAYPYDVPDYA
  • K7 DGNV-QSHT-QSSRl-DGHR-Gal4 includes the following amino acid sequence: MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKP FQCRICMRNFSDSGNLRVHIRTH TGEKP YKC (SEQ ID NO:227) EECGKAFRQSSHLTTHKIIHT TGEKP YKCPDCGKSFSQSSSLIRHQRTH TGEKP FQCRICMRNFSDPGH LVRHIRTH AAAA NFNQSGNIADSSLSFTFTNSSNGPNLITTQTNSQALSQPIASSNVHDNFMNNETTASKIDDGNNSK PLSPGWTDQTAYNAFGITTGMFNTTTMDDVYNYLFDDEDTPPNPKKEISMAYPYDVPDYA
  • K8 DGAR-RDTN-QTHQ-RDTN includes the following amino acid sequence: MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKP FQCRICMRNFSDPGALVRHIRTH TGEKP FQC (SEQ ID NO:228) RICMRNFSRSDTLSNHIRTH TGEKP YECHDCGKSFRQSTHLTQHRRIH TTGEKP FQCRICMRNFSRSDT LSNHIRTH AAAARGMHLEGRIM
  • K9 RDHT-QTHQ-QSHT-DGNV includes the following amino acid sequence: (SEQ ID NO:229) MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKP FQCKTCQRKFSRS DHLKTHTRTHTGEKP YECHDCGKSFRQSTHLTQH RRIHTGEKP YKCEECG KAFRQSSHLTTHKIIH TGEKP FQCRICMRNFSDSGNLRVHIRTH AAAARG MHLEGRIM
  • K10: RDHT-QTHQ-QSHT includes the following amino acid sequence: (SEQ ID NO:230) MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKP FQCKTCQRKFSRS DHLKTHTRTH TGEKP YECHDCGKSFRQSTHLTQHRRIH TGEKP YKCEECG KAFRQSSHLTTHKIIH AAAARGMHLEGRIM
  • K11: RDHT-QSHV-QSHV includes the following amino acid sequence: (SEQ ID NO:231) MGKPIPNPLLGLNSTQAMGAPPKKKRKVGIRIPGEKP FQCKTCQRKFSRS DHLKTHTRTH TGEKP YECDHCGKSFSQSSHLNVHKRTH TGEKP YECDHCG KSFSQSSHLNVHKRTH AAAARGMHLEGRIM
  • zinc finger proteins that include two, three, or more zinc finger domains that have the same general motifs (i.e., set of four DNA contacting residues) and that are arranged in the same consecutive order as any of the proteins described above may also confer drug resistance to a fungal cell.
  • K2, K3, and K4 ZFPs may bind to the same target site in vivo and regulate the same target genes.
  • the zinc finger protein encoded by the K10 clone is closely related to those encoded in the K9 and K11 clones.
  • the QTHQ, QSHT, and QSHV fingers found in K9, K10, and K11 can recognize the same 3-bp DNA site:5′-HGA-3′.
  • the K9, K10, and K11 ZFPs may bind to the same target site in vivo and regulate the same target genes.
  • all the ZFPs include the same type of regulatory features.
  • the K2, K3, and K4 clones each include the Ume6 repression domain.
  • the K9, K10, and K11 clones each function without a dedicated transcriptional regulatory domain.
  • ZFP mutants were constructed that alter a DNA contacting residue or that remove or replace a regulatory domain.
  • the asparagine DNA contacting residue of the second zinc finger of K5 was mutated to alanine. Gel shift assays demonstrated that this mutated ZFP had at least 10-fold decrease in DNA-binding affinity for its expected DNA site.
  • This mutant K5 protein does not confer drug resistance to yeast cells.
  • the Gal4 activation domain of the K5 zinc finger protein was deleted by inserting a stop codon in front of the DNA sequence encoding the activation domain. This protein also does not confer resistance to ketoconazole. Similar results were obtained for the other ketoconazole resistance ZFPs.
  • replacements that switch functional directionality include replacing one type of regulatory domain with another type or regulatory domain and removing regulatory domain.
  • K5 ZFP a protein that increases drug sensitivity was obtained by screening for a protein that increased drug resistance and replacing its transcriptional activation domain with a transcriptional repression domain.
  • DNA microarray analysis was used to identify genes associated with the drug resistance phenotype. We reasoned that different ZFPs conferring the identical phenotype may regulate identical gene sets whose differential expression is directly or indirectly associated with the phenotype. Three ZFPs—K5, K6, and K7—were chosen for expression profiling analyses. All three transcription factors contain the Gal4 activation domain. Out of 6,400 yeast open reading frames, ten were activated over 2 fold by at least two different ZFP transcription factors, and four open reading frames were activated by all three tested ZFP transcription factors. The four activated open reading frames are: YLL053C, YJR147W, YLL052C, and YPL091W.
  • Table 7 lists the number of genes that are regulated by more than one chimeric ZFP. TABLE 7 Coordinately Regulated Genes Number of Commonly K5 K6 K7 Regulated Genes + ⁇ ⁇ 39 + + ⁇ 1 + ⁇ + 2 + + + 4 ⁇ + ⁇ 95 ⁇ + + 3 ⁇ ⁇ + 126
  • PDR5 a gene known to pump out ketoconazole
  • YLL053C induced ketoconazole resistance when overexpressed on its own. See Table 6, above. YLL053C is homologous to plasma membrane and water channel proteins from Candida albicans.
  • amino acid sequence of YLL053C is as follows: (SEQ ID NO:232) MWFPQIIAGMAAGGAASAMTPGKVLFTNALGLGCSRSRGLFLEMFGTAVL CLTVLMTAVEKRETNFMAALPIGISLFMAHMALTGYTGTGVNPARSLGAA VAARYFPHYHWIYWISPLLGAFLAWSVWQLLQILDYTTYVNAEKAAGQKK ED
  • amino acid sequence of an exemplary homologous channel protein (AQY1) from Candida albicans is as follows: (SEQ ID NO:233) MVAESSSIDNTPNDVEAQRPVYEPKYDDSVNVSPLKNHMIAFLGEFFGTF IFLWVAFVIAQIANQDPTIPDKGSDPMQLIMISFGFGFGVMMGVFMFFRV SGGNLNPAVTLTLVLAQAVPPIRGLFMMVAQMIAGMAAAGAASAMTPGPI AFTNGLGGGASKARGVFLEAFGTCILCLTVLMMAVEKSRATFMAPFVIGI SLFLGHLICVYYTGAGLNPARSFGPCVAARSFPVYHWIYWVGPILGSVIA FAIWKIFKILKYETCNPGQDSDA
  • the YLL053C gene product may confer resistance by pumping out ketoconazole as does the PDR5 gene product.
  • yeast cells containing nucleic acids from the 3-fingered libraries were cultivated on SD synthetic liquid medium with 2% galactose for 3 hrs at 30° C. and then incubated at 52° C. (air temperature) with slow gyration for 2 hrs. After heat treatment, the culture were plated on galactose media and incubated for 5 days at 30° C. Growing yeast colonies were suspended in galactose liquid media and incubated at 52° C. for 2 hrs to confirm the thermotolerant phenotype. Plasmids were isolated from these cells and retransformed into yeast as describe above. The retransformants were cultivated in galactose liquid media for 3 hrs at 30° C. and incubated at 52° C.
  • thermotolerance was induced by the expression of zinc finger protein in the mutants.
  • retransformants were cultivated in SD glucose liquid media with the same condition described above and then spotted onto SD glucose agar plates.
  • the transformants with pYTC vector and plasmid encoding randomly-picked 3-fingered proteins were used as controls for the procedure.
  • Thermotolerant yeast cells were identified from the cells transformed with zinc finger protein expression plasmids. Wild-type cells carrying the pYTC-Lib plasmid or plasmid encoding a randomly-selected zinc finger protein were used as negative controls. A total of 10 7 cells were grown in galactose liquid media. Galactose induces zinc finger protein expression. Cells were heat treated at 52° C. for 2 hrs and then plated on galactose minimal agar plates. After five days of incubation at 30° C., 26 colonies grew on the plates. We rescued plasmids from these colonies and retransformed them into YPH499a. We isolated nine clones that induced varying degrees of thermotolerance.
  • ZFP-Tf libraries were constructed using 40 and 25 zinc finger domains, respectively.
  • the three-finger and four-finger libraries recognize approximately 9- and 12-bp DNA binding sites, respectively.
  • ZFPs were expressed as fusion proteins to the p65 transcriptional activation domain and to the KRAB repression domain.
  • Mouse neuroblastoma Neuro2a cells were maintained in MEM-a media with 10% FBS and antibiotics at 37° C., in a humidified atmosphere containing 95% air and 5% CO 2 .
  • Cells were seeded at 8.0 ⁇ 10 3 cells per 96-well culture plate and then were transfected using LIPOFECTAMINE PLUSTM reagent (Invitrogen,CA) according to the manufacturer's protocol, with 50 ng of ZFP, together with 20 ng of LacZ reporter plasmid.
  • LIPOFECTAMINE PLUSTM reagent Invitrogen,CA
  • In vitro differentiation was carried out with or without retinoic acid (RA) (10 ⁇ M) respectively, and G418 (1 mg/ml) was treated 24 hours after transfection to reduce the number of untransfected cells.
  • RA retinoic acid
  • Neuro2A cells were transiently transfected with library plasmids and a reporter plasmid that includes the LacZ gene. LacZ expression was used to visualize the morphology of transfected cells. Because the differentiating cells grow slower than non-differentiating cells and the latter will dominate the cell population, we treated the cell culture with G418 24 hr after transfection to reduce the number of untransfected cells. After five days, cells were fixed and LacZ-stained. We then characterized the cells by their morphological characteristics. In particular, we identified cells with increased neurite length and thickness.
  • ZFP-TFs that alter neuritogenesis. These ZFP-TFs affect neuritogenesis to varying degrees.
  • the ZFP-TF named Neuro1-p65, had the most prominent effect on differentiation, as measured by the length and thickness of neurites that it produced.
  • a marked acceleration of neuritogenesis in the Neuro1-p65-transfected Neuro2A cell was also observed when cells were treated with 10 ⁇ M RA (retinoic acid).
  • the nucleic acid sequence encoding Neuro1-p65 and its amino acid sequence is shown in FIG. 14.
  • the Neuro1-p65 nucleic acid encodes a chimeric ZFP that includes the zinc finger domains QSNR1-QSNK-CSNR1.
  • Other ZFPs with the same motifs and/or ZFPs that binding to a site that at least partially overlaps the Neuro1-p65 binding site are also expected to modulate differentiation.
  • the predicted binding site for Neuro1 is 5′-GACGAAGAA-3′.
  • Neuro1-p65 requires the p65 activation domain to induce neurite formation.
  • the same zinc finger domains fused to the KRAB transcriptional repression domain or without any effector domain do not drive neuritogenesis. See FIG. 8 and Table 9.
  • the DNA binding ability of Neuro1-p65 is critical, since mutations that are predicted to disable its DNA binding ability of the zinc finger domains of Neuro1-p65 (Neuro1-p65mut) abolished its ability to support neuritogenesis.
  • Real-time PCR is used to characterize the expression level of neuronal marker genes during the course of Neuro1-p65 expression in cells.
  • nucleic acid microarrays are used to compare the pattern of gene expression between RA-treated cells and ZFP-TF treated cells at various time points during differentiation.
  • Neuro1-p65 may activate a pathway that is required for neuroblastoma cell differentiation at least in vitro.
  • the C2C12 cell line is derived from the myoblast lineage, but can differentiate into osteoblasts upon the addition of bone morphogenic protein-2 (BMP-2) (Katagiri, T. et al., (1994) J. Cell. Biol. 127, 1755).
  • BMP-2 bone morphogenic protein-2
  • Several naturally-occurring transcription factors have been identified as candidates for controlling this process (Lee, K.-S. et al., (2000) Mol. Cell. Biol. 20:8783; Nakashima, K. et al., (2002) Cell 108:17).
  • Osteo1-p65 is a four-finger protein composed of the RDKR-QTHR1-VSTR-RDKR zinc finger domains (from N- to C-terminus). See FIG. 15. This protein is predicted to recognize the DNA element 5′-GGGGCWRGAGGG-3′ (SEQ ID NO:234).
  • the mouse myoblast cell line, C2C12 was maintained in Dulbecco's Modified Eagle's Medium (DMEM) with 4.5 g/L glucose, 10% FBS, and antibiotics, at 37° C., in a humidified atmosphere containing 95% air and 5% CO 2 .
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS FBS
  • antibiotics at 37° C.
  • Cells were seeded at 1.0 ⁇ 10 4 cells per 96-well culture plates.
  • the cells were transfected with 50 ng of library nucleic acid using LIPOFECTAMINE PLUSTM (Invitrogen) according to the manufacturer's protocol. Twenty-four hours after transfection, the growth medium was replaced with DMEM containing 2% FBS, and the cells were cultured for an additional six days.
  • 08_D04-p65 its derivatives, and similarly functional zinc finger proteins can be used as therapeutics for diabetes.
  • DNA encoding 08_D04-p65 or a similarly functional zinc finger protein can be delivered into diabetic patients by viral delivery or in encapsulated form (e.g., a liposome). Once DNA is delivered into cells, the zinc finger protein can be expressed to induce the production of insulin.
  • the nucleic acid encoding the zinc finger protein can be operably linked to an inducible promoter, e.g., a Tet-inducible promoter. The use of doxycycline as an inducer enables the level of insulin production to be regulated by a small chemical. Because insulin-inducing zinc finger proteins, such as 08_D04-p65, can function in different human cell lines, it may work in both pancreatic cells (e.g., beta cells and non-beta cells) and non-pancreatic cells.
  • pancreatic cells e.g., beta cells and non-beta cells
  • Cells can be engineered to express such a transcription factor and a transcription factor that induces the insulin gene, e.g., 08_D04-p65.
  • insulin-inducing zinc finger proteins can be also used for ex vivo cell therapy.
  • cells from a patient are modified in vitro by the introduction of a nucleic acid encoding an insulin-inducing zinc finger protein. The modified cells are then transplanted back into patients or into another subject.
  • Nucleic acids encoding zinc finger proteins were stably introduced into FlpTRex-293 cell lines (Invitrogen) essentially as described in the manufacturer's protocols. Briefly, the HindIII-XhoI fragment from the pLFD-p65 or pLFD-Kid vectors was subcloned into pCDNA5/FRT/TO (Invitrogen CA). This fragment includes a sequence encoding the zinc finger proteins. Resulting plasmids were cotransfected into FlpTRex-293 cells with pOG44 (Invitrogen). Stable integrants that express ZFP-TFs upon doxycycline induction were obtained.
  • DNA microarrays containing 7458 human EST clones were obtained from Genomictree (Korea). FlpTRex-293 cells stably expressing ZFP-TFs were grown with (+Dox) or without ( ⁇ Dox) 1 ⁇ g/ml Doxycycline for 48 hours. Total RNA was prepared from each sample. RNA from the ⁇ Dox sample was used as a reference (Cy3), and +Dox as the experimental sample (Cy5). Microarray analysis was performed according to the manufacturer's protocols. Spots with sum of median less than 500 were colored red and not analyzed.
  • Nucleic acids encoding certain zinc finger proteins were also expressed in HeLa cells.
  • pLFD-p65 containing 08_D04 ZFP was transiently transfected into HeLa cells using LIPOFECTAMINETM 2000 (Invitrogen).
  • the pLFD-p65 vector, which does not encode a zinc finger protein, was also transfected into cells in parallel as a control.
  • Two ZFP libraries were constructed as four finger proteins and three finger proteins in P3, a modified pcDNA3 vector. Construction of these libraries is described above.
  • pSEAP2-control (Clontech) plasmid was co-transfected with 50 ng of plasmids containing zinc finger protein gene fused to the functional domains into cells cultured at 1 ⁇ 10 4 cells/well. The transfected cells were incubated at 37° C. for three days before assays.
  • luciferase assay 5 ng of both CMV-luciferase and pRL-SV40 plasmid were co-transfected with 50 ng of ZFP coding plasmids to 293 cells seeded in wells of a 96 well plate. After 3 days of incubation, cells were harvested and the luciferase assay was done by a Dual Luciferase reporter assay system (Promega) as recommended by the manufacturer.
  • MTT assay is used to measure cell growth. Tetrazolium MTT reduced by metabolically active cells changes to a yellow color. Measurement of light absorbance by the yellow color indicates the number of viable cells. MTT assay was done using a MTT assay kit (Trevigen). The MTT assay was done at three days after transfection. Briefly, 10 ⁇ l of MTT solution was added directly to the culture medium and incubated for two hours in a C0 2 incubator. Then, 100 ⁇ l of detergent solution from the MTT assay kit was added and incubated in the dark for two hours before measurement of absorbance at 570 nm was measured using a Power-Wave 340 ⁇ (Bio-Tek Instrument).
  • the P_B08 a zinc finger protein fused to the p65 domain showing the highest SEAP activity. This ZFP was retested by transfection with the SEAP plasmid in three separate transfections. Table 11 illustrates one such retesting. The P value for P_B08 in this assay is 0.008. P_B08 exhibited approximately 16-fold increase of SEAP activity compared to that of the parental plasmid. TABLE 11 SV40-SEAP reporter in 293 cells. Expression Plasmid average st. dev. P_B08 16.51 0.54 Vector(HANLS) 1 0.17
  • SEAP assay was used to assay the production of foreign proteins, reasons for the observed increased SEAP activity could be various, for example, the direct or indirect activation of a SV40 promoter used for the SEAP gene expression in pSEAP2-Control plasmid. Other reasons include: increased activity of proteins functioning in secretion pathway or activation of the general machinery for the protein production.
  • the mechanism of enhanced activity of SEAP can be elucidated by determining the effect of P_B08 on the production of other proteins, e.g, using other reporter proteins (e.g., unsecreted reporters) and reporters linked to other promoters.
  • K44 — 11_D01 showed activation of SV40-SEAP but not the CMV-Luciferase, suggesting that the activation may be related to mechanisms specific to SEAP such as secretion.
  • K44 — 11_G12 showed approximately 2-3 fold activation for both CMV-luciferase and SV40-SEAP, suggesting that the mechanism is specific to neither promoter nor reporter.
  • K44 — 11_G12 may be used increase the production of proteins generally in cells, e.g., eukaryotic cells. TABLE 12 CMV-SEAP, CMV-Luc and SV40-SEAP reporter in 293F cells. average st.dev.
  • Zinc finger proteins K44-16-E12 (FIG. 25) and K12_A11 (FIG. 24) may also alter protein production, e.g., as indicated by CMV-SEAP and SMV-Luc expression.
  • cyclin D1 has been reported to cause to increase production of some proteins (U.S. Pat. No. 6,210,924 B1).
  • a zinc finger protein that can directly or indirectly activate the cyclin D1 expression may up-regulate protein production, although the effects of a zinc finger protein may be different, far more broad ranging, or more potent.
  • Other zinc finger proteins resulting in a physiological effect similar to cyclin D1 overexpression can be isolated by this assay.
  • Such zinc finger proteins can be used to enhance production of the protein of interest.
  • zinc finger proteins that can alter the rate of cell growth and proliferation. These important phenotypes are linked to diseases such as cancer and viral infection and to developmental processes.
  • the ability to use zinc finger protein to control cell growth and proliferation can include, for example, controlling apoptosis, cell differentiation, host cell defenses (e.g., against viral infection), and p53-mediated signaling
  • a zinc finger protein that produces one or more of these phenotypes may directly or indirectly regulate a gene that regulates cell growth or signaling.
  • the specific zinc finger proteins describe here produce detectable effects to cellular growth as detected by the MTT assay. For example, two zinc finger proteins showed at least a two-fold difference of cell growth compared to the controls. Considering that this assay was based on observations of transiently transfected cells three days after transfection, the two-fold difference is significant. In addition, the change in the cell number confirms that that the difference detected using the MTT assay is not particular to the assay.
  • the cDNA microarrays can be used profile the gene expression patterns of cells that expressing each of these zinc finger proteins. The profiles can identify novel genes and pathways that participate in cell proliferation or apoptosis.
  • the expression plasmids for these zinc finger proteins include an IPTG-inducible promoter. Transformants that express each of the hexane resistance inducing ZFPs were characterized in the presence and absence of IPTG. Transformants expressing HT-2 showed higher hexane tolerance in the presence of IPTG, whereas HT-1 or HT-3 expressing cells were hexane tolerant even in the absence of IPTG. TABLE 14 Zinc finger proteins that confer hexane tolerance in E . coli No.
  • F1 F2 F3 F4 DNA target (##) HT-1 RSHR HSSR ISNR GAH GTT GGG 5 HT-2 ISNR RDHT QTHR VSTR GCT GRA NGG GAH 3 (SEQ ID NO:235) HT-3 QNTQ CSNR ISNR GAH GAV ATA 1
  • Plac-UV5 was amplified from pBT-LGF2 by PCR (forward primer:5′-GACA ACC GGT CAT CGA TAA GCT AAT TCT CAC-3′ (SEQ ID NO:236); reverse primer: 5′-TTG TCC ATG GAC GCT GTT TCC TG GTG AAA-3′ (SEQ ID NO:237)).
  • the PCR product was cloned into the pYTC Lib vector between Age1 and NcoI site.
  • the vector was named as pYTC-lac.
  • pYTC-lac was digested with ClaI and NotI to subclone the DNA fragment containing the following elements: Plac promoter-V5 epitope-MCS.
  • the digested DNA fragment was purified after gel-electrophoresis and subcloned into the ClaI and NotI sites of pBT-LGF2.
  • the resulting vector was named as pZL1.
  • the 3F- or 4F-ZFP library constructed in pYTC-Lib was digested with EcoRI and NotI.
  • the digested DNA fragments were gel-purified and subcloned into EcoRI and NotI site of pZL1, thus providing a library for E. coli expression.
  • Plasmids were purified from the pool of growing colonies and transformed into DH5 ⁇ . The transformants were treated with hexane as described above. Selection for hexane tolerance was repeated two additional times. Plasmids were recovered from 20 individual colonies that could grow on LB plates with chloramphenicol (34 ⁇ g/ml) after the third round of selection. These plasmids were retransformed into DH5 ⁇ . Each transformant was retested for hexane-tolerance as described above. Plasmids that induce hexane tolerance were sequenced to characterize the encoded zinc finger protein.
  • thermotolerance phenotype that is, the percentage of cells expressing ZFP-TFs that survive under stress conditions (6.3%) divided by the percentage of C1 that survived under the same conditions (0.0085%). Since the expression of ZFP is induced by IPTG, transformants of ZFPs that induce thermo-tolerance were analyzed for their phenotype in the presence or absence of IPTG. Transformants of T-1 or T-10 showed higher thermo-tolerance in the presence of IPTG. TABLE 15 ZFPs that confer thermotolerance.
  • Plasmids were purified from the pool of growing colonies and transformed into DH5 ⁇ . Selection for thermotolerance was repeated with retransformants. Plasmid was purified from 30 individual colonies that could grow on LB+chloramphenicol plate (34 ⁇ g/ml) after third round of selection and retransformed into DH5 ⁇ . Each transformant was analyzed for thermo-tolerance as described above. Plasmids that could induce thermo-tolerance were sequenced to identify ZFP.
  • the zinc finger protein F121_p65 was expressed in human embryonic kidney 293 cells. Transcripts from the cells were profiled and compared transcripts profiled from a corresponding control cell that does not express F121_p65. Examples of transcripts up-regulated regulated by F121_p65 are listed in Table 16. F121_p65 increases transcription of insulin-like growth factor 2, among other genes.
  • Akt1 expressed in E. coli.
  • the mammalian Akt1 gene was cloned in pET21b vector and GFP was ligated to the C-terminal of the Akt1 ORF.
  • the nucleic acid library encoding different zinc finger proteins was transformed into E. coli cells.
  • the Akt1:GFP construct was introduced into ZFP transformants. Increased solubility of Akt1 was analyzed by FACS sorting, since soluble Akt1 expressing cells produce more fluorescent GFP.
  • Sorted cell were overnight cultured in LB media with chloramphenicol (34 ⁇ g/ml) and ampicillin (50 ⁇ g/ml). Plasmids were purified from the culture and transformed into DH5 ⁇ . Screening for enhanced solubility of recombinant protein was repeated with retransformants. Plasmid was purified from individual colonies that could grow on LB+chloramphenicol (34 ⁇ g/ml)+ampicillin (50 ⁇ g/ml) plate after eighth round of selection and retransformed into DH5 ⁇ . Each transformant was analyzed for enhanced solubility of Akt1 with western blot analysis. Plasmids that could increase solubility of Akt1 were sequenced to identify ZFP.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Cell Biology (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Bioinformatics & Computational Biology (AREA)
  • Crystallography & Structural Chemistry (AREA)
  • Botany (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Peptides Or Proteins (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
US10/314,669 2001-12-07 2002-12-09 Phenotypic screen of chimeric proteins Abandoned US20030194727A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US10/314,669 US20030194727A1 (en) 2001-12-07 2002-12-09 Phenotypic screen of chimeric proteins
US10/669,861 US7514257B2 (en) 2001-12-07 2003-09-24 Zinc finger transcription factor differentiation proteins
US10/746,864 US20040259258A1 (en) 2001-12-07 2003-12-23 Regulation of prokaryotic gene expression with zinc finger proteins

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US33844101P 2001-12-07 2001-12-07
US37605302P 2002-04-26 2002-04-26
US40090402P 2002-08-02 2002-08-02
US40108902P 2002-08-05 2002-08-05
US10/314,669 US20030194727A1 (en) 2001-12-07 2002-12-09 Phenotypic screen of chimeric proteins

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US10/669,861 Continuation-In-Part US7514257B2 (en) 2001-12-07 2003-09-24 Zinc finger transcription factor differentiation proteins

Publications (1)

Publication Number Publication Date
US20030194727A1 true US20030194727A1 (en) 2003-10-16

Family

ID=27502584

Family Applications (2)

Application Number Title Priority Date Filing Date
US10/314,669 Abandoned US20030194727A1 (en) 2001-12-07 2002-12-09 Phenotypic screen of chimeric proteins
US10/669,861 Expired - Fee Related US7514257B2 (en) 2001-12-07 2003-09-24 Zinc finger transcription factor differentiation proteins

Family Applications After (1)

Application Number Title Priority Date Filing Date
US10/669,861 Expired - Fee Related US7514257B2 (en) 2001-12-07 2003-09-24 Zinc finger transcription factor differentiation proteins

Country Status (8)

Country Link
US (2) US20030194727A1 (enExample)
EP (1) EP1451297A4 (enExample)
JP (1) JP2005511049A (enExample)
KR (1) KR20040065235A (enExample)
CN (1) CN100463962C (enExample)
AU (1) AU2002365796A1 (enExample)
CA (1) CA2469477A1 (enExample)
WO (1) WO2003048345A1 (enExample)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030165997A1 (en) * 2001-08-17 2003-09-04 Kim Jin-Soo Zinc finger domain libraries
US20050032186A1 (en) * 2002-12-09 2005-02-10 Kim Jin-Soo Regulatory zinc finger proteins
US20070042378A1 (en) * 2003-12-23 2007-02-22 Kim Jin-Soo Regulation of prokaryotic gene expression with zinc finger proteins
US20080138416A1 (en) * 2006-06-13 2008-06-12 Fmc Biopolymer As Method and systems for using biopolymer-based beads and hydrogels
US7514257B2 (en) 2001-12-07 2009-04-07 Toolgen, Inc. Zinc finger transcription factor differentiation proteins
US20100136663A1 (en) * 2006-10-24 2010-06-03 Korea Advanced Institute Of Science And Technology preparation of an artificial transcription factor comprising zinc finger protein and transcription factor of prokaryote, and a use thereof
CN113643758A (zh) * 2021-09-22 2021-11-12 华南农业大学 面向肠杆科细菌获得抗β-内酰胺类耐药性基因的预测方法

Families Citing this family (77)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030180777A1 (en) * 2002-03-12 2003-09-25 Victor Bartsevich Rapid identification of transcriptional regulatory domains
WO2005005662A2 (en) * 2003-07-08 2005-01-20 Axiogenesis Ag Secreted proteins as markers for cell differentiation
BRPI0514343A (pt) * 2004-08-13 2008-06-10 Anormed Inc combinações de quimiocinas para mobilizar células progenitoras/tronco
US7855279B2 (en) 2005-09-27 2010-12-21 Amunix Operating, Inc. Unstructured recombinant polymers and uses thereof
US20090099031A1 (en) * 2005-09-27 2009-04-16 Stemmer Willem P Genetic package and uses thereof
US20070212703A1 (en) * 2005-09-27 2007-09-13 Stemmer Willem P Proteinaceous pharmaceuticals and uses thereof
US7846445B2 (en) * 2005-09-27 2010-12-07 Amunix Operating, Inc. Methods for production of unstructured recombinant polymers and uses thereof
WO2007058833A2 (en) 2005-11-10 2007-05-24 Pioneer Hi-Bred International, Inc. Dof (dna binding with one finger) sequences and methods of use
AR060523A1 (es) 2006-04-19 2008-06-25 Pioneer Hi Bred Int Moleculas de polinucleotidos aislados que corresponden a alelos mutantes y tipo salvaje del gen de maiz d9 y metodos para usarlas
WO2007137114A2 (en) 2006-05-17 2007-11-29 Pioneer Hi-Bred International, Inc. Artificial plant minichromosomes
BRPI0715393A2 (pt) * 2006-08-07 2013-06-25 Genzyme Corp terapia de combinaÇço
US7642436B2 (en) 2007-04-13 2010-01-05 Ball Horticultural Company Petunia mutant allele
CA2687211A1 (en) 2007-05-25 2008-12-04 Cropdesign N.V. Yield enhancement in plants by modulation of maize alfins
EP2164863A4 (en) 2007-06-15 2010-07-28 Univ Arkansas METHOD FOR THE ADMINISTRATION OF MOLECULES IN CELLS USING A RICIN SUB-UNIT AND RELATED COMPOSITIONS THEREOF
BRPI0815416A2 (pt) * 2007-08-15 2014-10-21 Amunix Inc Composições e métodos para modificar propriedades de polipeptídeos biologicamente ativos
US8829282B2 (en) 2008-05-14 2014-09-09 Monsanto Technology, Llc Plants and seeds of spring canola variety SCV425044
US7964774B2 (en) 2008-05-14 2011-06-21 Monsanto Technology Llc Plants and seeds of spring canola variety SCV384196
US7935870B2 (en) 2008-05-14 2011-05-03 Monsanto Technology Llc Plants and seeds of spring canola variety SCV354718
US7947877B2 (en) 2008-05-14 2011-05-24 Monosanto Technology LLC Plants and seeds of spring canola variety SCV328921
AU2009329893A1 (en) * 2008-12-23 2011-06-30 Vivoscript, Inc. Compositions and methods for re-programming cells without genetic modification
EP2387567B1 (en) 2009-01-14 2014-06-11 The Salk Institute for Biological Studies Compounds that protect against amyloid diseases
WO2010091122A1 (en) * 2009-02-03 2010-08-12 Amunix, Inc. Extended recombinant polypeptides and compositions comprising same
WO2010118477A1 (en) 2009-04-17 2010-10-21 Molecular Plant Breeding Nominees Ltd Plant promoter operable in endosperm and uses thereof
CN102439032B (zh) 2009-05-04 2015-07-22 先锋国际良种公司 通过调节ap2转录因子增加植物中的产量
US8071848B2 (en) 2009-06-17 2011-12-06 Monsanto Technology Llc Plants and seeds of spring canola variety SCV218328
BR112012004094A2 (pt) 2009-08-24 2016-03-08 Amunix Operating Inc composições de fator vii de coagulação e métodos para fazer e usar as mesmas
US8440891B2 (en) 2009-09-22 2013-05-14 Board of Trustees of the University of Akransas, N.A. Rice cultivar CL 142-AR
US8440892B2 (en) 2009-10-15 2013-05-14 Board Of Trustees Of The University Of Arkansas, N.A. Rice cultivar CL 181-AR
EP2529018B1 (en) 2009-12-30 2016-06-22 Pioneer Hi-Bred International, Inc. Methods and compositions for the introduction and regulated expression of genes in plants
AU2010339481B2 (en) 2009-12-30 2016-02-04 Pioneer Hi-Bred International, Inc. Methods and compositions for targeted polynucleotide modification
US20130189780A1 (en) * 2009-12-31 2013-07-25 Fate Therapeutics, Inc. Reprogramming compositions
US8148611B2 (en) 2010-02-26 2012-04-03 Monsanto Technology Llc Plants and seeds of spring canola variety SCV453784
US8143488B2 (en) 2010-02-26 2012-03-27 Monsanto Technoloy LLC Plants and seeds of spring canola variety SCV470336
US8138394B2 (en) 2010-02-26 2012-03-20 Monsanto Technology Llc Plants and seeds of spring canola variety SCV431158
US8581048B2 (en) 2010-03-09 2013-11-12 Monsanto Technology, Llc Plants and seeds of spring canola variety SCV119103
US8153865B2 (en) 2010-03-11 2012-04-10 Monsanto Technology Llc Plants and seeds of spring canola variety SCV152154
BR112012028052A2 (pt) 2010-05-06 2019-09-24 Du Pont polipeptídeo, polinucleotídeo, cassete de expressão,planta e método de melhoramento de tolerância a seca em uma planta.
US8513487B2 (en) 2011-04-07 2013-08-20 Zenon LISIECZKO Plants and seeds of spring canola variety ND-662c
US8513494B2 (en) 2011-04-08 2013-08-20 Chunren Wu Plants and seeds of spring canola variety SCV695971
US8507761B2 (en) 2011-05-05 2013-08-13 Teresa Huskowska Plants and seeds of spring canola variety SCV372145
US8513495B2 (en) 2011-05-10 2013-08-20 Dale Burns Plants and seeds of spring canola variety SCV291489
US8785729B2 (en) 2011-08-09 2014-07-22 Nunhems, B.V. Lettuce variety redglace
US8754293B2 (en) 2011-09-09 2014-06-17 Nunhems B.V. Lettuce variety intred
WO2013096810A1 (en) 2011-12-21 2013-06-27 The Curators Of The University Of Missouri Soybean variety s05-11482
WO2013096818A1 (en) 2011-12-21 2013-06-27 The Curators Of The University Of Missouri Soybean variety s05-11268
US9380756B2 (en) 2012-01-04 2016-07-05 Nunhems B.V. Lettuce variety multigreen 50
LT2822577T (lt) 2012-02-15 2019-03-25 Bioverativ Therapeutics Inc. Rekombinantiniai faktoriaus viii baltymai
HRP20221531T1 (hr) 2012-02-15 2023-02-17 Bioverativ Therapeutics Inc. Pripravci faktora viii i postupci dobivanja i korištenja istih
US8859857B2 (en) 2012-04-26 2014-10-14 Monsanto Technology Llc Plants and seeds of spring canola variety SCV259778
US8835720B2 (en) 2012-04-26 2014-09-16 Monsanto Technology Llc Plants and seeds of spring canola variety SCV967592
US8878009B2 (en) 2012-04-26 2014-11-04 Monsanto Technology, LLP Plants and seeds of spring canola variety SCV318181
US8802935B2 (en) 2012-04-26 2014-08-12 Monsanto Technology Llc Plants and seeds of spring canola variety SCV942568
CN102816214B (zh) * 2012-07-27 2014-08-13 中国人民解放军军事医学科学院生物工程研究所 乙型肝炎病毒HBx基因的人工转录因子及其应用
CN102994437B (zh) * 2012-11-06 2014-04-02 大连理工大学 高产木质纤维素降解酶的链霉菌人工锌指蛋白突变菌株及其获得方法
EP3033097B1 (en) 2013-08-14 2021-03-10 Bioverativ Therapeutics Inc. Factor viii-xten fusions and uses thereof
WO2016007640A1 (en) 2014-07-10 2016-01-14 Benson Hill Biosystems, Inc. Compositions and methods for increasing plant growth and yield
MX2018001497A (es) 2015-08-03 2018-05-15 Bioverativ Therapeutics Inc Proteinas de fusion de factor ix y metodos para producirlas y usarlas.
IL293963A (en) 2015-09-01 2022-08-01 Univ California Modular polypeptide libraries and methods of making and using same
US10947554B2 (en) 2015-10-23 2021-03-16 Donald Danforth Plant Science Center Expression of dicarboxylate transporter from setaria italica in transgenic plants to increase yield
EP3372985B1 (en) 2015-10-28 2025-06-25 The University Of Tokyo Analysis device
US11330776B2 (en) 2015-10-30 2022-05-17 Pioneer Hi-Bred International, Inc. Methods and compositions for rapid plant transformation
WO2017078836A1 (en) 2015-11-06 2017-05-11 Pioneer Hi-Bred International, Inc. Methods and compositions of improved plant transformation
WO2018102743A1 (en) 2016-12-02 2018-06-07 Bioverativ Therapeutics Inc. Methods of treating hemophilic arthropathy using chimeric clotting factors
AU2019234474B2 (en) 2018-03-12 2025-05-15 Pioneer Hi-Bred International, Inc. Methods for plant transformation
RS66972B1 (sr) 2018-05-18 2025-07-31 Bioverativ Therapeutics Inc Metode lečenja hemofilije a
WO2019241443A1 (en) 2018-06-13 2019-12-19 Thinkcyte Inc. Methods and systems for cytometry
US20210277409A1 (en) 2018-06-28 2021-09-09 Pioneer Hi-Bred International, Inc. Methods for selecting transformed plants
CN113226333A (zh) 2018-10-02 2021-08-06 桑格摩生物治疗股份有限公司 用于调控Tau蛋白的方法和组合物
WO2020081819A1 (en) * 2018-10-18 2020-04-23 Thinkcyte Inc. Methods and systems for target screening
JP2022512817A (ja) 2018-10-31 2022-02-07 パイオニア ハイ－ブレッド インターナショナル， インコーポレイテッド オクロバクテリウム（Ｏｃｈｒｏｂａｃｔｒｕｍ）媒介植物形質転換のための組成物及び方法
EP4083603A4 (en) 2019-12-27 2024-04-03 ThinkCyte, Inc. FLOW CYTOMETER PERFORMANCE EVALUATION METHODS AND STANDARD PARTICLE SUSPENSION
WO2021138560A2 (en) 2020-01-02 2021-07-08 The Trustees Of Columbia University In The City Of New York Programmable and portable crispr-cas transcriptional activation in bacteria
WO2021200911A1 (ja) 2020-04-01 2021-10-07 シンクサイト株式会社 フローサイトメーター
WO2021200960A1 (ja) 2020-04-01 2021-10-07 シンクサイト株式会社 観察装置
CN113077849B (zh) * 2021-03-16 2023-03-31 华南农业大学 一种大肠杆菌β-内酰胺类获得性耐药表型预测复合方法
CN115873900A (zh) * 2021-12-28 2023-03-31 武汉爱博泰克生物科技有限公司 胞外表达人源Ly6d蛋白的重组表达质粒、重组细胞及构建方法
CN114360652B (zh) * 2022-01-28 2023-04-28 深圳太力生物技术有限责任公司 细胞株相似性评价方法及相似细胞株培养基配方推荐方法

Citations (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5773583A (en) * 1988-04-08 1998-06-30 Arch Development Corporation Methods and materials relating to the functional domains of DNA binding proteins
US5789538A (en) * 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
US5866941A (en) * 1995-02-23 1999-02-02 Silicon Systems, Inc. Ultra thin, leadless and molded surface mount integrated circuit package
US5882941A (en) * 1994-05-04 1999-03-16 Massachusette Institute Of Technology Programmable genotoxic agents and uses therefor
US6007988A (en) * 1994-08-20 1999-12-28 Medical Research Council Binding proteins for recognition of DNA
US6107059A (en) * 1992-04-29 2000-08-22 Affymax Technologies N.V. Peptide library and screening method
US6140466A (en) * 1994-01-18 2000-10-31 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6242568B1 (en) * 1994-01-18 2001-06-05 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6326166B1 (en) * 1995-12-29 2001-12-04 Massachusetts Institute Of Technology Chimeric DNA-binding proteins
US20020045158A1 (en) * 2000-02-08 2002-04-18 Casey Case Cells for drug discovery
US20020061512A1 (en) * 2000-02-18 2002-05-23 Kim Jin-Soo Zinc finger domains and methods of identifying same
US20020081614A1 (en) * 1999-09-14 2002-06-27 Sangamo Biosciences, Inc. Functional genomics using zinc finger proteins
US6453242B1 (en) * 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
US20020164575A1 (en) * 1999-09-14 2002-11-07 Sangamo Biosciences, Inc., A Delaware Corporation Gene identification
US20020173006A1 (en) * 1998-03-02 2002-11-21 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US6492117B1 (en) * 2000-07-12 2002-12-10 Gendaq Limited Zinc finger polypeptides capable of binding DNA quadruplexes
US6503717B2 (en) * 1999-12-06 2003-01-07 Sangamo Biosciences, Inc. Methods of using randomized libraries of zinc finger proteins for the identification of gene function
US6511808B2 (en) * 2000-04-28 2003-01-28 Sangamo Biosciences, Inc. Methods for designing exogenous regulatory molecules
US20030021776A1 (en) * 2000-12-07 2003-01-30 Sangamo Biosciences, Inc. Regulation of angiogenesis with zinc finger proteins
US20030044787A1 (en) * 2000-05-16 2003-03-06 Joung J. Keith Methods and compositions for interaction trap assays
US20030044404A1 (en) * 2000-12-07 2003-03-06 Edward Rebar Regulation of angiogenesis with zinc finger proteins
US6534261B1 (en) * 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US20030165997A1 (en) * 2001-08-17 2003-09-04 Kim Jin-Soo Zinc finger domain libraries
US20030166131A1 (en) * 1997-09-18 2003-09-04 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20040209277A1 (en) * 2001-12-07 2004-10-21 Toolgen, Inc Differentiation proteins
US20040214766A1 (en) * 2001-10-01 2004-10-28 Kari Alitalo VEGF-C or VEGF-D materials and methods for treatment of neuropathologies
US20050239203A1 (en) * 1999-01-12 2005-10-27 Case Casey C Regulation of endogenous gene expression in cells using zinc finger proteins

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB9710809D0 (en) * 1997-05-23 1997-07-23 Medical Res Council Nucleic acid binding proteins
ATE355368T1 (de) * 2000-01-24 2006-03-15 Gendaq Ltd Nucleinsäure bindende polypeptide gekennzeichnet durch flexible linker verbundene nucleinsäuredomäne

Patent Citations (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5866325A (en) * 1988-04-08 1999-02-02 Arch Development Corporation Methods and materials relating to the functional domains of DNA binding proteins
US5773583A (en) * 1988-04-08 1998-06-30 Arch Development Corporation Methods and materials relating to the functional domains of DNA binding proteins
US6107059A (en) * 1992-04-29 2000-08-22 Affymax Technologies N.V. Peptide library and screening method
US20030059767A1 (en) * 1994-01-18 2003-03-27 Barbas Iii Carlos F. Zinc finger protein derivatives and methods therefor
US6242568B1 (en) * 1994-01-18 2001-06-05 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US6140466A (en) * 1994-01-18 2000-10-31 The Scripps Research Institute Zinc finger protein derivatives and methods therefor
US5882941A (en) * 1994-05-04 1999-03-16 Massachusette Institute Of Technology Programmable genotoxic agents and uses therefor
US6013453A (en) * 1994-08-20 2000-01-11 Medical Research Council Binding proteins for recognition of DNA
US6007988A (en) * 1994-08-20 1999-12-28 Medical Research Council Binding proteins for recognition of DNA
US5789538A (en) * 1995-02-03 1998-08-04 Massachusetts Institute Of Technology Zinc finger proteins with high affinity new DNA binding specificities
US5866941A (en) * 1995-02-23 1999-02-02 Silicon Systems, Inc. Ultra thin, leadless and molded surface mount integrated circuit package
US6326166B1 (en) * 1995-12-29 2001-12-04 Massachusetts Institute Of Technology Chimeric DNA-binding proteins
US20030166131A1 (en) * 1997-09-18 2003-09-04 Genentech, Inc. Secreted and transmembrane polypeptides and nucleic acids encoding the same
US20020173006A1 (en) * 1998-03-02 2002-11-21 Massachusetts Institute Of Technology Poly zinc finger proteins with improved linkers
US20050215502A1 (en) * 1999-01-12 2005-09-29 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6453242B1 (en) * 1999-01-12 2002-09-17 Sangamo Biosciences, Inc. Selection of sites for targeting by zinc finger proteins and methods of designing zinc finger proteins to bind to preselected sites
US20050130304A1 (en) * 1999-01-12 2005-06-16 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6824978B1 (en) * 1999-01-12 2004-11-30 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US20040204345A1 (en) * 1999-01-12 2004-10-14 Sangamo Biosciences, Inc. Modulation of endogenous gene expression in cells
US6607882B1 (en) * 1999-01-12 2003-08-19 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US6534261B1 (en) * 1999-01-12 2003-03-18 Sangamo Biosciences, Inc. Regulation of endogenous gene expression in cells using zinc finger proteins
US20050239203A1 (en) * 1999-01-12 2005-10-27 Case Casey C Regulation of endogenous gene expression in cells using zinc finger proteins
US20020164575A1 (en) * 1999-09-14 2002-11-07 Sangamo Biosciences, Inc., A Delaware Corporation Gene identification
US20020081614A1 (en) * 1999-09-14 2002-06-27 Sangamo Biosciences, Inc. Functional genomics using zinc finger proteins
US6599692B1 (en) * 1999-09-14 2003-07-29 Sangamo Bioscience, Inc. Functional genomics using zinc finger proteins
US6503717B2 (en) * 1999-12-06 2003-01-07 Sangamo Biosciences, Inc. Methods of using randomized libraries of zinc finger proteins for the identification of gene function
US6689558B2 (en) * 2000-02-08 2004-02-10 Sangamo Biosciences, Inc. Cells for drug discovery
US20020045158A1 (en) * 2000-02-08 2002-04-18 Casey Case Cells for drug discovery
US20020061512A1 (en) * 2000-02-18 2002-05-23 Kim Jin-Soo Zinc finger domains and methods of identifying same
US6511808B2 (en) * 2000-04-28 2003-01-28 Sangamo Biosciences, Inc. Methods for designing exogenous regulatory molecules
US20030044787A1 (en) * 2000-05-16 2003-03-06 Joung J. Keith Methods and compositions for interaction trap assays
US6492117B1 (en) * 2000-07-12 2002-12-10 Gendaq Limited Zinc finger polypeptides capable of binding DNA quadruplexes
US20030044404A1 (en) * 2000-12-07 2003-03-06 Edward Rebar Regulation of angiogenesis with zinc finger proteins
US20030021776A1 (en) * 2000-12-07 2003-01-30 Sangamo Biosciences, Inc. Regulation of angiogenesis with zinc finger proteins
US20030165997A1 (en) * 2001-08-17 2003-09-04 Kim Jin-Soo Zinc finger domain libraries
US20040214766A1 (en) * 2001-10-01 2004-10-28 Kari Alitalo VEGF-C or VEGF-D materials and methods for treatment of neuropathologies
US20040209277A1 (en) * 2001-12-07 2004-10-21 Toolgen, Inc Differentiation proteins

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030165997A1 (en) * 2001-08-17 2003-09-04 Kim Jin-Soo Zinc finger domain libraries
US7514257B2 (en) 2001-12-07 2009-04-07 Toolgen, Inc. Zinc finger transcription factor differentiation proteins
US20050032186A1 (en) * 2002-12-09 2005-02-10 Kim Jin-Soo Regulatory zinc finger proteins
US20070042378A1 (en) * 2003-12-23 2007-02-22 Kim Jin-Soo Regulation of prokaryotic gene expression with zinc finger proteins
US20080138416A1 (en) * 2006-06-13 2008-06-12 Fmc Biopolymer As Method and systems for using biopolymer-based beads and hydrogels
WO2007147014A3 (en) * 2006-06-13 2008-07-31 Fmc Biopolymer As Method and systems for using biopolymer-based beads and hydrogels
US20100136663A1 (en) * 2006-10-24 2010-06-03 Korea Advanced Institute Of Science And Technology preparation of an artificial transcription factor comprising zinc finger protein and transcription factor of prokaryote, and a use thereof
US8242242B2 (en) * 2006-10-24 2012-08-14 Korea Advanced Institute Of Science And Technology Preparation of an artificial transcription factor comprising zinc finger protein and transcription factor of prokaryote, and a use thereof
CN113643758A (zh) * 2021-09-22 2021-11-12 华南农业大学 面向肠杆科细菌获得抗β-内酰胺类耐药性基因的预测方法

Also Published As

Publication number Publication date
CN1610737A (zh) 2005-04-27
AU2002365796A1 (en) 2003-06-17
WO2003048345A1 (en) 2003-06-12
CA2469477A1 (en) 2003-06-12
CN100463962C (zh) 2009-02-25
US7514257B2 (en) 2009-04-07
EP1451297A1 (en) 2004-09-01
JP2005511049A (ja) 2005-04-28
KR20040065235A (ko) 2004-07-21
EP1451297A4 (en) 2006-06-28
US20040209277A1 (en) 2004-10-21

Similar Documents

Publication Publication Date Title
US7514257B2 (en) Zinc finger transcription factor differentiation proteins
JP4156840B2 (ja) ジンクフィンガードメイン及びその同定方法
Strunnikov et al. SMC2, a Saccharomyces cerevisiae gene essential for chromosome segregation and condensation, defines a subgroup within the SMC family.
Kortschak et al. ARID proteins come in from the desert
van Essen et al. Two modes of transcriptional activation at native promoters by NF-κB p65
Freire Translation initiation factor (iso) 4E interacts with BTF3, the β subunit of the nascent polypeptide-associated complex
WO1998053059A1 (en) Nucleic acid binding proteins
US20090176653A1 (en) Zinc finger domain libraries
AU2002324352A1 (en) Zinc finger domain libraries
CN100398652C (zh) 调节性锌指蛋白
KR100648842B1 (ko) 약리학적 활성 물질을 동정하는 방법 및 이러한 방법에서 사용하기 위한 dna 주형
US20070042378A1 (en) Regulation of prokaryotic gene expression with zinc finger proteins
US20040259258A1 (en) Regulation of prokaryotic gene expression with zinc finger proteins
WO2004022575A2 (en) Bioinformatics analysis of cellular effects of artificial transcription factors
Smith Investigating the role of protein-protein and protein-DNA interactions in the function of Isl1
Park Screening new cytokinesis genes and investigation of regulation of Hof1 in cytokinesis
Strunnikov et al. and condensation, defines a subgroup within the SMC family

Legal Events

Date Code Title Description
AS Assignment

Owner name: TOOLGEN, INC., KOREA, REPUBLIC OF

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KIM, JIN-SOO;PARK, KYUNG SOON;LEE, DONG-KI;AND OTHERS;REEL/FRAME:014042/0825

Effective date: 20030226

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION