WO2005026718A1 - Banques de cellules souches - Google Patents
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- WO2005026718A1 WO2005026718A1 PCT/US2004/011270 US2004011270W WO2005026718A1 WO 2005026718 A1 WO2005026718 A1 WO 2005026718A1 US 2004011270 W US2004011270 W US 2004011270W WO 2005026718 A1 WO2005026718 A1 WO 2005026718A1
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- 235000019786 weight gain Nutrition 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 230000037314 wound repair Effects 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N5/0602—Vertebrate cells
- C12N5/0603—Embryonic cells ; Embryoid bodies
- C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/61—Growth hormone [GH], i.e. somatotropin
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- 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
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
- C12N9/1211—Thymidine kinase (2.7.1.21)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/03—Animal model, e.g. for test or diseases
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2510/00—Genetically modified cells
Definitions
- the present invention relates to libraries of embryonic stem cells that stably express transgenes encoding heterologous polypeptides, and methods of detecting the expressed polypeptides. These transgenes are "under the control of promoters that respond when a signal transduction pathway is activated, and they comprise easily detectable reporter systems for monitoring tlieir expression.
- the invention also relates to chimeric animals, tissues, and cells derived from embryonic stem cells that express heterologous polypeptides, and methods of using these animals, tissues, and cells to determine the components of signal transduction pathways operating in health and disease.
- the invention further relates to the effects of therapeutic agents which act upon signal transduction pathways.
- Stem cells are transfected with heterologous nucleic acids that express heterologous polypeptides.
- the stem cells have the capacity to differentiate into a plurality of cell types that express the heterologous polypeptides.
- the stem cells can be inco ⁇ orated into a blastocyst which develops into a chimeric embryo, fetus, or adult non-human animal.
- the heterologous nucleic acid is introduced into the stem cell at certain loci, e.g., the ROSA 26 locus, the chimeric non-human animal expresses the heterologous polypeptide in most or all of its tissues.
- the invention provides an embryonic stem cell with a nucleic acid molecule introduced at a gene locus that permits its expression in more than one cell type upon differentiation.
- the nucleic acid molecule can encode a secreted protein, a transcription factor, and/or a receptor.
- the nucleic acid molecule can be under the regulatory control of an inducible promoter, e.g., a tetracycline-inducible promoter or an ecdysone inducible promoter, or a tissue specific promoter, e.g., a brain- specific promoter, a breast-specific promoter, a prostate-specific promoter, a lung-specific promoter, a kidney-specific promoter, a bone tissue-specific promoter, a cartilage- specific promoter, a pancreatic cell-specific promoter, a liver-specific promoter, a monocyte-specific promoter, a T cell-specific promoter, a B cell-specific promoter, an epithelial cell-specific promoter, a dendritic cell-specific promoter, an NK cell- specific promoter, a cardiomyocyte-specif ⁇ c promoter, a muscle-specific promoter, a bone marrow-specific promoter, an ovarian tissue-specific promoter, a spermato
- the nucleic acid molecule can be involved in a signal transduction pathway, for example, one or more of a Wnt pathway, a growth factor receptor pathway, e.g., CSF, GMCSF, PDGF, KGF, FGF, and EGF, an erbB2 pathway, an apoptosis pathway, a NFKB pathway, a STAT activation pathway, a cytokirie receptor pathway, an endothelin pathway, a survival signaling pathway, an adrenergic receptor signaling pathway, a TNF receptor pathway, a pathway regulating glucose homeostasis, a pathway regulating food intake, a pathway that regulates atherosclerosis, a pathway that alters peripheral nerve function, a pathway that alters central nervous system function, a T cell activation pathway, a B cell activation pathway, a monocyte activation pathway, an NK cell activation pathway, a dendritic cell activation pathway, a pathway regulating bone homeostasis, a pathway
- the invention also provides a blastocyst of a non-human animal implanted with an embryonic stem cell as described above, as well as a non-human animal developed from the blastocyst, or a progeny thereof.
- the progeny can include a next-generation animal produced by crossing a first animal developed from the blastocyst with a second animal developed from the blastocyst.
- Progeny also include an animal produced by crossing an animal developed from the blastocyst with another animal, or a progeny thereof.
- Suitable animals include laboratory animals, e.g. , mice, and farm animals. Any of these animals can be either homozygous or heterozygous for the introduced nucleic acid molecule.
- the invention encompasses tissues and cells derived from these animals, as well as derivatives of these tissues and derivatives and progeny of these cells.
- the invention further provides an animal that is a model of a b man disease. For example, it may model a CNS disorder, a glucose imbalance disorder, an inflammatory disorder, an autoimmune disorder, a bone disorder, a cardiovascular disorder, a cancer, or another proliferative disorder.
- the invention provides a method of determining an effect of a therapeutic or prophylactic on a non-human animal, or a progeny thereof, developed from a blastocyst implanted with an embryonic stem cell as described above.
- the invention provides a method of determining an effect of a therapeutic or prophylactic on the tissue of a non-human animal, or a progeny thereof, developed from a blastocyst implanted with an embryonic stem cell as described above. This method involves administering the therapeutic or prophylactic to the tissue or a derivative of the tissue; and observing the tissue for one or more physiological changes.
- the invention provides a method of determining an effect of a therapeutic or prophylactic on the cell of a non-human animal, or a progeny thereof, developed from a blastocyst implanted with an embryonic stem cell as described above.
- This method involves administering the therapeutic or prophylactic to the cell or a derivative or progeny of the cell; and observing the cell for one or more physiological changes.
- BRIEF DESCRIPTION OF THE DRAWINGS [014]
- Figure 1 is a schematic representation depicting an example of the generation of a targeting vector for a secreted factor. It is described in more detail in Example 1.
- FIG. 2 illustrates the expression of a transmembrane protein, the EGF receptor, on the cell surface of several embryonic stem cell clones.
- Six positive clones expressed EGF receptor on the cell surface as demonstrated by Western hlot in the left panel and by fluorescence activated cell sorting (FACS) in the right panel. The Western blot shows the immunoreactivity of EGF receptor in cellular lysates.
- Lane 1 contained molecular weight markers.
- Lane 2 contained 40 ⁇ g clone 13 lysate.
- Lane 3 contained 40 ⁇ g clone 14 lysate.
- Lane 4 contained 40 ⁇ g clone 38 lysate.
- Lane 5 contained 40 ⁇ g clone 64 lysate.
- FIG. 3 illustrates the functionality of proteins expressed by embryonic stem cells.
- Figure 3 A shows the appearance of the differentiation marker CD235 in human bone marrow CD34+ cells in response to commercially available recombinant erythropoietin (Epo).
- Epo erythropoietin
- the right panel shows the FACS profile of the cells in the presence of Epo.
- Figure 3B shows the appearance of the differentiation marker CD235 in human bone marrow CD34+ cells in response to Epo expressed and secreted by the embryoid body of the invention.
- the left panel shows the FACS profile of the cells in the presence of the negative control IL-5, which was expressed and secreted from the embryoid body of the invention, but which does not induce differentiation of CD34+ cells.
- the right panel shows the FACS profile of the cells in the presence of Epo expressed and secreted from the embryoid body of the invention.
- the expressed, secreted EPO induced the appearance of the differentiation marker CD235 in CD34+ cells.
- the percent differentiated cells is shown in each of the four panels.
- Figure 3C shows the FACS profile of the TER 119 marker in the CD34+ cells, confirming that the cells are of human origin.
- Figure 4 illustrates the ability of proteins expressed and secreted from embryonic stem cells to induce mitosis in TF-1 cells.
- the dark bars show the mitogenic activity of commercially available recombinant IL-5 and Epo.
- the light bars show the mitogenic activity of conditioned medium from embryonic stem cells secreting IL-5 and Epo.
- a "stem cell” is a pluripotent or multipotent cell with the abilities to self-renew, to remain undifferentiated, and to become differentiated. Stem cells can divide without limit, for at least the lifetime of the animal in which they naturally reside. Stem cells are not terminally differentiated, i.e., they are not at the end of a pathway of differentiation. When a stem cell divides, each daughter cell can either remain a stem cell or it can embark on a course that leads to terminal differentiation. [019] An “embryonic stem cell” is a stem cell that is present in or isolated from an embryo.
- a "blastocyst" is an embryo at an early stage of development in which the fertilized ovum has undergone cleavage, and the trophectoderm and a spherical layer of cells surrounding a fluid-filled cavity is forming, or has formed. Inside the trophectoderm is a cluster of cells termed the inner cell mass.
- the trophectoderm is the precursor of the placenta, and the inner cell mass is the precursor of the embryo.
- Cells of the early mammalian embryo are pluripotent.
- Progeny are those born of or derived from another. Progeny include all descendents of the first, second, and all subsequent generations. Progeny include those taken, received, or obtained from a parent organism.
- a "cell line” is a population of cultured cells that has undergone a change that allows the cells to grow indefinitely. Cell lines can result from transformation by, for example, any other chemical or viral systems.
- nucleic acid molecule refers to polymeric forms of nucleotides of any length.
- the polynucleotides can contain deoxyribonucleotides, ribonucleotides, and/or their analogs or derivatives.
- nucleic acids can be naturally occurring DNA or RNA, or can be synthetic analogs, as known in the art.
- the terms also encompass genomic DNA, genes, gene fragments, exons, introns, regulatory sequences or regulatory elements (such as promoters, enhancers, initiation and termination regions, other control regions, expression regulatory factors, and expression controls), DNA comprising one or more single-nucleotide polymorphisms (SNPs), allelic variants, isolated DNA o f any sequence, and cDNA.
- SNPs single-nucleotide polymorphisms
- allelic variants isolated DNA o f any sequence
- cDNA cDNA.
- the terms also encompass mRNA, tRNA, rRNA, ribozymes, splice variants, antisense RNA, antisense conjugates, RNAi, and isolated RNA of any sequence.
- the terms also encompass recombinant polynucleotides, heterologous polynucleotides, branched polynucleotides, labeled polynucleotides, hybrid DNA/RNA, polynucleotide constructs, vectors comprising the subject nucleic acids, nucleic acid probes, primers, and primer pairs.
- the polynucleotides can comprise modified nucleic acid molecules, with alterations in the backbone, sugars, or heterocyclic bases, such as methylated nucleic acid molecules, peptide nucleic acids, and nucleic acid molecule analogs, which may be suitable as, for example, probes if they demonstrate superior stability and/or binding affinity under assay conditions.
- Analogs of purines and pyrimidines, including radiolabeled and fluorescent analogs, are known in the art.
- the polynucleotides can have any three-dimensional structure, and can perform any function, known or as yet unknown.
- the terms also encompass single-stranded, double-stranded and triple helical molecules that are either DNA, RNA, or hybrid DNA/RNA and that may encode a full-length gene or a fragment thereof. Fragments of polynucleotides may be biologically active and can encode the polypeptides herein, as well as anti-sense and RNAi molecules.
- the full length polynucleotides herein may be treated with enzymes, such as Dicer, to generate a library of short RNAi fragments which are within the scope of the present invention.
- the term "gene” or “genomic sequence” as used herein is an open reading frame encoding specific proteins and polypeptides, for example, an mRNA, cDNA, or genomic DNA, and also may or may not include intervening introns, or adjacent 5' and 3 'non-coding nucleotide sequences involved in the regulation of expression up to about 20 kb beyond the coding region, and possibly further in either direction.
- a gene can be introduced into an appropriate vector for extrachromosomal maintenance or for integration into a host genome.
- Nucleic acid composition is a composition comprising a nucleic acid sequence, including one having an open reading frame that encodes a polypeptide and is capable, under appropriate conditions, of being expressed as a polypeptide.
- the term includes, for example, vectors, including plasmids, cosmids, viral vectors (e.g., retrovirus vectors such as lentivirus, adenovirus, and the like), human, yeast, bacterial, PI -derived artificial chromosomes (HAC's, YAC's, BAC's, PAC's, etc), and mini-chromosomes, in vitro host cells, in vivo host cells, tissues, organs, allogenic or congenic grafts or transplants, multicellular organisms, and chimeric, genetically modified, or transgenic animals comprising a subject nucleic acid sequence.
- vectors including plasmids, cosmids, viral vectors (e.g., retrovirus vectors such as lentivirus, a
- an "isolated,” “purified,” or “substantially isolated” polynucleotide, or a polynucleotide in “substantially pure form,” in “substantially purified form,” in “substantial purity,” or as an “isolate,” is one that is substantially free of the sequences with which it is associated in nature, or other nucleic acid sequences that do not include a sequence or fragment of the subject polynucleotides.
- substantially free is meant that less than about 90%, less than about 80%, less than about 70%, less than about 60%, or less than about 50% of the composition is made up of materials other than the isolated polynucleotide.
- the isolated polynucleotide is at least about 50%), at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% free of the materials with which it is associated in nature.
- an isolated polynucleotide may be present in a composition wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%), at least about 90%, at least about 95%), at least about 97%, at least about 99% of the total macromolecules (for example, polypeptides, fragments thereof, polynucleotides, fragments thereof, lipids, polysaccharides, and oligosaccharides) in the composition is the isolated polynucleotide. Where at least about 99% of the total macromolecules is the isolated polynucleotide, the polynucleotide is at least about 99% pure, and the composition comprises less than about 1% contaminant.
- the total macromolecules for example, polypeptides, fragments thereof, polynucleotides, fragments thereof, lipids, polysaccharides, and oligosaccharides
- an "isolated,” “purified” or “substantially isolated” polynucleotide, or a polynucleotide in “substantially pure form,” in “substantially purified form,” in “substantial purity,” or as an “isolate,” also refers to recombinant polynucleotides, modified, degenerate and homologous polynucleotides, and chemically synthesized polynucleotides, which, by virtue of origin or manipulation, are not associated with all or a portion of a polynucleotide with which it is associated in nature, are linked to a polynucleotide other than that to which it is linked in nature, or do not occur in nature.
- the subject polynucleotides are generally provided as other than on an intact chromosome, and recombinant embodiments are typically flanked by one or more nucleotides not normally associated with the subject polynucleotide on a naturally-occurring chromosome.
- a "gene locus" is the position of a DNA segment, e.g., a gene, on a chromosome.
- the G3BP (BT-5) locus is the position of the ras-GTPase- activating protein-binding protein (G3BP) in the BT-5 mouse cell line.
- the Rosa 26 locus is the position at which the ROSA ⁇ geo retrovirus integrates into the genome of the ROSA ⁇ geo26 (ROSA26) mutant strain of mice.
- a "promoter,” as discussed in detail infra, is a region of DNA that binds RNA polymerase before initiating the transcription of DNA into RNA.
- the nucleotide at which transcription begins is designated +1; nucleotides are numbered from this reference point. Negative numbers indicate upstream nucleotides and positive numbers indicate downstream nucleotides.
- the promoter directs the RNA polymerase to bind to DNA, to open the DNA helix, and to begin RNA synthesis. Some promoters are "constitutive,” and initiate transcription in the absence of regulatory influences.
- promoters are "tissue specific,” and initiate transcription exclusively or selectively in one or a few tissue types. Some promoters are “inducible,” and initiate gene transcription under the influence of an inducer. Induction can occur, e.g., as the result of a physiologic response, a response to outside signals, or as the result of artificial manipulation.
- a promoter may be operably linked to a coding sequence and be capable of effecting the expression of the coding sequence when the proper factors are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct its expression.
- transcription factor is a factor that initiates or regulates transcription in eukaryotes. Transcription factors include gene regulatory proteins, which turn specific sets of genes on or off, and general transcription factors, which assemble at the promoter region to enable and regulate transcription of many genes. They also include transcription elongation factors, which are proteins that add amino acids to growing polypeptide chains on ribosomes (Alberts et al., 1994).
- Transcription factors may interact with a wide variety of molecules, including DNA binding proteins, polymerases, regulatory molecules such as kinases, and specific regions of DNA, e.g., promoters, and enhancers (Alberts et al., 1994; Vallejo et al., 1993).
- polypeptide refers to a polymeric form of amino acids of any length, which can include naturally-occurring amino acids, coded and non-coded amino acids, chemically or biochemically modified, derivatized, or designer amino acids, amino acid analogs, peptidomimetics, and depsipeptides, and polypeptides having modified, cyclic, bicyclic, depsicyclic, or depsibicyclic peptide backbones.
- the term includes single chain protein as well as multimers.
- the term also includes conjugated proteins, fusion proteins, including, but not limited to, GST fusion proteins, fusion proteins with a heterologous amino acid sequence, fusion proteins with heterologous and homologous leader sequences, fusion proteins with or without N-terminal methionine residues, pegolyated proteins, and immunologically tagged proteins. Also included in this term are variations of naturally occurring proteins, where such variations are homologous or substantially similar to the naturally occurring protein, as well as corresponding homologs from different species. Variants of polypeptide sequences include insertions, additions, deletions, or substitutions compared with the subject polypeptides. The term also includes peptide aptamers.
- Depsipeptides are compounds containing a sequence of at least two alpha-amino acids and at least one alpha-hydroxy carboxylic acid, which are bound through at least one normal peptide link and ester links, derived from the hydroxy carboxylic acids.
- Linear depsipeptides can comprise rings formed through S-S bridges, or through an hydroxy or a mercapto group of an hydroxy-, or mercapto- amino acid and the carboxyl group of another amino- or hydroxy-acid but do not comprise rings formed only through peptide or ester links derived from hydroxy carboxylic acids.
- Cyclic depsipeptides are peptides containing at least one ring formed only through peptide or ester links, derived from hydroxy carboxylic acids.
- the term "bicyclic” refers to a peptide with two ring closures formed by covalent linkages between amino acids. A covalent linkage between two nonadjacent amino acids constitutes a ring closure, as does a second covalent linkage between a pair of adjacent amino acids which are already linked by a covalent peptide linkage.
- the covalent linkages forming the ring closures can be amide linkages, i.e., the linkage formed between a free amino on one amino acid and a free carboxyl of a second amino acid, or linkages formed between the side chains or "R" groups of amino acids in the peptides.
- bicyclic peptides can be "true” bicyclic peptides, i.e., peptides cyclized by the formation of a peptide bond between the N-terminus and the C-terminus of the peptide, or they can be "depsi-bicyclic" peptides, i.e., peptides in which the terminal amino acids are covalently linked through their side chain moieties.
- an "isolated,” “purified,” or “substantially isolated” polypeptide, or a polypeptide in “substantially pure form,” in “substantially purified form,” in “substantial purity,” or as an “isolate,” is one that is substantially free of the materials with which it is associated in nature or other polypeptide sequences that do not include a sequence or fragment of the subject polypeptides.
- substantially free is meant that less than about 90%, less than about 80%, less than about 70%, less than about 60%, or less than about 50% of the composition is made up of materials other than the isolated polypeptide.
- the isolated polypeptide is at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% free of the materials with which it is associated in nature.
- an isolated polypeptide may be present in a composition wherein at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, or at least about 99% of the total macromolecules (for example, polypeptides, fragments thereof, polynucleotides, fragments thereof, lipids, polysaccharides, and oligosaccharides) in the composition is the isolated polypeptide.
- the total macromolecules for example, polypeptides, fragments thereof, polynucleotides, fragments thereof, lipids, polysaccharides, and oligosaccharides
- the polypeptide is at least about 99%o pure, and the composition comprises less than about 1%> contaminant.
- an "isolated,” “purified,” or “substantially isolated” polypeptide, or a polypeptide in “substantially pure form,” in “substantially purified form,” in “substantial purity,” or as an “isolate,” also refers to recombinant polypeptides, modified, tagged and fusion polypeptides, and chemically synthesized polypeptides, which by virtue or origin or manipulation, are not associated with all or a portion of the materials with which they are associated in nature, are linked to molecules other than that to which they are linked in nature, or do not occur in nature.
- a “signal peptide,” or a “leader sequence,” comprises a sequence of amino acid residues, typically, at the N terminus of a polypeptide, which directs the intracellular trafficking of the polypeptide.
- Polypeptides that contain a signal peptide or leader sequence typically also contain a signal peptide or leader sequence cleavage site. Such polypeptides, after cleavage at the cleavage sites, generate mature polypeptides, for example, after extracellular secretion or after being directed to the appropriate intracellular compartment.
- Secreted proteins also referred to as secreted factors or secreted polypeptides, include polypeptides, or active portions thereof, that are produced by cells and exported extracellularly; extracellular fragments of transmembrane proteins that are proteolytically cleaved; or extracellular fragments of cell surface receptors, which fragments may be soluble.
- Secreted proteins include extracellular domains of membrane proteins that may act as targets for antibody production or that may be cleaved to become a soluble receptor or a ligand for a receptor.
- Some secreted proteins have the ability to act as ligands for binding to receptors on cell surfaces in ligand/receptor interactions, to bind to ligands, soluble or otherwise, to inhibit ligand/receptor interactions, to trigger certain intracellular responses, such as inducing signal transduction to activate cells or inhibit cellular activity, to induce cellular growth, proliferation, or differentiation, to induce the production of other factors that, in turn, mediate such activities, and/or to inhibit cell activation or signaling.
- a "library" of polynucleotides comprises a collection of sequence information for a plurality of polynucleotide sequences, which information is provided in either biochemical form (e.g., as a collection of polynucleotide molecules), or in electronic form (e.g., as a collection of polynucleotide sequences stored in a computer-readable form, as in a computer-based system, a computer data file, and/or as part of a computer program).
- a "library" of polypeptides comprises a collection of sequence information for a plurality of polypeptide sequences, which information is provided in either biochemical form (e.g., as a collection of polynucleotide molecules), or in electronic form (e.g., as a collection of polynucleotide sequences stored in a computer-readable form, as in a computer-based system, a computer data file, and/or as part of a computer program).
- the terms "individual,” “host,” “patient,” and “subject,” used interchangeably herein, refer to a mammal, including, but not limited to, murines, simians, humans, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian farm animals, mammalian sport animals, and mammalian pets.
- mammals or “mammalian,” are used broadly to describe organisms which are within the class mammalia, including the orders carnivore (e.g., dogs and cats), rodentia (e.g., mice, guinea pigs, and rats), and other mammals, including cattle, goats, sheep, cows, horses, rabbits, and pigs, and primates (e.g., humans, chimpanzees, and monkeys).
- candidate agent “subject agent,” or “test agent,” used interchangeably herein, encompass numerous chemical classes, typically synthetic, semi-synthetic, or naturally occurring inorganic or organic molecules, small molecules, or macromolecular complexes.
- Candidate agents can be small organic compounds having a molecular weight of more than about 50 and less than about 2,500 daltons.
- Candidate agents can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and can include at least an amine, carbonyl, hydroxyl or carboxyl group, and can contain at least two of the functional chemical groups.
- the candidate agents can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
- Candidate agents are also found among biomolecules, including oligonucleotides, polynucleotides, and fragments thereof, depsipeptides, polypeptides and fragments thereof, oligosaccharides, polysaccharides and fragments thereof, lipids, fatty acids, steroids, purines, pyrimidines, derivatives thereof, structural analogs, modified nucleic acids, modified, derivatized or designer amino acids, or combinations thereof.
- the term "agonist” refers to a substance that mimics the function of an active molecule. Agonists include, but are not limited to, drugs, hormones, antibodies, and neurotransmitters, as well as analogues and fragments thereof.
- Antagonist refers to a molecule that competes for the binding sites of an agonist, but does not induce an active response. Antagonists include, but are not limited to, drugs, hormones, antibodies, and neurotransmitters, as well as analogues and fragments thereof.
- receptor refers to a polypeptide that binds to a specific extracellular molecule and may initiate a cellular response.
- ligand refers to any molecule that binds to a specific site on another molecule.
- modulate encompasses an increase or a decrease, a stimulation, inhibition, or blockage in the measured activity when compared to a suitable control.
- Modulation of expression levels includes increasing the level and decreasing the level of an mRNA or polypeptide encoded by a polynucleotide of the invention when compared to a control lacking the agent being tested.
- agents of particular interest are those which inhibit a biological activity of a subject polypeptide, and/or which reduce a level of a subject polypeptide in a cell, and/or which reduce a level of a subject mRNA in a cell and/or which reduce the release of a subject polypeptide from a eukaryotic cell.
- agents of interest are those that increase a biological activity of a subject polypeptide, and/or which increase a level of a subject polypeptide in a cell, and or which increase a level of a subject mRNA in a cell and/or which increase the release of a subject polypeptide from a eukaryotic cell.
- the term "over-expressed” refers to a state wherein there exists any measurable increase over normal or baseline levels. For example, a molecule that is over-expressed in a disease is one that is manifest in a measurably higher level compared to levels in the absence of the disease.
- a "disease” is a pathological, abnormal, and/or harmful condition of an organism.
- Signal transduction is the conversion of a signal from one physical or chemical form into another. It can refer in particular to the sequential process initiated by interaction of an extracellular signal, e.g., a hormone, growth factor, or neurotransmitter, with a receptor, causing a change in the level of an intracellular second messenger, e.g., calcium or cyclic AMP, and culminating in one or more specific cellular response, often mediated by the activation of a transcription factor.
- a “signal transduction pathway” is the collection of molecules and/or the cascade of processes by which signal transduction takes place.
- “Differentiation” is a progressive developmental change to a more specialized form or function.
- Cell differentiation is the process a cell undergoes as it matures to become an overtly specialized cell type. Differentiated cells have distinct characteristics, perform specific functions, and typically are less likely to divide than their less differentiated counterparts.
- An undifferentiated cell e.g., an immature, embryonic, or primitive cell, typically has a non-specific appearance, may perform multiple, non-specific activities, and may perform poorly, if at all, in functions typically performed by differentiated cells.
- “Dedifferentiation” is a process by which a mature cell returns to a less mature state.
- a “dedifferentiated cell” is one that has fewer characteristics of differentiation than it possesses at an earlier point in time.
- a “dedifferentiated state” is one in which a mature cell has returned or is returning to a less differentiated state, e.g., as in some cancers.
- a “differentiation factor” is a factor that induces a cell to undergo a change in the direction of an overtly specialized cell type.
- An “anti-differentiation factor” is a factor that prevents or inhibits a cell from undergoing a change in the direction toward an overtly specialized cell type.
- a “therapeutic” is an agent that is palliative, curative, or otherwise useful in treating or ameliorating a disease, disorder, syndrome, or condition.
- a “prophylactic” is an agent that prevents the occurrence or recurrence of a disease, disorder, syndrome, or condition, e.g., a drug or vaccine.
- Methods Embryonic Stem Cells can be pluripotent; they can differentiate into any of the cells present in the organism. When they divide in vivo, pluripotent stem cells can maintain their pluripotency while giving rise to differentiated progeny. Thus, stem cells can produce replicas of themselves which are pluripotent, and are also able to differentiate into lineage-restricted committed progenitor cells. Stem cells can also reproduce and differentiate in vitro.
- Embryonic stem cells have been directed to differentiate into cardiac muscle cells in vitro and, alternatively, into early progenitors of neural stem cells, and then into mature neurons and glial cells in vitro (Trounson, 2002).
- the embryonic stem cells of the invention can be derived from a variety of animal species, including mammalian species such as, but not limited to, mouse, rat, guinea pig, sheep, goat, bovine, rabbit, canine, feline, porcine, ovine, and equine.
- Embryonic stem cells of the invention transfected with erythropoietin or IL-5 have been demonstrated to stably express physiologically functional erythropoietin or IL-5, respectively (PCT/US03/34811).
- Transfected embryonic stem cells of the invention also have been demonstrated to express other secreted proteins which are involved in signal transduction, such as the parathyroid hormone-like protein, FrizB or sFRP3, myostatin, bone morphogenetic protein 4, insulin-like growth factor 1, neuropeptide Y, growth hormone, Wnt 2, and Wnt 11 (PCT/US03/34811).
- Mouse embryonic stem cells are derived from the inner cell mass, the cells which give rise to the embryo.
- Inner cell mass of a donor mouse at the blastomere stage of development can be transferred into the embryo of a second mouse, and the donor mouse can contribute genes to every organ of the host embryo.
- Inner cell mass blastomeres can be isolated from the embryo and cultured in vitro to produce ES cell cultures and cell lines.
- ES cells retain their totipotency in vitro, and each of them can contribute to all the organs of a host embryo, e.g., following injection into the host embryo.
- ES cell cultures and cell lines can incorporate new DNA as transgenes.
- Embryonic stem cells can be transformed with nucleic acids encoding a protein or a fragment of a protein, e.g., a secreted protein or an extracellular domain of a transmembrane protein.
- the activity of the proteins or fragments thereof encoded can be assayed.
- the gene encoding the protein is expressed, and the modulation of the proliferation and/or differentiation of the stem cell transformed with the gene can be observed.
- Changes in a parameter associated with a disease e.g., the rate of proliferation, the lack of proliferation, and/or differentiation of the genetically-altered stem cells can be compared with the wild type, non-genetically- altered stem cells.
- mouse ES cells such as the mouse 129/SvJ cell line, are derived from the early mouse embryo and grown under culture conditions well known in the art.
- the vector is introduced into ES cells by transformation methods such as electroporation, liposome delivery, microinjection, and the like, which are well known in the art.
- the vector can contain genes for a secreted protein, for example, such as growth factors, cytokines, or hormones.
- the endogenous rodent gene is replaced by the disrupted disease gene through homologous recombination and integration during cell division.
- transformed ES cells are selected and used to study the proliferation and differentiation into various cell types.
- the differentiated embryonic stem cells can form various cell types and tissues in vitro, such as neural cells, hematopoietic lineages, and cardiomyocytes (Bain et al, 1995; Wiles and Keller, 1991; Klug et al, 1996).
- a mouse embryonic stem cell comprises a reporter system with a readout signal under the regulatory control of a promoter responsive to the activation of a signal transduction pathway.
- the reporter system is inserted into the ROSA26 or the G3BP(BT5) locus.
- the embryonic stem cell can then be cultured, induced to differentiate, be incorporated into an ES cell library, or be introduced into a blastocyst.
- the transformed stem cells can be induced to differentiate into different cell types by adding factors that promote differentiation.
- the differentiation factors can be lineage specific or non-lineage specific, and can be supplied individually, in a formulation containing a combination of factors, or by the addition of a cell or cells that are capable of providing the differentiation factors to the genetically-modified stem cell.
- the differentiated cell can be identified by the marker on the surface of the cell or by its phenotype.
- the transformed ES cells described above are selected, exposed to an exogenously added factor, and the proliferation and differentiation of the stem cell populations into various cell types and tissues in vitro, such as neural cells, hematopoietic lineages, and cardiomyocytes can be studied (Bain et al, 1995; Wiles and Keller, 1991; Klug et al, 1996).
- ES Cell Libraries [059] Libraries of transfected stem cells can be compiled to express selected polypeptides known or hypothesized to modulate selected in vivo or in vitro cellular functions, and can be used to screen, test, or compare potentially therapeutic or otherwise modulatory agents (PCT/US03/34811).
- the invention provides a library of transformed mouse ES cells comprising nucleic acid molecules encoding polypeptides which are targeted to the ROSA 26 locus of the ES cells.
- the libraries comprise a plurality of cells located in an addressable matrix. The matrix contains a plurality of spots or wells, each of which has an address, such as, i.e., column 2, row 4.
- the number of addressable spots in the matrix can range from between 5-50, 10-100, 20-200, 30-300, 40-400, 50-500, 60-600, 70- 700, 80-800, 90-900, 100-1000, 250-2000, 350-3000, 450-4000, 550-5000, 650-6000, 750-7000, 850-8000, 950-9000, 1050-10000, and 10000-50000, or more.
- At least one cell, and preferably more, is located in one or more spots or wells of the matrix.
- Each address in the matrix can contain either the same or a different number of cells, and the same or different type of cells.
- At least some of the stem cells are transformed with at least one, and optionally 2, or 3, or 4, or 5, or more introduced nucleic acid molecules.
- the invention provides this cell library on any suitable substrate or support, such as a 96-well plate, a 384-well or larger plate, a glass slide containing depressions or wells in rows and columns, and such similar substrates that are suitable for high throughput analysis, or can be adapted for a robotics system.
- the nucleic acid molecules introduced into the stem cells of the library can encode secreted molecules, transmembrane molecules, or intracellular molecules, comprising, e.g., transcription factors, nuclear receptors, kinases, phosphatases, proteases, and ion channels. These molecules may have either a stimulatory or an inhibitory effect on the transformed cells.
- the stem cells of the library may exhibit a gain of function, for example, the cells may acquire the ability to secrete certain proteins whose secretion is known to be mediated by particular signal transduction pathways.
- the stem cells of the library may also exhibit a loss of function, for example, the cells may lose the ability to secrete certain proteins whose secretion is known to be mediated by particular signal transduction pathways.
- Cells exhibiting such a gain of function can be further transformed by introducing additional nucleic acid molecules that affect the gain of function, for example, by introducing nucleic acid molecules that knock out function.
- This twice-transformed library can comprise inhibitory molecules, such as RNAi molecules to knock out function.
- the stem cells of the library provide a source of secreted molecules; they can be placed in contact with other stem cells to determine the effect of the secreted molecules on the signal transduction pathways of the other stem cells.
- the invention also provides a library of stem cells that are differentiated into cells of different lineages, including but not limited to cardiomyocytes, T cells, B cells, leukocytes, other cells of the hematopoietic system, neurons, astrocytes, glia cells, other cells of the CNS, liver cells, bone cells, cartilage cells, pancreatic islet cells, kidney cells, muscle cells, and other cells of the body.
- the stem cell library can comprise a first stem cell transformed with a first nucleic acid molecule that encodes a first protein, a second stem cell transformed with a second nucleic acid molecule that encodes a second protein, a third stem cell transformed with a third nucleic acid molecule that encodes a third protein, a fourth stem cell transformed with a fourth nucleic acid molecule that encodes a fourth protein, and so on, up to tens, hundreds, thousands, or tens of thousands of stem cells, each transformed with a different nucleic acid molecule encoding a different protein.
- Each of the nucleic acid molecules of the library can encode a polypeptide.
- the medium in which the stem cells are suspended can comprise added proteins or fragments of proteins, which may come into contact with the stem cells.
- the added proteins or fragments may be present in the form of other cells in the medium. These cells can express the proteins or fragments on their cell surface, and/or secrete them.
- the medium can comprise (a) a stem cell transformed with a first different nucleic acid molecule; (b) two different stem cells: one transformed with a first different nucleic acid molecule and the other transformed with a second different nucleic acid molecule, respectively; (c) three different stem cells: one transformed with a first different nucleic acid molecule, a second transformed with a second different nucleic acid molecule, and a third transformed with a third different nucleic acid molecule, respectively; (d) four different stem cells: one transformed with a first different nucleic acid molecule, a second transformed with a second different nucleic acid molecule, a third transformed with a third different nucleic acid molecule, and a fourth transformed with a fourth different nucleic acid molecule, respectively; and/or (e) five different stem cells: one transformed with a first different nucleic acid molecule, a second transformed with a second different nucleic acid molecule, a third transformed with a third different nucleic acid molecule, a fourth transformed
- the library includes readout cells, also referred to as reporter cells, that are capable of exhibiting an observable biological effect or phenotype when placed in contact with a biological molecule that has either stimulatory or inhibitory function.
- readout cells can be embryonic or adult stem cells, and the introduced nucleic acid molecules may encode a factor that causes them to differentiate into cells of different lineages.
- the stem cell readout cells can be placed in contact with proteins or fragments of protein having biological activity, such that the readout cells determine the function and/or effect of one or more polypeptides on the signal transduction pathways, e.g., as reflected in the growth and/or differentiation of the stem cells.
- the readout cells can also be selected from T cells, B cells, CNS cells, cartilage cells, bone cells, pancreatic islet cells, fat cells, and oocytes.
- Stem cell readout cells may differentiate to produce cells selected from CNS cells including brain cells, neurons, astrocytes, and glial cells; T cells; B cells; cartilage cells; bone cells; pancreatic islet cells; fat cells; heart cells; liver cells; kidney cells; lung cells; muscle cells; and eye cells.
- the readout cells can be derived from animal species other than the stem cells, such as, for example, frogs, rabbits, cows, pigs, and the like.
- the invention provides a combined library of transformed cells and readout cells that are in physical contact with each other, and can be used, for example, to study molecular interaction.
- the transformed cells can express polypeptides involved in signal transduction.
- the transformed cells can express a ligand, and a nucleic acid molecule introduced into the readout stem cells can encode a receptor.
- the invention provides a method of determining the function of a first protein encoded by a first nucleic acid molecule, where the method comprises allowing a first transformed stem cell to grow, where the first transformed stem cell is transformed with a first nucleic acid molecule that is targeted to a first locus; and observing one or more signal transduction pathways of the first transformed stem cell to determine a function of the first protein.
- This method can be used to determine the function of a library of proteins by transforming a stem cell library with a plurality of nucleic acid molecules encoding a plurality of polypeptides, allowing the stem cells in the library to grow or differentiate; and observing signal transduction in the stem cells of the library.
- the invention also provides a method of massively parallel screening for signal transduction activity by providing a combinatorial library of a plurality of stem cells in an addressable matrix transformed with a plurality of distinguishable nucleic acid molecules encoding a plurality of proteins and monitoring the library of stem cells for signal transduction activity.
- Chimeras from ES Cells [069] An ES cell with exogenous DNA can be injected into an early-stage mouse embryo, and will integrate into the host embryo, resulting in a chimeric mouse. Some of the chimera's cells will be derived from the host embryonic stem cells, but some portion of its cells will be derived from the embryonic stem cell with the exogenous gene.
- the treated cells become part of the germ line of the mouse, some of its gametes will be derived from the donor cell. If such a chimeric mouse is mated with a wild-type mouse, some of its progeny will carry one copy of the inserted gene. When these heterozygous progeny are mated to one another, about 25% of the resulting offspring are predicted to carry two copies of the inserted gene in every cell of their bodies (Gossler et al, 1986). Thus, in three generations: the chimeric mouse, the heterozygous mouse, and the homozygous mouse, an exogenous gene will be present in both copies of the chromosomes of the mouse genome.
- the transformed stem cells of the invention can be used to develop in vivo mouse models of human disease.
- a gene construct encoding a polypeptide is inserted into the mouse embryonic stem cells to produce transfected stem cells.
- One or more than one polypeptide can be encoded by the construct.
- the polypeptide can be, e.g., a secreted protein, a fragment of a secreted protein, a transmembrane protein, an extracellular domain of a transmembrane protein, or a combination of these.
- the gene construct can be inserted in the ROSA26 or G3BP locus to allow gene expression in most or all tissues of the mouse. [072]
- the resulting transfected stem cells are inserted into a blastocyst, e.g., at the 64 cell stage, to form a chimeric blastocyst. Normal mice, knockout mice, or mouse models of human disease can provide a source for these blastocysts.
- the blastocysts When implanted into a pseudo-pregnant mouse, the blastocysts can develop into chimeric embryos, fetuses, and mice.
- the chimeric mice can also be produced by breeding, e.g., by crossing a mouse carrying a gene of interest with a mouse model of human disease.
- Mouse models that are useful for practicing the invention include, but are not limited to, models for a central nervous system (CNS) disorder; a peripheral nerve disorder; a glucose imbalance disorder, e.g., a disorder of insulin resistance; an inflammatory disorder, an autoimmune disorder; a bone disorder; a cartilage disorder; a cardiovascular disorder, e.g., atherosclerosis; cancer, or other proliferative disorders; disorders of food intake; epithelial cell disorders; and disorders mediated by the state of activation of B cells, T cells, monocytes, NK cells, and/or dendritic cells.
- Mouse models that are useful for practicing the invention also include disorders of signal transduction, including cancers and inflammatory diseases.
- mice that overexpress A ⁇ peptide or TGF ⁇ peptide examples include mice that overexpress A ⁇ peptide or TGF ⁇ peptide.
- Other useful mouse models include the severe combined immunodeficiency (SCID) mouse, non-obese diabetic mouse, Rb-/- mouse, and p53 -/- mouse. These models provide an opportunity to observe whether an inserted gene corrects the deficiency of each.
- SCID severe combined immunodeficiency
- the invention provides a system for conducting in vivo and in vitro testing of signal transduction protein function, for expression or manufacture of proteins.
- the system provides targeting a gene to a locus, e.g., the ROSA 26 locus in mouse ES cells and allowing the transfected DNA to proliferate and differentiate in vitro.
- the ROSA 26 locus directs the ubiquitous expression of the heterologous gene (U.S. Patent No. 6,461,864).
- the effect of the transfected DNA on healthy or diseased cells can be monitored in vitro. Differentiation of cells, e.g., cardiomyocytes, hepatocytes, skeletal myocytes, etc. can be monitored by morphologic, histologic, and/or physiologic criteria.
- the tissues of the chimeric mice or their progeny can be isolated and studied, or cells and/or cell lines can be isolated from the tissues and studied.
- Tissues and cells from any organ in the body including heart, liver, lung, kidney, spleen, thymus, muscle, skin, blood, bone marrow, prostate, breast, stomach, brain, spinal cord, pancreas, ovary, testis, eye, and lymph node are suitable for use.
- the invention includes the observation that ES cells transfected with interleukin-5 and incorporated into a blastocyst produced a chimeric mouse that expressed a greater than normal number of eosinophils in the liver.
- Eosinophilia is a previously observed effect of interleukin-5, and demonstrates that the ES cell mouse expression system (ESpresso mouse) " can be used to determine the function of unknown and novel secreted polypeptides. Mice possessing phenotypic changes as a result of transgene expression may physically appear only slightly chimeric.
- Gene Trap Vectors [077] The invention provides gene trap vectors to identify the discrete expression pattern of genes during signal transduction. Constructs with a reporter gene but lacking a promoter are designed so that activation of the reporter gene depends on its insertion within an active transcription unit. Following insertion, the tagged gene can be detected in space and time by assaying for the reporter gene product.
- the trap vectors contain a reporter gene that is not expressed unless it integrates into an intron or exon of a transcription unit. Integration results in an expression pattern that reflects the pattern of the endogenous transcription unit.
- the reporter gene provides a molecular tag for cloning the "trapped" gene of the transcription unit. All of the above-described reporter systems can be used with the gene trap vectors described below.
- Gene trap vectors can be constructed in retroviral vectors.
- ROSAP geo26 ROSAP geo26
- GAP ras-GTPase-activating protein
- G3BP ras-GTPase-binding protein
- the stem cells of the invention can express nucleic acids under the regulatory control of inducible promoters.
- inducible promoter is the tetracycline-inducible promoter, which is induced to initiate transcription by tetracycline. This promoter has been demonstrated, with a luciferase reporter, to activate and inactivate its target transgenes (Canete-Soler et al, 1998).
- an inducible promoter is the ecdysone-inducible promoter, which is induced to initiate transcription by ecdysone. This promoter has also been demonstrated to provide a useful tool for studying gene function, and has been used with the lacZ and green fluorescent protein reporters (Luers et al., 2000).
- the stem cells of the invention can also express nucleic acids under the regulatory control of tissue-specific promoters.
- tissue-specific promoters include the astrocyte-specific (CNS) promoter for glial fibrillary acidic protein (GFAP), a brain-specific promoter; kidney androgen regulated protein (KAP), the kidney-specific promoter for kidney androgen regulated protein (KAP); the adipocyte-specific promoter for adipocyte specific protein (ap2), the blood vessel endothelium-specific promoter for vascular endothelial growth factor receptor 2 (VEGFR2), the liver-specific promoter for albumin, the pancreas-specific promoter for pancreatic duodenal homeobox 1 (PDX1), the muscle-specific promoter for muscle creatine kinase (MCK), the bone-specific promoter for osteocalcin, the cartilage-specific promoter for type II collagen, the lung-specific promoter for surfactant protein C (SP-C), the cardiac-specific promoter alpha-myosin heavy chain ( ⁇ -MHC), and the intestinal epithelial-specific promoter fatty acid binding
- the astrocyte-specific (CNS) promoter for glial fibrillary acidic protein (GFAP) has been described by Miura et al., 1990.
- the promoter sequence and transcriptional startpoint of the GFAP gene have been characterized; the cis elements for astrocyte specific expression are located within 256 base pairs from the transcription startpoint.
- DNase I footprinting has shown three trans-acting factor binding sites, GFI, GFII, and GFIII, which have AP-2, NFI, and cyclic AMP- responsive element motifs, respectively (Miura et al., 1990).
- the kidney-specific promoter for kidney androgen regulated protein (KAP) has been described by Ding et al., 1997.
- KAP kidney androgen-regulated protein
- adipocyte-specific promoter for adipocyte specific protein (ap2) which is dysregulated in various forms of obesity, has structural similarity to TNFalpha, and is involved in whole body energy homeostasis. It has been described by Hunt et al. to contain sequence information necessary for differentiation-dependent expression in adipocytes (Hunt et al., 1986).
- VEGFR2 vascular endothelial growth factor receptor 2
- RNase protection and primer extension analyses they revealed a single transcriptional start site located 299 base pairs upstream from the translational start site in an initiator-like pyrirnidine-rich sequence.
- the 5 -flanking region was found to be rich in GC residues and lacking a typical TATA or CAAT box.
- a luciferase reporter construct containing a fragment from nucleotides -1900 to +299 showed strong endothelium-specific activity in transfected bovine aortic endothelial cells.
- the liver-specific promoter for albumin was described by Power et al., 1994, who cloned the bovine serum albumin (bSA) promoter. It functions efficiently in the differentiated, but not dedifferentiated, liver cells. Footprint analysis of the promoter revealed seven sites of DNA protein interaction extending from -31 to -213. The deletion of one of these sites, extending from -170 to -236, results in a four fold increase in promoter activity (Power et al., 1994). [091] The pancreas-specific promoter for pancreatic duodenal homeobox 1 (PDX1) was described by Melloul et al., 2002.
- PDX1 pancreatic duodenal homeobox 1
- Upstream sequences of the gene up to about -6 kb were demonstrated to show islet-specific activity in transgenic mice, and several distinct sequences that conferred beta-cell-specific expression were identified.
- a conserved region localized to the proximal promoter around an E-box motif was found to bind members of the upstream stimulatory factor family of transcription factors (Melloul et al., 2002).
- MCK muscle creatine kinase
- the interferon-gamma response was retained in the type II collagen core promoter region spanning -45 to +11 base pairs, containing the TATA-box and GC-rich sequences (Osaki et al., 2003).
- the intestinal epithelial-specific fatty acid binding protein promoter (FABP) was described by Sweetser et al. as both cell-specific and exhibiting regional differences in its expression within continuously regenerating small intestinal epithelium. Sequences located within 277 nucleotides of the start site of intestinal FABP transcription were reported to be sufficient to limit reporter gene (human growth hormone) expression to the intestine.
- Nucleotides -278 to -1178 of the intestinal FABP gene mediated its expression in the distal jejunum and ileum (Sweetser et al., 1988).
- the lung-specific promoter for surfactant protein C was described by Glasser et al. This group identified the transcriptional start site and a TATAA consensus element located 29 base pairs five prime to exon 1 (Glasser et al., 1990).
- the cardiac-specific promoter alpha-myosin heavy chain ( ⁇ -MHC) was described by Molkentin et al.
- Promoters specific for expression in B-cells include the IgM promoter. Promoters specific for expression in T-cells include the CD2, CD4, and CD8 promoters.
- Promoters specific for expression in NK cells include the NKG2D and natural cytotoxicity receptor promoters. Promoters specific for expression in macrophages include the Mac-1 and myeloperoxidase promoters. [099] It has been reported that CD Id presents lipid antigens to a specific population of NK T cells which are involved in host immune defense, suppression of autoimmunity and rejection of tumor cells (Chen and Jackson, 2004). However, the transcriptional regulation and tissue distribution of the expression of CD Id is unknown. The authors herein have identified dual promoters upstream of the open reading frame encoding the CD Id gene.
- the proximal promoter was found to be located within the region -106 to +24, and the distal promoter in the region -665 to - 202 with the A of the start codon located at position +1.
- the region covering the proximal promoter produced a much higher luciferase activity in Jurkat cells (T-cells) than in K562 cells (erythroleukemia cells), whereas the reverse was found with the distal promoter, indicating a cell type specific activity of the two promoters.
- T-cells T-cells
- K562 cells erythroleukemia cells
- the stem cells are transformed with a gene or part of a gene encoding a protein.
- the protein can be any protein, for example, a secreted protein or an extracellular domain of a transmembrane protein.
- the gene or part of a gene, and the protein can be chosen from known databases, e.g., the National Center for Biotechnology Information database.
- the gene or part of a gene, and the protein can also be chosen from PCT/US03/27,107, PCT/US03/27,106, PCT/US03/26,864, and/or PCT/US03/26,780, filed in the United States Receiving Office on August 28, 2003; PCT/US03/33,657, PCT/US03/33725, PCT/US03/33947, and PCT/US03/33948, filed in the United States Receiving Office on October 24, 2003; PCT/US04/002655, filed in the United States Receiving Office on January 30, 2004, PCT/US04/#pending, "Human Polypeptides Encoded by Polynucleotides and Methods for Their Use," filed in the United States Receiving Office on April 19, 2004, PCT/US04/#pending, "Novel Human Polypeptides Encoded by Polynucleotides," filed in the United States Receiving Office on April 19, 2004, and PCT/US04/#pending, "Methods of Use for
- proteins suitable for use in the invention include, but are not limited to, ghrelin, insulin-like growth factor 1, insulin, resistin, tumor necrosis factor alpha, BLyS, and endothelin. These proteins, their ligands or receptors, and active fragments or portions of these are included as among the proteins with which the stem cells can be transformed to produce animal models useful for studying disease processes and screening for therapeutics.
- Growth hormone secretagogues stimulate the release of growth hormone from the pituitary. They are reported to act through the growth hormone secretagogue receptor, a G protein-coupled receptor (Kojima et al., 1999). Ghrelin is an endogenous ligand for the growth hormone secretagogue receptor, reported to be a peptide of 28 amino acids in which the serine-3 residue is n-octanoylated, and known to be involved in regulating growth hormone release both in vivo and in vitro (Kojima et al., 1999).
- the expression of ghrelin by the stem cells of the invention can provide an animal model for the study of energy homeostasis, fat utilization, obesity, and insulin secretion.
- Insulin is synthesized by the beta cells of the islets of Langerhans as proinsulin, which is converted to insulin by the enzymatic removal of a segment that connects the amino end of the A chain to the carboxyl end of the B chain.
- disorders of the insulin gene, insulin secretion, insulin processing, and insulin sensitivity are well known to be causes of diabetes in humans.
- most of the monogenic diabetic syndromes in rodent models, such as ob, db, agouti, tubby, and fat mice also have accompanying obesity.
- the responsible genes are involved in the regulation of body weight, and their alterations result in increased insulin resistance in peripheral tissues, with the exception of "fat" mice.
- the monogenic Akita mouse model for diabetes does not have associated obesity or insulinitis; it has a pancreatic beta cell dysfunction, specifically, a secretory defect, observed as a reduction in secretory granules and enlargement of the endoplasmic reticulum (Yoshioka, et al., 1997). Therefore, the expression of insulin by the stem cells of the invention can provide an animal model for the study of diabetes both in ES chimeras made from normal mice, and ES chimeras prepared from a mouse with a diabetic syndrome.
- Insulin-like growth factor 1 also known as somatomedin C, has a structural homology to insulin. It is recognized to stimulate skeletal muscle hypertrophy by activating the calcium calmodulin-dependent phosphatase calcineurin and inducing the nuclear translocation of the transcription factor NFATCl (Semsarian et al., 1999). In Duchenne muscular dystrophy, the normal regenerative capacity of skeletal muscle cannot compensate for increased susceptibility to damage, leading to repetitive cycles of degeneration and regeneration that ultimately result in the replacement of muscle fibers with fibrotic tissue.
- IGF1 enhances muscle regeneration and protein synthetic pathways
- Barton et al., 2002 hypothesized that muscle-specific expression of IGF 1 could preserve muscle function in the mdx mouse model of Duchenne muscular dystrophy.
- Transgenic mdx mice overexpressing IGF1 in muscle showed increased muscle mass, increased force generation, reduced f ⁇ brosis, and decreased myonecrosis compared with mdx mice.
- signaling pathways associated with muscle regeneration and protection against apoptosis showed significantly elevated activities.
- IGF1 was observed to cause tyrosine phosphorylation and stabilization of beta-catenin in human colorectal cancer cells, contributing to transformation, cell migration, and a propensity for metastasis in vivo (Playford et al., 2000). Low IGF1 levels have been demonstrated to be associated with type 2 diabetes and cardiovascular disease (Arends et al., 2002). Therefore, the expression of IGF 1 by the stem cells of the invention can provide an animal model for the study of signaling pathways and pathological processes, e.g., colon cancer, type 2 diabetes, and cardiovascular disease both in ES chimeras made from normal mice, and ES chimeras prepared from mice with syndromes caused by or affected by IGF1.
- signaling pathways and pathological processes e.g., colon cancer, type 2 diabetes, and cardiovascular disease both in ES chimeras made from normal mice, and ES chimeras prepared from mice with syndromes caused by or affected by IGF1.
- Tumor necrosis factor alpha is recognized in the art as a multifunctional proinflammatory cytokine, with effects on lipid metabolism, coagulation, insulin resistance, and endothelial function. It demonstrates antitumor effects, has been implicated in the pathogenesis of psoriasis, and has been observed to mediate hepatocellular apoptosis (Garcia-Ruiz et al., 2003). TNF ⁇ has also been implicated in the pathophysiology of insulin resistance; circulating TNF ⁇ levels correlate with insulin resistance, and it is associated with a predisposition to progress to insulin dependency (Obayshi et al., 2000).
- B-lymphocyte stimulator (BlyS) is a member of the TNF ligand superfamily that has been reported to have both a soluble and a transmembrane form (Moore et al., 1999). BlyS has also been reported to activate B cells, inducing them to increase their immunoglobulin concentration (Moore et al., 1999). BlyS has been implicated in systemic lupus erythrematosis (SLE), an autoimmune disease; patients with SLE have increased serum levels of BlyS (Zhang et al., 2001).
- SLE systemic lupus erythrematosis
- BlyS may play an antiapoptotic role in B-cell tolerance and that inhibiting BlyS can provide therapeutic benefit for SLE and other autoimmune diseases. It has also been reported that patients with SLE displayed marked heterogeneity with 50% to 61% of patients manifesting persistently or intermittently elevated serum BLyS and blood BLyS mRNA phenotypes, respectively. Surface BLyS and expression by SLE peripheral blood mononuclear cells was also often increased (Stohl et al., 2003). Overexpressing BLyS protein in BLyS-transgenic mice was observed to result in B cell hype ⁇ lasia, hypergammaglobulinemia, and development of autoimmune-like disease (Nardelli et al., 2002).
- the expression of BLyS by the stem cells of the invention can provide an animal model for the study of B-cell function and autoimmune disease, e.g., SLE.
- Resistin resistance to insulin
- Resistin gene expression has been observed to be induced during adipocyte differentiation, and its level is increased in both genetic and diet-induced obesity. As a result, it has been hypothesized that resistin is a hormone that links obesity to diabetes (Steppan et al., 2001). Therefore, the expression of resistin by the stem cells of the invention can provide an animal model for the study of obesity and diabetes.
- Endothelin is a vasoconstrictor peptide produced by vascular endothelial cells. It has been reported to have effects on the central nervous system and on neuronal excitability (Giaid et al., 1989). Endothelin is a signaling ligand that has been reported to act upon multiple receptor subtypes; some of which signal via an increase in intracellular calcium and/or tyrosone kinase (Maggi et al., 2000). Tumor- produced endothelin- 1 was observed to stimulate new bone formation in vitro and osteoblastic metastases in vivo via the endothelin-A receptor.
- stem cells can be transformed with one or more nucleic acid constructs encoding the protein of interest and transformed stem cells that express the protein on their surfaces can be used to immunize a host to produce antibodies.
- the protein may be derived from the same species as the stem cell or from a different species.
- fragments of the stem cell such as membrane fragments that include a transmembrane protein, can be used to immunize the subject of interest to produce antibodies.
- antibodies can be recovered from the subject, for example from blood.
- spleen cells can be obtained from the vaccinated organism, and the recovered cells can be fused to immortalized cells to produce hybridomas, as described further below.
- mRNA can be extracted from the spleen cells and mRNA encoding an antibody of interest can be selected and used to generate cDNA molecules or a cDNA library, or to produce recombinant antibodies, also as described below.
- Proteins can be produced in mini-libraries that provide sets of related proteins. For example, a mini-library of extracellular protein domains, or of secreted proteins are provided by the invention. These mini-libraries provide an efficient way of screening stem cell factors, and for producing proteins for other uses, e.g., screening.
- the nucleic acids expressed by the embryonic stem cells of the invention can be associated with the intracellular pathways that mediate cellular function, physiological homeostasis, and/or disease processes. Examples of some of these pathways, and the genes and gene products that comprise these pathways, are provided below.
- Wnt genes encode a family of conserved signaling molecules and/or growth factors implicated in oncogenesis and developmental processes, such as regulating cell behavior during embryogenesis. Members of the Wnt family activate receptor-mediated signal transduction pathways, resulting in changes in gene expression, which in turn lead to changes in cell behavior, cell adhesion, and cell polarity.
- Wnt genes and Wnt signaling pathways are implicated in pathologies as diverse as cancer, epilepsy, bipolar disorder, and hypersecretory lung diseases.
- Wnt proteins are signaling molecules and are generally secreted. They adhere to the plasma membrane of the cells from which they are secreted, and thus, are likely to signal over relatively short distances from their origin.
- Wnt proteins are ligands for receptors with seven transmembrane regions that comprise the "frizzled" gene family. Wnt protein ligands bind frizzled receptors, resulting in the generation of an intracellular signal.
- This signal can diversify into interconnecting pathways. There is cross-talk among these pathways, and these pathways can also interact with other, non- Wnt, signaling pathways (Polakis, 2000). Such interactions among signaling pathways provide a mechanism for upstream components of one pathway to modulate downstream events in another pathway.
- the pathogenic expression of genes in growth factor signaling pathways can contribute to altered cell growth associated with proliferative disorders. The constitutive activation of growth factor signaling pathways through genetic alterations affecting these genes contributes to the development and progression of most, if not all, human cancers (Bast et al., 2000). Oncogenes can be abnormal counte ⁇ arts of membrane- spanning growth factor receptors.
- the v-sis oncogene of simian sarcoma virus encodes a growth factor homologous to the B chain of human platelet-derived growth factor (PDGF-B).
- PDGF-B platelet-derived growth factor
- the cytokine colony stimulating factor 1 (CSF-1), also known as macrophage-colony stimulating factor (M-CSF) is another growth factor in the PDGF family.
- M-CSF promotes the growth and maturation of monocytes and macrophage precursors and enhances the phagocytic and tumoricidal activity of human macrophage/monocytes, inducing them to secrete a variety of different cytokines.
- GM-CSF granulocyte-macrophage colony-stimulating factor
- cytokines examples include interleukins, interferons, and colony stimulating factors of the hematopoeitic system.
- Cytokine signaling pathways can effect cytoskeletal changes in response to events in the extracellular matrix (Hirsh et al., 2001).
- Some cytokine signaling pathways e.g., interferons and interleukins, can be induced by viral activity, and possess antiviral activity (Sheppard et al., 2003).
- EGF Epidermal growth factor
- EGFR epidermal growth factor
- the EGFR was identified and isolated by biochemical techniques and shown to be the cellular homologue of the v- erbB, a retroviral oncogene member of the EGF/ErbB receptor family, which includes, e.g., the EGFR and ErbB-2; the latter is also known as homologue of the human EGF receptor (HER-2) (Khazaie et al., 1994).
- the fibroblast growth factors (FGF) and their receptors comprise another multigene family of signaling pathways that exhibits mitogenic activity toward a wide variety of cells of mesenchymal, neuronal, and epithelial origin.
- FGF fibroblast growth factors
- An example of a member of the FGF family is keratinocyte growth factor (KGF or FGF- 7); it is specifically expressed in stromal cells of most major epithelial tissues, where its signaling pathways play a role in epithelial renewal during wound repair, as stromal mediators of epithelial cell proliferation/differentiation, and in the normal mesenchymal stimulation of epithelial cell growth (Rubin et al., 1989).
- Apoptosis or programmed cell death, is a regulated signaling process leading to cell death via a series of well-defined mo ⁇ hological changes. Signaling pathways that lead to apoptosis are balanced with those that lead to cell survival. This maintains a balance between cell growth and multiplication, and the necessary elimination of unnecessary cells. The default state of the cell is to remain alive. A cell enters the apoptotic pathway when an essential factor is removed from the extracellular environment or when an internal signal is activated. Genes and proteins of the invention that suppress the growth of tumors by activating cell death provide the basis for treatment strategies for hype ⁇ roliferative disorders and conditions.
- NF- ⁇ B transcription factor NF- ⁇ B control a wide range of genes and are involved in disease processes.
- Many different intracellular signals induce activation of NF- ⁇ B for example, hormones, stress, bacterial or viral infection, UV irradiation, B or T-cell activation, LPS, and certain cytokines, e.g., TNF or IL-1.
- NF- ⁇ B is a heterodimer of two related proteins of 65 kDa and 50 kDa (p65 and p50) that share a region of homology at their N-termini required for DNA binding and dimerization.
- p65 and p50 a region of homology at their N-termini required for DNA binding and dimerization.
- NF- ⁇ B In resting cells, NF- ⁇ B is found in the cytoplasm. In response to an extracellular signal, NF- ⁇ B translocates to the nucleus, where it binds to specific sites in DNA and regulates transcription (Lodish et al, 1999).
- NF- B is sequestered in an inactive state in the cytoplasm by direct binding to its inhibitor I- ⁇ B, which masks the nuclear localization sequences.
- I- ⁇ B In response to an extracellular signal, I- ⁇ B is phosphorylated at two N-terminal serine residues, targeted for ubiquitination, and degraded in the proteasome. NF- ⁇ B then translocates to the nucleus, where it binds to specific DNA sequences and regulates gene expression. For example, NF- ⁇ B has been described to activate the transcription of immunoglobulin kappa light chains in B lymphocytes (Lodish et al., 1999). [0124] Both peptide hormones and steroid hormones can regulate signal transduction pathways that utilize NF- ⁇ B.
- peptide hormones may activate protein kinase C via the inositol pathway, releasing NF- ⁇ B from I- ⁇ B, thus permitting its translocation to the nucleus.
- Steroid hormones may inhibit NF- ⁇ B- regulated gene transcription, e.g., an anti-inflammatory glucocorticoid can bind to its receptor and induce a heat shock protein that mediates the translocation of NF- ⁇ B to the nucleus, but prevents the transcription of certain pro-inflammatory proteins, such as interleukin-6.
- STAT Signal transducers and activators of transcription
- STAT can follow cytokine, growth factor, and/or hormone signaling, and can be mediated by tyrosine phosphorylation, normally a transient and tightly regulated process.
- Signaling pathways that comprise STAT proteins participate in such normal cellular events as differentiation, proliferation, cell survival, apoptosis, and angiogenesis.
- Constitutively activated STATs have been observed in a wide number of human cancer cell lines and primary tumors, including blood malignancies, e.g., leukemias, and solid neoplasias, e.g., breast cancer.
- STAT 5 is one of a number of STATS involved in interferon signaling and activated by a series of ligands.
- STATS play a role in controlling cell-cycle progression and apoptosis. They are involved in cellular responses to growth hormone, the development of mammary glands, and embryogenesis, and they contribute to oncogenesis.
- STAT 5 is considered an oncogene, since it can activate genes involved in promoting cell-cycle progression, cellular transformation, and in preventing apoptosis, e.g., cyclin Dl, c- Myc, and bcl-xl (Calo et al., 2003).
- STAT 5 is activated during myeloid differentiation in a cell-type and maturation state-dependent manner.
- STAT 3 and/or STAT 5 results in enhanced transcription of anti- apoptotic cell-cycle progression genes, thereby contributing to the pathogenesis of myeloid leukemias (Coffer et al., 2000).
- Endothelin (ET) signaling pathways have been described as vasoconstrictors, and to evoke diverse responses such as contraction, secretion, and cell growth in glomerular mesangial cells (Simonson et al., 1992).
- Components of ET signaling pathways include several G-protein linked endothelin receptors, one or more ion channel, one or more cytosolic protein kinase, one or more mitogen-activated protein kinase, and one or more inducible transcription factor, e.g., activator protein- 1 (Simonson et al., 1991).
- endothelins are produced as pro-proteins with specific sites of cleavage.
- Endothelin converting enzyme produces active endothelins by metallopeptidase cleavage of pro-endothelins.
- ET-1 is the specifically expressed in endothelial cells, smooth muscle cells, and cardiac myocytes (Simonson et al., 1992).
- Adrenergic receptor signaling pathways comprise catecholamines, e.g., epinephrine and norepinephrine as ligands, and various subtypes of ⁇ and ⁇ - adrenergic receptors. These pathways are present, inter alia, in smooth muscles that are innervated by sympathetic autonomic motor neurons. Stimulation of these peripheral neurons increases the activity of the heart and internal organs in "fight or flight" reactions. Norepinephrine is also found at synapses in the central nervous system.
- Adrenergic receptors are generally coupled to G proteins; different receptors are linked to different G proteins, and their activation leads to changes in the levels of different intracellular second messengers. For instance, binding of norepinephrine to ⁇ -adrenergic receptors on nerve cells can activate G s and increase cAMP synthesis. Other neuronal adrenergic receptors can activate G;, G 0 , or other types of G proteins, and decrease cAMP levels or increase levels of other intracellular second messengers, such as cGMP, inositol 1,4,5-trisphosphate (IP 3 ), diacylglycerol, and arachidonic acid (Lodish et al. 1999).
- cGMP inositol 1,4,5-trisphosphate
- IP 3 inositol 1,4,5-trisphosphate
- diacylglycerol diacylglycerol
- arachidonic acid Lidish et al. 1999.
- Tumor necrosis factor (TNF) ligand superfamily e.g., LT-alpha
- TNF tumor necrosis factor
- LT-alpha Tumor necrosis factor
- TNF receptor superfamily e.g., LT-alpha
- the receptor activator of NF- ⁇ B ligand/osteoclast differentiation factor (RANKL/ODF) a member of the TNF ligand superfamily, mediates its signal transduction via TNF receptor-associated factor (TRAF) proteins, which are involved in osteoclast differentiation and activation (Gravallese et al., 2001 ).
- Signal transduction molecules form pathways that convert extracellular signals into cellular responses.
- Cells use myriads of signal-transduction pathways to regulate cellular metabolism, function, and development.
- Signal transduction pathways comprise many different classes of proteins, among them ligands, receptors, kinases, phosphatases, proteases, transcription factors, GTPase switch proteins, which are in their active conformation only when bound to GTP, and adapter proteins, which hold together multiprotein signaling pathways.
- the ability of cells to respond appropriately to extracellular and intracellular signals depends on proper regulation of signal transduction pathways. Abnormalities in signal transduction underlie many different diseases, including the majority of cancers and many inflammatory conditions.
- Stimuli that activate intracellular signal transduction pathways activate specific, known promoters. This activation can be measured by reporter systems linked to specific promoters, each of which can be linked to a "readout" signal, such as the expression of an easily detectable protein or the expression of a selectable marker.
- the reporter is generally introduced into a cell by transfecting DNA encoding the promoter and the "readout" signal. In current practice, a reporter is transfected into one cell type at a time.
- the ES cells are transformed with . gene trap vectors, e.g., ROSA26 and G3BP, which are ubiquitously expressed and encode proteins involved in disease processes.
- the animal models of the invention can provide information regarding the components, function, and effects of intracellular pathways. They can also be used to identify pathway components that are rationally targeted in drug development and to test the effect of therapeutic agents on the target pathway components.
- the animal models of the invention can provide this information for many disease processes; by way of example, they can be used to provide information about the roles of secreted proteins, transcription factors, and receptors in disease processes.
- Transformed stem cells can also be used in methods of determining gene function in vivo. For example, a gene of interest can be used to target a specific locus in an ES cell, e.g., a locus described above, such as the Rosa 26 locus.
- the transformed stem cell can be injected into an embryonic precursor, such as a blastocyst, using standard techniques, and the blastocyst can be implanted into the uterus of an animal, e.g., a non-human animal, such as a mouse, by methods well known in the art.
- the blastocyst can then be allowed to develop into a chimeric embryo and chimeric fetus in vivo, and ultimately, a chimeric animal can be produced, such as a chimeric mouse.
- the chimeric embryo, fetus, or animal produces the product encoded by the gene of interest in multiple tissues, such that the effect of the gene product on the embryo, fetus or animal, can be determined.
- Cell lines can be produced from cells or tissues obtained from the chimeric embryo, fetus or animal above.
- the ES cells can be used to make chimeric animals that have the reporter expressed in various specified tissues, such as by use of tissue specific promoters. Tissues removed from these animals, and/or cell derived thereof, provide a means to study these processes. A gene or gene product with restricted tissue distribution can be studied using various biochemical and localization methods well known in the art by applying these methods to the tissue upon its removal from the animal.
- This in vivo reporter system can be used to test drug efficacy, toxicity, pharmacokinetics, and metabolism.
- a gene of interest can be used to target a specific locus in an ES cell, e.g., a locus described above, such as the Rosa 26 locus, and the transformed embryonic stem cell can be provided to a tissue of an animal, for example, delivered to an immunocompromised animal such as a nude mouse.
- the embryonic stem cell can then develop into a chimeric neoplasm, such as a teratoma, and the effect of the gene product on the neoplasm can be determined, thus providing information on the action of particular agents on cancerous and precancerous cells.
- the chimeras of the invention can be used in assays for screening, testing and comparing agents or libraries of agents.
- the agents can be genes, proteins, peptides, small molecules, and the like, and any convenient multiplex testing configuration can be used.
- the chimera can be used to study the effect of an agent on signal transduction.
- the chimeras, their progeny, and their tissues and cells can be used to detect combination effects, that is, the effect of the gene of interest as well as any additional factors or cells, on signal transduction.
- the additional factors include, for example, factors in solution, and factors secreted by cells or present as extracellular portions of transmembrane proteins.
- the activity of the signal transduction polypeptides encoded by the nucleic acids that transfect the embryonic stem cell can be assayed.
- the gene encoding the protein is expressed, and the modulation of the chimera transformed with the gene are observed.
- changes in function e.g., by direct observation of the signal transduction pathways, or by observation of a phenotype, e.g., the rate of proliferation, a lack of proliferation, and/or differentiation of the genetically-altered mice can be observed.
- This aspect provides a method of determining an in vivo effect of a therapeutic by administering the therapeutic to a chimeric animal or its progeny and determining the tissue or cell type in which the reporter system is activated by a signal transduction pathway.
- the therapeutic can, e.g., be a protein therapeutic or a small molecule therapeutic.
- the invention also provides a method of determining one or more components of a signal transduction pathway by administering a molecule or compound to a chimeric animal or its the progeny and determining the one or more genes activated by the molecule or compound.
- references to “a subject polypeptide” includes a plurality of such polypeptides and reference to “the agent” includes reference to one or more agents and equivalents thereof known to those skilled in the art, and so. forth.
- all numbers expressing quantities of ingredients, reaction conditions, % purity, polypeptide and polynucleotide lengths, and so forth, used in the specification and claims, are modified by the term "about,” unless otherwise indicated. Accordingly, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties of the present invention.
- each numerical parameter should at least be construed in light of the number of reported significant digits, applying ordinary rounding techniques. Nonetheless, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors from the standard deviation of its experimental measurement. [0141] With respect to ranges of values, the invention encompasses each intervening value between the upper and lower limits of the range to at least a tenth of the lower limit's unit, unless the context clearly indicates otherwise. Further, the invention encompasses any other stated intervening values.
- the invention also encompasses ranges excluding either or both of the upper and lower limits of the range, unless specifically excluded from the stated range.
- Examples [0142] The examples, which are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way, also describe and detail aspects and embodiments of the invention discussed above. The examples are not intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
- a targeting vector for secreted or other molecules targeting to the ROSA26 locus was constructed as shown in Fig. 1.
- the PGKneobpA fragment was made by combining PGKneo from New England Biolabs (Beverly, MA) and bovine growth hormone poly A (bpA) from BD Biosciences Clontech (Palo Alto, CA).
- the adenovirus major late transcript splicing acceptor (SA) was PCR amplified from adenovirus genomic DNA.
- TK gene was PCR amplified from a cosmid vector svPHEP from ATCC (Manassas, VA).
- the 5' and 3' homologous arms were PCR amplified and cloned from genomic DNA according to a public genomic database, e.g., NCBI.
- the basic targeting vector without the gene of interest was made by inserting a fragment containing SA, the Gateway cassette (Invitrogen, Carlsbad, CA), polyA, and PGKneo between the 5' and 3' homologous arms of the ROSA 26 targeting arms.
- a gene of interest such as a secreted factor of interest, can be cloned into a master ROSA26 targeting vector, as shown in Figure 1.
- the endogenous ROSA26 promoter will drive the expression of the secreted factor.
- the ROSA26 promoter resides in the 5' homologous arm.
- the 5' and 3' homologous arms and the TK (HSV thymidine kinase) gene can be used to target the secreted factor to the ROSA 26 locus in the mouse ES cells.
- the PGKneobpA can be used as a selection marker for the targeting experiment.
- the S A and bpA sequences facilitate the expression of the secreted factor.
- the above described targeting fragment can be cloned into the multiple cloning site of a plasmid (e.g. pBluescript from Stratagene, La Jolla, California).
- the plasmid backbone is not shown in the figure.
- the master targeting vector was constructed by cloning the SA (PCR amplified) and bpA fragment into pBluescript. Then the PGKneobpA was cloned 3 ' to the bpA fragment.
- the Gateway conversion cassette (Invitrogen, Carlsbad, CA) was then cloned between SA and bpA. Then the whole fragment containing the SA, Gateway cassette, polyA, and PGKneo was cloned between the 5' and 3' homologous arms of the ROSA 26 targeting arms (the 5' and 3' homologous arms were PCR amplified and cloned from genomic DNA according to the public genomic database). Only the targeted clones were demonstrated to have a PCR product.
- the genes of interest can be cloned into a Gateway entry vector first and subsequently cloned into the ROSA 26 targeting vector by the Gateway cloning technology (Invitrogen, Carlsbad, California).
- Example 2. Targetability as a Screening Method for Identifying Potent Factors that Inhibit ES Cell Proliferation or Induce Differentiation
- the same homologous arms as above are used for targeting all the secreted molecules to the ROSA 26 locus.
- the initial number of secreted molecules selected for targeting/expression is about 100-200.
- the 'potent' factors that inhibit ES cell growth or induce differentiation can be discovered by the fact that no targeted clones can be obtained solely for these clones.
- ES cells can differentiate into mature hematopoietic cells under defined experimental conditions. For example, erythropoietin and/or interleukin I a (IL-la) plus IL 3 can induce this differentiation.
- Example 3 The multiple pools of ES cells generated as described in Example 3 are used to test the ability of the secreted factor combinations to induce ES cells' differentiation potential to pancreatic beta cells.
- a number of beta cell markers e.g. insulin, PDX-1, PAX-4, PAX-6, Nkx2.2 andNkx ⁇ .l, insulin I, insulin II, glucose transporter 2 are used to track the differentiation.
- Example 6. In Vivo Use of ES Cells Containing Introduced Nucleic Acid Molecules [0151] A pool of ES cells were transformed with a nucleic acid molecule of interest using standard techniques and maintained in culture in vitro under conditions that allow proliferation but not differentiation.
- Transformed ES cells were obtained from the pool and introduced into a normal mouse blastocyst to produce a chimeric blastocyst.
- a normal mouse blastocyst typically from about 4-50 transformed ES cells, more typically, about 10- 40 ES cells and particularly about 20 ES cells were injected into a blastocyst, such that about 1 out of 10 to about 6 out of 8 cells in the blastocyst represent transformed ES cells.
- the chimeric blastocyst can then be implanted into the uterus of a suitable surrogate female mouse for further embryonic development.
- One or more of such blastocysts for example up to eight, can be implanted per surrogate female mouse.
- a chimeric mouse Upon birth of a chimeric mouse from a chimeric blastocyst, the chimeric mouse can be studied to determine the function of the introduced nucleic acid molecule. Standard methods implantation are known in the art. See, e.g., C.L. Stewart, p. 823 in Wassarman and DePamphilis, 1993).
- any suitable function of the introduced nucleic acid molecule can be examined using the chimeric mouse, including but not limited to, for example, ligand function or receptor function; its function in tissue mo ⁇ hogenesis; stimulation of differentiation, stimulation of proliferation, inhibition, or activation of different cellular systems, including: (1) hematopoietic system including T cells, B cells, NK cells, dendritic cells, monocytic, other cells of the hematopoietic system; (2) beta islet cells of the pancreas; (3) chondrocytes; (4) bone marrow system including osteoclasts, osteoblasts, and stromal cells; (5) cardiovascular system including cardiomyocytes; (6) CNS and spinal cord including neuronal cells; and so forth.
- the chimeric mouse, tissues and cells thereof can be dissected for observation of their functional states, including but not limited to: number of the target cell type that is of interest, such as T cells, for example, the stage of development of the cell, whether it is mature or immature, the functional status of the cell, and the activation status of the cell, etc.
- Example 7 Animal Models for Obesity and Diabetes
- Transformed stem cells can be used to determine gene function in vivo. A gene of interest in the pathogenesis of diabetes is targeted to the Rosa 26 locus of an ES cell, and the transformed stem cell is then injected into a blastocyst using standard techniques.
- the blastocyst is implanted into the uterus of a mouse, by methods well known in the art.
- the blastocyst develops into a chimeric embryo then chimeric fetus in vivo, and ultimately, a chimeric mouse is produced.
- the chimeric embryo, fetus, and/or animal produces the product encoded by the gene of interest in multiple tissues, such that the effect of the gene product on the embryo, fetus or animal, can be determined.
- Cell lines are produced from cells or tissues obtained from the chimeric embryo, fetus or animal.
- the ES cells can be used to make chimeric mice with a reporter gene expressed in various specified tissues, by tissue-specific promoters.
- mice are useful in testing or determining which tissues respond to protein factors or small molecules administered to the animals. Such tests can be performed in vivo, or in vitro from tissues or cells removed from the animal. A gene or gene product with restricted tissue distribution can be studied using various biochemical and localization methods well known in the art by applying these methods to the tissue upon its removal from the animal. Cells and their progeny can be studied in a similar manner.
- the chimeric mice can be produced by inserting ES cells into normal mouse blastocysts, or, alternatively, into blastocysts of mouse disease models for human diabetes and/or obesity, as described above.
- Example 8 Animal Model for Post-translationally Modified Proteins
- S ome proteins need to be processed or modified post-translationally in order to function in their differentiated or mature state.
- insulin and IGF-1 are cleaved by two processing enzymes PCI/3 and PC2.
- the processing enzymes have a tissues restricted expression pattern.
- PC 1/3 and PC2 together are specifically expressed in insulin producing beta cells of the pancreas.
- the gene trap vectors of the invention described above, provide a method to express the protein of interest in the relevant cell and tissue types.
- Ghrelin is another example of a post-translationally modified protein; it is modified (n-octanoylated) on the serine-3 residue. The modification is necessary for it biological function.
- the gene trap vectors of the invention, described above, provide a method to express the protein of interest in the cells of the stomach and the neurons to which its localization is restricted. [0159] Accordingly, stem cell libraries and methods of using the same are disclosed.
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Abstract
Selon l'invention, une banque de cellules souches est créée par modification génétique de cellules souches avec des acides nucléiques codant pour des polypeptides pouvant favoriser la différenciation des cellules souches en types de cellules spécifiques. Selon une autre variante, la banque de cellules souches est exposée à un facteur ajouté par voie externe favorisant la différenciation des cellules souches en une lignée cellulaire souhaitée, telle qu'une lignée de cellules neuronales ou musculaires. Cette banque est utilisée pour déterminer l'effet de la protéine codée sur le processus de différenciation. Ladite banque est également utilisée pour produire des acides nucléiques destinés à être insérés dans des cellules souches embryonnaires en vue de la production de cellules souches embryonnaires transfectées. Les acides nucléiques sont insérés dans un locus permettant l'expression étendue du polypeptide codé chez des animaux produits à partir de blastocystes comprenant les cellules transfectées. Des animaux chimériques non humains produits par combinaison de blastocystes dérivés de modèles animaux d'une maladie humaine et de cellules souches embryonnaires transfectées avec des molécules issues de cette banque permettent d'obtenir un système in vivo pour la conception thérapeutique.
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USPCT/US03/26780 | 2003-08-28 | ||
US60/505,144 | 2003-09-24 | ||
USPCT/US03/33948 | 2003-10-24 | ||
USPCT/US03/33725 | 2003-10-24 | ||
USPCT/US03/33657 | 2003-10-24 | ||
USPCT/US03/33947 | 2003-10-24 | ||
USPCT/US03/34811 | 2003-10-31 | ||
PCT/US2003/034811 WO2004042023A2 (fr) | 2002-11-01 | 2003-10-31 | Bibliotheques de cellules souches |
US60/534,403 | 2004-01-07 | ||
USPCT/US04/02655 | 2004-01-30 | ||
US60/548,191 | 2004-03-01 | ||
USPCT/US04/11912 | 2004-04-19 | ||
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WO2006076288A2 (fr) * | 2005-01-11 | 2006-07-20 | Five Prime Therapeutics, Inc. | Constructions d'adn pour l'expression a long terme d'adn nu en injection intravasculaire |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2005116070A2 (fr) * | 2004-04-28 | 2005-12-08 | Five Prime Therapeutics, Inc. | Systeme rapporteur destine a detecter l'activation d'une voie de signalisation dans de multiples types de cellules |
WO2005116070A3 (fr) * | 2004-04-28 | 2006-06-15 | Five Prime Therapeutics Inc | Systeme rapporteur destine a detecter l'activation d'une voie de signalisation dans de multiples types de cellules |
WO2006076288A2 (fr) * | 2005-01-11 | 2006-07-20 | Five Prime Therapeutics, Inc. | Constructions d'adn pour l'expression a long terme d'adn nu en injection intravasculaire |
WO2006076288A3 (fr) * | 2005-01-11 | 2006-12-21 | Five Prime Therapeutics Inc | Constructions d'adn pour l'expression a long terme d'adn nu en injection intravasculaire |
US11377637B2 (en) | 2016-04-15 | 2022-07-05 | Memorial Sloan Kettering Cancer Center | Transgenic T cell and chimeric antigen receptor T cell compositions and related methods |
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