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OwnerJin-Soo Kim, Kyung-Soon Park +7inventors
InventorsJin-Soo Kim, Kyung-Soon Park, Dong-Ki Lee, Wongi Seol, Horim Lee, Seong-Il Lee, Hyo-Young Yang, Yangsoon Lee, Young-Soon Jang
Estimating the term…
In one aspect, 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. Some chimeric proteins include multiple zinc finger domains and can induce, for example, thermotolerance, solvent-tolerance, altered cellular growth, insulin production, differentiation, and drug resistance.
A modified cell comprising 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.
A target protein-expressing cell that contains (a) a coding nucleic acid that encodes a target protein and (b) a heterologous nucleic acid that comprises a sequence encoding an artificial protein chimera that (1) increases the amount of the protein produced by the cell relative to an otherwise-identical cell that does not include the heterologous nucleic acid, and (2) binds to DNA, but (3) does not bind to a transcriptional regulatory region that directly regulates transcription of the coding nucleic acid encoding the target protein.
An artificial transcription factor that (1) increases the amount of a target protein produced by a eukaryotic test cell relative to an otherwise-identical cell that does not include the artificial transcription factor, and (2) does not bind to a transcriptional regulatory region that directly regulates transcription of a DNA encoding the target protein in the test cell.
An artificial transcription factor comprising a plurality of zinc finger domains, wherein the artificial transcription factor alters the rate of cell cycle progression in a eukaryotic cell and at least two zinc finger domains are heterologous to each other.
A transfected cell that contains an endogenous gene encoding a secreted protein and a heterologous nucleic acid that comprises a sequence encoding an artificial transcription factor that increases the amount of the secreted protein produced by the cell relative to an otherwise identical cell that does not include the heterologous nucleic acid.
An artificial transcription factor comprising at least two zinc finger domain, 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.
An artificial transcription factor that alters sensitivity of a reference cell to a toxic agent relative to an otherwise identical cell that does not contain the nucleic acid.
A transfected cell that contains and expresses a nucleic acid encoding an artificial transcription factor that alters sensitivity of the cell to a toxic agent relative to an identical cell that does not contain the nucleic acid.
A method of altering the drug resistance of a fungal cell, the method comprising:
A method of identifying an artificial chimeric protein that alters the sensitivity of a cell to a toxic agent, the method comprising:
A nucleic acid comprising a sequence encoding an artificial transcription factor comprising three zinc finger domains, wherein expression of the artificial transcription factor in a vertebrate cell induces a neuronal phenotype in at least one vertebrate cell.
A nucleic acid comprising a sequence encoding an artificial transcription factor comprising three zinc finger domains, wherein expression of the artificial transcription factor in a vertebrate cell induces osteogenesis in at least one vertebrate cell.
A method of altering the differentiative capacity of a stem cell, the method comprising:
A method of identifying an artificial transcription factor, the method comprising:
A nucleic acid library comprising a first plurality of nucleic acids and a second plurality of nucleic acids, wherein
A method of identifying a plurality of protein chimeras, the method comprising:
A method of preparing a modified cell, the method comprising
A method of preparing a library encoding protein chimeras, the method comprising:
A method of identifying a target of artificial transcription factors, the method comprising:
A method of evaluating a cell, the method comprising:
A method of evaluating an artificial transcription factor, the method comprising:
A method of identifying a protein chimera, the method comprising:
A method of identifying a protein chimera, the method comprising:
A method of identifying a protein chimera, the method comprising:
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.
A method of transferring an altered trait from a first cell to a second cell, the method comprising:
An isolated polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 260, 262, and 264.
A nucleic acid comprising a sequence encoding an artificial transcription factor comprised of three zinc finger domains, wherein (a) expression of the artificial transcription factor alters a property of at least one eukaryotic cell, the property being selected from the group consisting of viral replication, virus production, and viral infectivity;
Dependencies are inferred from the claim text.
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Isolating an individual clone by screening libraries: screening libraries by altering the phenotype or phenotypic trait of the host
Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor: plant cells…
Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor: animal cells…
Genetically modified cells
Term end not estimated
Most 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. For example, 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.
In one aspect, 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. Typically, 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.
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.
In one aspect of the invention, 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.
In one example, 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.
Although this method is generally described below in the context of chimeras of zinc finger domains, it is easily adapted to other structural domains, including other DNA binding domains and cell signaling domains.