WO2005073385A1 - 遺伝子導入効率を上昇させるための組成物および方法 - Google Patents
遺伝子導入効率を上昇させるための組成物および方法 Download PDFInfo
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- WO2005073385A1 WO2005073385A1 PCT/JP2005/001148 JP2005001148W WO2005073385A1 WO 2005073385 A1 WO2005073385 A1 WO 2005073385A1 JP 2005001148 W JP2005001148 W JP 2005001148W WO 2005073385 A1 WO2005073385 A1 WO 2005073385A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2839—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily
- C07K16/2842—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the integrin superfamily against integrin beta1-subunit-containing molecules, e.g. CD29, CD49
Definitions
- compositions and methods for increasing gene transfer efficiency are provided.
- the present invention relates to the field of cell biology. More specifically, the present invention relates to compounds, compositions, devices, methods and systems that increase the efficiency of introducing substances into cells. Background art
- Transfusion is a technique used to transiently express a gene in cells such as animal cells and observe the effects thereof. It is a technique that is frequently used to elucidate the functions of the genes that it encodes.
- the present invention provides a target substance (for example, DNA, polypeptide, sugar, or a complex thereof) that is conventionally difficult to be introduced into cells by diffusion or hydrophobic interaction.
- the task is to develop a method that can improve the efficiency of introduction (in particular, transfer excursion) in any situation.
- the present invention provides the following inventions.
- a composition containing a cell adhesion-related factor for increasing the efficiency of introducing a target substance into cells (1) A composition containing a cell adhesion-related factor for increasing the efficiency of introducing a target substance into cells.
- composition for increasing the efficiency of introducing a target substance into a cell wherein the cell adhesion-related factor includes an interacting substance that interacts with a cell adhesion molecule.
- composition according to item 2 wherein the cell adhesion molecule is an extracellular matrix.
- composition according to item 2 wherein the cell adhesion molecule contains an integrin receptor.
- composition according to item 2 wherein the cell adhesion molecule contains an integrin.
- composition according to item 1 wherein the interacting substance reacts with the partner of the cell adhesion molecule through an antigen-antibody reaction.
- composition according to item 1 wherein the interacting substance is an antibody or a derivative thereof.
- composition according to item 1 wherein the interacting substance is a monoclonal antibody or a polyclonal antibody.
- composition according to item 1, wherein the interacting substance comprises an antibody selected from the group consisting of an anti-CD49a antibody, an anti-CD49b antibody, an anti-CD49c antibody, an anti-CD49e antibody and an anti-CD49f antibody.
- composition according to item 1 wherein the target substance includes a nucleic acid molecule.
- composition according to item 4 wherein the integrin receptor is selected from the group consisting of CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, and CD29.
- composition according to item 4 wherein the integrin receptor is selected from the group consisting of CD29, CD49a, CD49c, CD49d, CD49e and CD49f.
- composition according to item 4 wherein the integrin receptor interacts with a molecule selected from the group consisting of collagen, fibronectin, vitronectin and laminin.
- composition according to item 1 wherein the cells include at least one cell selected from the group consisting of a stem cell and a differentiated cell.
- composition according to item 1 wherein the cell adhesion molecule is specifically expressed in the cell.
- composition according to item 1 wherein the target substance is a genetic substance and further contains a gene introduction reagent.
- composition according to item 18, wherein the gene introduction reagent is selected from the group consisting of a cationic polymer, a cationic lipid, and calcium phosphate.
- composition according to item 1 further comprising particles.
- composition according to item 20 wherein the particles include a gold colloid.
- composition according to item 1 The composition according to item 1, further comprising a salt.
- composition according to item 22 wherein the salt is selected from the group consisting of salts contained in a buffer and salts contained in a medium.
- a kit comprising:
- a composition comprising:
- composition according to item 25 wherein the target substance includes a substance selected from the group consisting of DNA, RNA, polypeptide, sugar, and a complex thereof.
- composition according to item 25 wherein the target substance contains DNA encoding a gene sequence to be transfused.
- composition according to item 25 further comprising a gene introduction reagent.
- composition according to item 25 wherein the cell adhesion-related factor includes an interacting substance that interacts with a cell adhesion molecule.
- composition according to item 25 wherein the cell adhesion-related factor includes an antibody of a cell adhesion molecule.
- composition according to item 25 which is present as a solid phase.
- the cell adhesion-related factor is fixed to a support.
- the target substance includes a substance selected from the group consisting of DNA, RNA, polypeptide, sugar, and a complex thereof.
- the support is selected from the group consisting of a plate, a microwell plate, a chip, a slide glass, a film, a bead, and a metal.
- the coating agent includes a substance selected from the group consisting of poly-L-lysine, silane, MAS, hydrophobic fluororesin, and metal.
- the target substance includes a substance selected from the group consisting of DNA, RNA, polypeptide, sugar, and a complex thereof.
- cell adhesion-related factor includes an interacting substance that interacts with a cell adhesion molecule.
- cell adhesion-related factor includes a cell adhesion molecule antibody.
- a method comprising:
- transfusion efficiency increasing reagent is particularly useful for carrying out a transfusion in a solid phase.
- FIG. 1 shows the relationship between integrin receptors and recognized extracellular matrices.
- FIG. 2A shows various cell adhesion-related factors and 5 shows the results of transfection efficiency using fibronectin.
- FIG. 2B shows the results of transfection efficiency using another cell adhesion-related factor in an experiment using HepG2 cells.
- Figure 3A shows the relationship between various integrin receptors (CD49a, d, f) expressed on HepG2 cells and the extracellular matrix recognized in Example 3, and the transfection on the surface coated with each extracellular matrix. Shows the exciton efficiency.
- Fig. 3B is a cell photograph showing a state of transfection in an experiment using HepG2. Uncoated is a control without coating,
- FIG. 3C is a cell photograph showing a state of transfection in an experiment using HepG2. CD44, CD46 and CD54 are shown.
- Fig. 3D is a cell photograph showing a state of transfection in an experiment using HepG2. CD49a, CD49c and CD49d are shown.
- FIG. 3E is a cell photograph showing a state of transfection in an experiment using HepG2. CD49e, CD49f and HLA are shown.
- Fig. 4 shows the relationship between various integrin receptors (CD49a, d, f) expressed on PC12 cells and the extracellular matrix recognized in Example 4, and the transfection on the surface coated with each extracellular matrix. Shows the exciton efficiency.
- FIG. 5A and 5B show the state of cell adhesion of PC12 cells in Example 4.
- 3 shows the inhibition of PC12 cell adhesion to the TypelV collagen-coated surface using cell adhesion-related factors (eg, CD antibody) and transfection.
- TypelV collagen coated on poly-L-lysine coated slide glass Thereafter, PC12 cells that had been brought into contact with the antibody solution in advance were seeded, and transfection was performed using Lipofectamin2000 according to a normal (liquid phase) protocol.
- Figure 5A shows that anti-CD49a markedly inhibited the adhesion of PC12 cells to the TypelV collagen-coated surface.
- FIG. 5B also shows the difference in transfection efficiency in the presence of anti-CD49f. The displayed values indicate how many times the antibody stock solution was diluted and brought into contact with PC12 cells in advance.
- FIG. 5B shows the state of cell adhesion of PC12 cells in Example 4.
- 3 shows the inhibition of PC12 cell adhesion to the TypelV collagen-coated surface and the transfection using a cell adhesion-related factor (eg, a CD antibody).
- a cell adhesion-related factor eg, a CD antibody
- TypelV collagen coating on poly-L-lysine coated slide glass Thereafter, PC12 cells that had been brought into contact with the antibody solution in advance were seeded, and transfection was performed using Lipofectamin2000 according to a normal (liquid phase) protocol.
- Figure 5A shows that anti-CD49a markedly inhibited the adhesion of PC12 cells to the TypelV collagen-coated surface. Anti-CD49d does not inhibit the adhesion of PC12 cells and shows the same transfection efficiency as Control without antibody.
- FIG. 5B also shows the difference in transfection efficiency in the presence of anti-CD49f. The displayed values indicate how many times the antibody stock solution was diluted and brought into contact with PC12 cells in advance.
- FIG. 6 shows the transfusion effect of the coating on the support of PC12 cells in Example 4. The test was performed using an anti-CD49a antibody, an anti-CD49c antibody, and an anti-CD49d antibody.
- FIG. 7 shows the transfection effect of the coating on the support of PC12 cells in Example 4. The test was performed using an anti-CD49e antibody, an anti-CD49f antibody, and an anti-HLA antibody.
- FIG. 8 shows an outline of a solid-phase transfection using a cell adhesion-related factor.
- the lower right is the same as Fig. 1.
- the upper right shows the results of fluorescence intensity distribution using three types of anti-CD antibodies.
- On the left the relative transfer efficiency index is shown.
- SEQ ID NO: 1 Nucleic acid sequence of fibronectin (human)
- SEQ ID NO: 2 Fibronectin amino acid sequence (human)
- SEQ ID NO 3 Amino acid sequence of CD29 (preform of isoform 1A)
- SEQ ID NO: 4 Amino acid sequence of CD49a
- SEQ ID NO: 5 Amino acid sequence of CD49b
- SEQ ID NO: 6 Amino acid sequence of CD49c (isoform a precursor)
- SEQ ID NO: 7 Amino acid sequence of CD49d (precursor)
- SEQ ID NO: 8 Amino acid sequence of CD49e (precursor)
- SEQ ID NO: 9 Amino acid sequence of CD49f
- SEQ ID NO: 10 Amino acid sequence of CD29 (preform isoform 1B)
- SEQ ID NO: 11 Amino acid sequence of CD29 (preform of isoform 1C-1)
- SEQ ID NO: 12 Amino acid sequence of CD29 (preform of isoform 1C-2)
- SEQ ID NO: 13 Amino acid sequence of CD29 (isoform 1D precursor)
- SEQ ID NO: 14 Amino acid sequence of CD49c (isoform b precursor)
- cell adhesion molecule or “adhesion molecule” is used interchangeably, and means that two or more cells approach each other (cell adhesion) or a substrate and a cell Refers to molecules that mediate adhesion between In general, a cell-cell adhesion (intercellular adhesion) on the molecular (ce ll_ ce ll adhesion molecule) , adhesion between a cell and an extracellular matrix - molecules involved in (cell substrate adhesion) (cell- substrate adhesion molecul e ). Cells usually contain such cell adhesion molecules. Therefore, in this specification, cell adhesion molecules include proteins on the substrate side during cell-substrate adhesion.
- a cell-side protein for example, an integrin receptor
- a cell-side protein for example, an integrin receptor
- a molecule other than a protein mediates cell adhesion
- the concept of the cell adhesion molecule or the adhesion molecule in the present specification is used. to go into.
- cadherin many molecules belonging to the immunoglobulin superfamily (NCAM, Ll, ICAM, facyclin II, III, etc.), selectins and the like are known, and cell membranes are formed by unique molecular reactions. It is also known to combine
- Whether a molecule is a cell adhesion molecule can be determined by biochemical quantification (SDS-PAG method, labeled collagen method), immunological quantification (enzyme antibody method, fluorescent antibody method, immunohistological examination), By determining that a positive result is obtained in an assay such as the PCR method or the hybridization method, the semi-IJ can be determined.
- Such cell adhesion molecules include collagen, integrins, fibronectin, laminin, vitronectin, fibrinogen, the immunoglobulin parfamily (eg, CD2, CD4, CD8, ICM1, ICAM2, VCAM1), selectin, cadherin, CD29. , CD49 family and the like. Many of such cell adhesion molecules transmit an auxiliary signal for cell activation by cell-cell interaction into the cell simultaneously with cell adhesion.
- cell adhesion molecules for example, cadherin is widely known as a cell adhesion molecule in tissue-fixing cell lines.
- cell adhesion molecules include, for example, immunoglobulin superfamily molecules (CD2, LFA_3, ICAM_1, CD2, CD4, CD8, ICM1, ICAM2, VCAM1, etc.) Integrin family molecules (LFA-1, Mac-1, gpllbllla, pl50, 95, VLA1, VLA2, VLA3, VLA4, VLA5, VLA6, etc.); selectin family molecules (L-selectin, E-selectin, P-selectin, etc.) ); CD29, CD49 family (CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, etc.), and the like, but are not limited thereto. Therefore, such minutes Offspring can be particularly useful for treating tissues or organs of the blood-immune system.
- Cell adhesion molecules are required for fixing non-fixed cells. In this case, it is thought that the adhesion between cells is gradually strengthened by the primary adhesion by a constantly expressed selectin molecule and the like, followed by the secondary adhesion of an activated intedarin molecule and the like.
- cell adhesion molecules contemplated in the present invention may include such factors that mediate primary adhesion, factors that mediate secondary adhesion, or both, with secondary adhesion being preferred. It seems.
- the cell adhesion molecule is a protein, its active site has been elucidated at the amino acid level, and RGD, YIGSR, etc. are known (these are also collectively referred to as the RGD sequence).
- cell adhesion-related factor refers to a factor that suppresses the activity of a cell to adhere to another substance (eg, a support, another cell, or the like).
- factors include, but are not limited to, interactors that interact with cell adhesion molecules.
- Such an interacting substance is confirmed to have an adhesion inhibitory effect due to the coexistence of the interacting substance when cells are seeded on a surface coated with an ECM substrate such as fibronectin.
- an increase in the adhesion of cells to the surface is confirmed.
- the interacting substance examples include a substance that interacts with a competitor in an allosteric manner, a partner in an antigen-antibody reaction (an antibody in the case of an antigen, an antigen in the case of an antibody), and a partner having a receptor-one ligand relationship (such as a receptor)
- a partner in an antigen-antibody reaction an antibody in the case of an antigen, an antigen in the case of an antibody
- a partner having a receptor-one ligand relationship such as a receptor
- RGD molecule refers to a protein molecule containing the amino acid sequence RGD (Arg-Gly-Asp) or a functionally identical sequence thereof.
- the RGD molecule is characterized by containing RGD, which is an amino acid sequence useful as the amino acid sequence of the cell adhesion active site of the cell adhesion protein, or another functionally equivalent amino acid sequence.
- RGD sequence was discovered as a cell adhesion site in fibular mouth nectin, and subsequently, many cell adhesion properties such as type I collagen, laminin, vitronectin, fibrinogen, von Willebrand factor, and entactin.
- the biomolecule in the present invention may be a chemically synthesized RGD molecule.
- RGD molecules include, but are not limited to, the GRGDSP peptide, for example, in addition to the naturally occurring molecules described above. Since the RGD sequence is recognized by integrin (eg, CD49 family, CD29, etc.), which is a cell attachment molecule (and also a receptor), a functionally equivalent molecule of RGD would recognize such an integrin. Can be identified by examining the interaction.
- integrin or "integrin receptor” is used interchangeably and refers to a transmembrane glycoprotein that is a receptor involved in cell adhesion. Integrins are present on the cell surface and function when cells adhere to the extracellular matrix. It is known that blood cells and the like are also involved in adhesion between cells. Such integrins include, for example, receptors such as fibronectin, vitronectin, and collagen, lib / Ilia of platelets, Mac-1 of macrophages, LFA-1 of lymphocytes, VLA-16, PSA of Drosophila. And the like, but are not limited thereto.
- Integrin usually takes the form of a heterodimer in which a ⁇ -chain with a molecular weight of 130 kDa-210 kDa and a chain with a molecular weight of 95 kDa-130 kDa are covalently and one-to-one associated with each other.
- the ⁇ chain is not limited to a powerful force S such as ⁇ , ⁇ 2, ⁇ 3, ⁇ 4, ⁇ 5, ⁇ 6, ct L, ⁇ , ⁇ , allb, aV, ⁇ . .
- Examples of the chain include, but are not limited to, 1, ⁇ 2, ⁇ 3, 4, ⁇ 5, ⁇ 6, 7, and the like.
- heterodimers examples include, for example, VLA-1, VLA-2, VLA-3, VLA-4, VLA-5, VLA-6, CD51 / CD29, CD49 Family, LFA-1, Mac-1, pl 50, 90, vitronectin receptor, / 34 subfamily, j35 subfamily, ⁇ 6 subfamily, LPAM_1, HML-1, and the like.
- the extracellular domain of the heavy chain has a divalent cation binding site
- the extracellular domain of the / 3 chain has a cysteine-rich region
- the intracellular domain of the ⁇ chain often has a tyrosine phosphorylation site.
- the recognition site in the binding ligand is often an RGD sequence.
- integrins may be RGD molecules.
- extracellular matrix protein refers to a protein in the "extracellular matrix”. It refers to what is Parkaceous.
- extracellular matrix ECM
- ECM extracellular matrix
- somatic cell somatic cell
- the extracellular matrix is involved in the organization of the internal environment necessary for the survival of all somatic cells, not just for tissue support. Extracellular matrices are generally produced from connective tissue cells, but are also partially secreted by the cells themselves that carry the basement membrane, such as epithelial or endothelial cells.
- the extracellular matrix is generally divided into a fibrous component and a matrix filling the fibrous component.
- the fibrous component includes collagen fibers and elastic fibers.
- the basic constituent of the substrate is glycosaminodalican (acid mucopolysaccharide), most of which binds to non-collagenous proteins to form macromolecules of proteodalican (acid mucopolysaccharide-protein complex).
- substrates include laminin in the basal membrane, microfibril fibers around the elastic fibers, and fibronectin on the cell surface.
- the basic structure is the same in specially differentiated tissues.
- chondroblasts produce characteristically large amounts of cartilage matrix containing proteoglycan in hyaline cartilage
- osteoblasts produce bone matrix in bone that causes calcification. Is done. Therefore, examples of the extracellular matrix used in the present invention include, but are not limited to, collagen, elastin, proteoglycan, glycosaminoglycan, fibronectin, vitronectin, laminin, elastic fiber, collagen fiber and the like.
- examples of the extracellular matrix protein include, but are not limited to, fibronectin, vitronectin, laminin and the like.
- any extracellular matrix protein may be considered as long as it is a factor related to cell adhesion.
- CD49a is integrin heavy chain subunit 1 (amino acid sequence is SEQ ID NO: 4)
- CD49b is integrin heavy chain subunit 2 (amino acid sequence is SEQ ID NO: 5)
- CD49c is Is the integrin heavy chain subunit HI3 (amino acid sequence 1J is SEQ ID NO: 6 and 14)
- CD49d is the integrin heavy chain subunit a4 (amino acid sequence is SEQ ID NO: 7).
- CD49e is integrin heavy chain subunit 5 (amino acid sequence is SEQ ID NO: 8), and CD49f is integrin heavy chain subunit (Amino acid sequence is SEQ ID NO: 9), and CD29 is integrin ⁇ -subunit i31 (amino acid sequence is SEQ ID NO: 3, 10, 11, 12, and 13).
- CD44 is a cell surface hyaluronan binding protein
- CD46 is a cell adhesion factor called membrane cofactor protein (MCP)
- CD54 is a cell-indirect adhesion molecule ICAM-1 that binds cells.
- HLA refers to the product of the major histocompatibility complex (HLA gene) complex.
- biomolecule refers to a molecule associated with a living body.
- the term “living body” refers to a biological organism, including, but not limited to, animals, plants, fungi, and vinoles. Therefore, in the present specification, the biomolecule includes, but is not limited to, a molecule that can be extracted from a living body. Therefore, molecules synthesized by combinatorial chemistry and small molecules that can be used as pharmaceuticals (eg, small molecule ligands) also fall within the definition of biomolecules as long as their effects on living organisms can be intended.
- biomolecules include proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, including DNA such as cDNA, genomic DNA, and RNA such as mRNA), polypeptides, and the like. Includes saccharides, oligosaccharides, lipids, small molecules (eg, hormones, ligands, signal transducers, small organic molecules, etc.), and their complex molecules (glycolipids, glycoproteins, lipoproteins, etc.). Not limited to them. Biomolecules can also include the cells themselves, as well as parts of tissues, as long as introduction into the cells is contemplated.
- the biomolecules include nucleic acids (DNA or RNA) or proteins.
- the biomolecule is a nucleic acid (eg, genomic DNA or cDNA, or DNA synthesized by PCR or the like).
- the biomolecule can be a protein.
- "genetic material” refers to any material that carries genetic information. Examples of such a substance include, but are not limited to, nucleic acids such as DNA and RNA, PNA, proteins and the like.
- the DNA can be in any form, such as cDNA, genomic DNA, artificial DNA, RNAi, and the like.
- the RNA may be mRNA, tRNA, rRNA, RNAi, or the like.
- protein As used herein, the terms "protein,” “polypeptide,” “oligopeptide,” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length. .
- This polymer may be linear or branched or cyclic.
- the amino acid may be a natural or non-natural amino acid or a modified amino acid.
- the term may also include those assembled into a complex of multiple polypeptide chains.
- the term also includes natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, dalycosylation, lipidation, acetylation, phosphorylation or any other manipulation or modification (eg, conjugation with a labeling component).
- This definition also includes, for example, polypeptides containing one or more analogs of an amino acid (eg, including unnatural amino acids, etc.), peptidomimetic compounds (eg, peptoids), and other modifications known in the art. Is included. Gene products of extracellular matrix proteins such as fibronectin usually take the form of polypeptides.
- polynucleotide As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein, and refer to a polymer of nucleotides of any length. The term also includes “derivative oligonucleotides” or “derivative polynucleotides.” The term “derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide containing a derivative of a nucleotide or having an unusual linkage between nucleotides, and is used interchangeably.
- oligonucleotide examples include, for example, 2,1-O-methyl-ribonucleotide, a derivative in which a phosphodiester bond in an oligonucleotide is converted to a phosphorothioate bond, and an oligonucleotide in a oligonucleotide.
- oligonucleotide in which phosphodiester bond is converted to N3'_P5, phosphoramidate bond, ribose and oligonucleotide in oligonucleotide Derivative oligonucleotide in which diester bond is converted to peptide nucleic acid bond, Derivative in which peracyl in oligonucleotide is substituted with C_5 propierperacyl, Derivative in which peracyl in oligonucleotide is substituted with C_5 thiazole peracyl Oligonucleotides, derivatives in which cytosine in the oligonucleotide is replaced with C-5 propynylcytosine Oligonucleotides, derivatives in which cytosine in the oligonucleotide is replaced with phenoxazine-modified cytosine, DNA Derivative oligonucleotides in which the ribose in the amino acid is substitute
- a particular nucleic acid sequence also includes conservatively modified variants (eg, degenerate codon substitutions) and complementary sequences thereof, as well as explicitly stated sequences. Is contemplated. Specifically, degenerate codon substitutions create a sequence in which the third position of one or more selected but not all codons has been replaced with a mixed base and / or deoxyinosine residue. (Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91. —98 (1994)). Genes such as extracellular matrix proteins such as fibronectin usually take this polynucleotide form. The molecule to be subjected to transfection is also this polynucleotide.
- nucleic acid molecule is also used interchangeably with nucleic acids, oligonucleotides, and polynucleotides, and includes cDNA, mRNA, genomic DNA, and the like.
- nucleic acids and nucleic acid molecules may be included in the concept of the term “gene”.
- a nucleic acid molecule encoding a gene sequence also includes "splice variants (variants).”
- a particular protein encoded by a nucleic acid includes any protein encoded by a splice variant of the nucleic acid.
- splice variants are the products of onoretanative splicing of a gene.
- an initial nucleic acid transcript can be spliced such that different (alternate) nucleic acid splice products encode different polypeptides.
- the mechanism of splice variant production involves alternative splicing of variable force exons.
- Another polypeptide derived from the same nucleic acid by read-through transcription It is also included in this definition. Any product of a splicing reaction, including recombinant forms of the splice product, is included in this definition.
- extracellular matrix proteins useful as cell adhesion-related factors may also include splice variants thereof.
- gene refers to a factor that defines a genetic trait. Usually they are arranged in a certain order on the chromosome. Those that define the primary structure of a protein are called structural genes, and those that control its expression are called regulatory genes (eg, promoters). As used herein, a gene includes a structural gene and a regulatory gene unless otherwise specified. Therefore, a fibronectin gene usually includes both a fibronectin structural gene and a fibronectin promoter. In this specification,
- Gene may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein”, “polypeptide”, “oligopeptide” and “peptide”
- gene product also refers to “polynucleotide”, “oligonucleotide” and “nucleic acid” and / or “protein”, “polypeptide”, “oligopeptide” and “peptide” expressed by a gene. ". Those skilled in the art can understand what a gene product is, depending on the situation.
- the term “homology” of a sequence refers to the degree of identity between two or more gene sequences. Therefore, the higher the homology between certain two genes, the higher the identity or similarity between their sequences. Whether the two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, the hybridization method under stringent conditions.
- the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% , 95%, 96%, 97%, 98% or 99% identical, the genes are homologous.
- similarity refers to two or more gene sequences when conservative substitutions are regarded as positive (identical) in the homology described above. , The degree of identity to each other. Thus, if there are conservative substitutions, identity and similarity will vary depending on the presence of the conservative substitution. When there is no conservative substitution, identity and similarity show the same numerical value.
- the comparison of the similarity, identity and homology between the amino acid sequence and the base sequence is calculated using FASTA, a sequence analysis tool, using default parameters.
- amino acid may be natural or non-natural as long as the object of the present invention is satisfied.
- “Derivative amino acids” or “amino acid analogs” are those that differ from naturally occurring amino acids but have the same function as the original amino acid. Such derived amino acids and amino acid analogs are well-known in the art.
- the term "natural amino acid” refers to the L-isomer of a natural amino acid.
- Natural amino acids include glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, pheninolealanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, ⁇ —carboxyglutamic acid, Arginine, ordinine, and lysine. Unless otherwise indicated, all amino acids referred to herein are L-forms, but forms using D-form amino acids are also within the scope of the present invention.
- the term “unnatural amino acid” refers to an amino acid not normally found in nature in proteins.
- Examples of unnatural amino acids include D- or L-forms of norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homoarginine, and D-phenylalanine.
- Amino acid analog refers to a molecule that is not an amino acid, but that is similar in physical properties and / or function to the amino acid.
- Amino acid analogs include, for example, etyonin, canavanine, 2-methyldaltamine and the like.
- Amino acid mimetics refers to compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
- amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC- IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by the generally recognized one-letter code.
- corresponding amino acid refers to a given amino acid in a protein or polypeptide as a reference for comparison in a certain protein molecule or polypeptide molecule. Refers to amino acids that have the same action as, or are predicted to have, the same effect as amino acids. Say. For example, if it is a binding site of the integrin used in the present invention, it may be a similar site (domain) in the onoresolog corresponding to the binding site of the molecule.
- nucleotide may be natural or non-natural.
- “Derivative nucleotides” or “nucleotide analogs” are those that are different from naturally occurring nucleotides, but that have the same function as the original nucleotides. Such derivative nucleotides and nucleotide analogs are well known in the art. Examples of such derivative nucleotides and nucleotide analogs include, but are not limited to, phosphorothioate, phosphonoreamidate, methylphosphonate, chiral methylphosphonate, 2-0-methylribonucleotide, peptide mononucleic acid (PNA). Not done.
- fragment refers to a polypeptide or a polynucleotide having a sequence length of up to 11-1 relative to a full-length polypeptide or polynucleotide (length is n). Polynucleotide.
- the length of the fragment can be appropriately changed depending on the purpose.For example, the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10 for a polypeptide. , 15,20,25,30,40,50 and more amino acids, and the lengths represented by specifically listed non-integer integers (eg, 11 and the like) are also lower limits. As appropriate.
- a length represented by a non-integer integer (eg, 11, etc.) may also be appropriate as a lower limit.
- the lengths of the polypeptide and the polynucleotide can be represented by the number of amino acids or nucleic acids, respectively, as described above. However, the above-mentioned numbers are not absolute, as long as they have the same function. The above number as an upper limit or adjustment is intended to include a few above and below (or, for example, 10% above and below).
- fragments have a certain size (for example, 5 kDa) or more. It is preferable. Without being bound by theory, it appears that some size is required to function as a cell adhesion-related factor.
- polynucleotide that hybridizes under stringent conditions refers to well-known conditions commonly used in the art. Using a polynucleotide selected from the polynucleotides of the present invention as a probe, a colony'no, hybridization method, a plaque'hybridization method, a Southern blot hybridization method, or the like is used. Thus, such a polynucleotide can be obtained.
- sequences containing only the A sequence or only the T sequence are preferably excluded from the sequences that hybridize under stringent conditions.
- polynucleotide that can be hybridized refers to a polynucleotide that can hybridize to another polynucleotide under the above hybridization conditions.
- the hybridizable polynucleotide is a polynucleotide having at least 60% or more homology with the nucleotide sequence of the DNA encoding the polypeptide having the amino acid sequence specifically shown in the present invention, preferably 80% Polynucleotides having the above homology, more preferably polynucleotides having a homology of 95% or more can be mentioned.
- RNAi is an abbreviation for RNA interference.
- Homologous mRNA is specifically expressed by introducing a factor that causes RNAi such as double-stranded RNA (also referred to as dsRNA) into cells.
- dsRNA double-stranded RNA
- RNAi may also be used synonymously with factors that cause RNAi in some cases.
- factor causing RNAi refers to any factor capable of causing RNAi.
- RNAi RNAi related to the gene and achieves the effect of RNAi (for example, suppression of expression of the gene).
- Means Factors that cause such RNAi include, for example, at least 10%, including sequences that have at least about 70% homology to a portion of the nucleic acid sequence of the target gene or that hybridize under stringent conditions. Examples include, but are not limited to, RNAs containing nucleotide-length double-stranded portions or variants thereof.
- the factor preferably includes a 3′-protruding end, and more preferably, the 3′-protruding end may be a DNA having a length of 2 nucleotides or more (eg, a DNA having a length of 24 nucleotides).
- RNAi when a molecule that causes RNAi, such as dsRNA, is introduced into a cell, it is relatively long (eg, 40 base pairs or more).
- dsRNA an R Naselll-like nuclease called Dicer with a helicase domain cuts out the molecule from the 3 'end by about 20 base pairs in the presence of ATP, producing short dsRNA (also called siRNA). .
- ⁇ siRN A '' is an abbreviation for short interfering RNA, which is artificially synthesized or biochemically synthesized, or synthesized in an organism, or Short-chain double-stranded RNA of 10 base pairs or more, which is formed by degrading double-stranded RNA of about 40 bases or more in the body, usually has the structure of 5'-phosphate and 3'-OH And the 3 'end protrudes about 2 bases.
- a specific protein binds to the siRNA to form RISC (RNA-induced d-silencing-complex).
- This complex recognizes and binds mRNA having the same sequence as the siRNA, and cleaves the mRNA at the center of the siRNA by RNaselll-like enzyme activity.
- 100% is preferable.
- Mutation of the base at a position outside the central force of the siRNA leaves partial activity that does not completely eliminate the RNAi cleavage activity.
- mutation of the base in the center of the siRNA has a large effect, and the mRNA cleavage activity by RNAi is extremely reduced. Utilizing such properties, for mRNA having a mutation, an siRNA having the mutation in the center can be synthesized and introduced into cells.
- siRNA itself can be used as a factor that causes RNAi, and a factor that generates siRNA (for example, dsRNA typically having about 40 bases or more) can be used as such a factor. Can be.
- siRNA acts as a primer for RNA-dependent RNA polymerase (RdRP) by binding the antisense strand of siRNA to mRNA. It is also contemplated that dsRNA is synthesized, and this dsRNA again becomes a substrate for Dicer, generating new siRNA and amplifying the action.
- RdRP RNA-dependent RNA polymerase
- the siRNA itself and the factor by which the siRNA occurs are also useful. In fact, in insects and the like, for example, 35 dsRNA molecules almost completely degrade mRNA in a cell having more than 1,000 copies, so it is reasonable to think that siRNA itself and factors that generate siRNA are useful. It is cornered.
- RNA having a length of about 20 bases (eg, typically about 21 to 23 bases) or less which is called siRNA
- siRNA can be used for the treatment, prevention, prognosis and the like of a disease because it suppresses gene expression by expressing it in a cell and suppresses expression of a pathogenic gene targeted by the siRNA.
- siRNA used in the present invention may take any form as long as it can induce RNAi.
- the factor that causes RNAi of the present invention may be a short hairpin structure (shRNA; short hairpin RNA) having an overhang at the 3 'end.
- shRNA refers to a single-stranded RNA containing a partially palindromic base sequence, thereby taking on a double-stranded structure in the molecule to form a hairpin-like structure. Molecules with more than base pairs. Such shRNAs are artificially chemically synthesized.
- such shRNAs can generate the DNA of a hairpin structure in which the DNA sequences of the sense strand and the antisense strand are ligated in opposite directions by synthesizing RNA in vitro with T7 RNA polymerase.
- T7 RNA polymerase T7 RNA polymerase
- shRNAs can be transfected into a cell and then transfected into the cell for about 20 bases (typically, for example, 21 bases, 22 bases, 23 salts). It should be understood that it is degraded to the length of the group, causing RNAi like siRNA, and has the therapeutic effect of the present invention. It is to be understood that such effects are exerted on a wide variety of organisms, such as insects, plants, animals (including mammals).
- shRNA causes RNAi similarly to siRNA, and thus can be used as an active ingredient of the present invention.
- the shRNA may also preferably have a 3 'overhang.
- the length of the double-stranded portion is not particularly limited, but may be preferably about 10 nucleotides or more, more preferably about 20 nucleotides or more.
- the 3 'protruding end may be preferably DNA, more preferably at least 2 nucleotides in length, and even more preferably 2 to 4 nucleotides in length.
- RNAi-causing factor used in the present invention may be artificially synthesized (for example, chemically or biochemically) or naturally-occurring. There is no essential difference in the effect of. For those chemically synthesized, it is preferable to purify them by liquid chromatography or the like.
- RNAi used in the present invention can also be synthesized in vitro.
- antisense and sense RNAs are synthesized from type I DNA using T7 RNA polymerase and T7 promoter. When these are annealed in vitro and then introduced into cells, RNAi is induced through the above-described mechanism, and the effects of the present invention are achieved.
- such RNA can be introduced into cells by the calcium phosphate method.
- Factors that cause RNAi of the present invention also include factors such as single strands that can hybridize with mRNA, or all similar nucleic acid analogs thereof. Such factors are also useful in the treatment methods and compositions of the present invention.
- salt is used in the same meaning as commonly used in the art, and includes both inorganic salts and organic salts.
- a salt is usually formed by a neutralization reaction between an acid and a base. Salts include NaCl, KSO, etc. generated by the neutralization reaction.
- a salt a normal salt (H of an acid or ⁇ H of a base is not contained in the salt Substances, for example, NaCl, NH Cl, CH C ⁇ ONa, Na CO), acidic salts (H
- salts are those constituting the medium (e.g., calcium chloride, sodium hydrogen phosphate, sodium hydrogen carbonate, sodium pyruvate, HEPES, calcium chloride, sodium chloride, lithium chloride, magnesium sulfide, iron nitrate, amino acid, etc.).
- Vitamins, and salts constituting buffer solutions eg, calcium chloride, magnesium chloride, sodium hydrogen phosphate, sodium chloride
- buffer solutions eg, calcium chloride, magnesium chloride, sodium hydrogen phosphate, sodium chloride
- These salts may be used alone or in a combination of two or more, preferably a plurality, since the affinity for cells tends to increase.
- the salt contained in the medium eg, Shii-dani calcium, Shii-dani magnesium, sodium hydrogen phosphate, chloride
- More preferably preferably be used a plurality of thorium) device, be used as they are all salts that is part of the culture medium may be advantageous In. Another preferred embodiment, it may be added glucose.
- search refers to another nucleobase sequence having a specific function and / or property, utilizing a certain nucleobase sequence, electronically or biologically or by another method.
- Means to find Electronic search includes BLAST (Altschul et al., J. Mol. Biol. 215: 403-410 (1990)), FASTA (Pearson & Lipman, Proc. Natl. Acad. Sci., USA 85: 2444). —2448 (1988)), Smith and Waterman method (Smith and Waterman, J. Mol. Biol. 147: 195—197 (1981)), and Needleman and Wunsch method (Needleman and Wunsch, J. Mol. Biol.
- Biological searches include stringent hybridization, macroarrays in which genomic DNA is attached to a nylon membrane, or microarrays (microarray assays) attached to a glass plate, PCR and in situ hybridization. But not limited to them. In the present specification, it is intended that Fnl should also include a corresponding gene identified by such electronic search and biological search. [0058] In the present specification, "introduction" of a substance into a cell means that the substance enters the inside of a cell membrane.
- Intracellular changes eg, gene expression, signal transduction, events due to binding to intracellular receptors, metabolic changes, etc.
- Intracellular changes can be physically (eg, visually), chemically (measuring secretions), biochemical, Biological measurements can be used to determine semi-IJ. Therefore, such “introduction” includes not only the simple transfer of a substance such as a protein into a cell, but also operations such as transfection, transformation, and transduction, which are usually called gene manipulation.
- target substance refers to a substance that is intended to be introduced into cells.
- the target substances contemplated by the present invention are substances that are not introduced into cells under normal conditions. Thus, substances that can be introduced into cells under normal conditions by diffusion or hydrophobic interaction are not considered as important aspects of the present invention.
- Target substances that are not introduced into cells under normal conditions include, for example, proteins (polypeptides), RNA, DNA, sugars (especially polysaccharides), and their complex molecules (eg, glycoproteins, PNAs, etc.), viruses Vectors, and other compounds, but are not limited thereto.
- the term "antibody” is used in a meaning commonly used in the art, and in this specification, all of antibodies and fragments and derivatives thereof, humanized antibodies, chimeric antibodies, And anti-idiotypic antibodies, and fragments thereof, such as F (ab ') 2 and Fab fragments, and other recombinantly produced conjugates.
- it is an antibody that recognizes the polypeptide used in the present invention, and more preferably, it is an antibody that specifically recognizes the polypeptide used in the present invention.
- Such antibodies may be either polyclonal or monoclonal. In one embodiment of the present invention, such antibodies are also included within the scope of the present invention.
- the term "antigen" refers to an antibody that binds to an antibody or binds to a specific receptor such as a B lymphocyte or T lymphocyte to induce an immune reaction such as antibody production and / or cytotoxicity.
- a substance that causes an inflammation for example, but not limited to proteins, lipids, sugars, etc.
- Binding to an antibody or lymphocyte receptor is called “antigenicity”.
- binding to an antibody or lymphocyte receptor is called "antigenicity”.
- Properties that induce an immune response, such as antibody production are called "immunogenicity”.
- the substance used as an antigen includes, for example, at least one substance of interest (eg, a protein).
- epitopes or “antigenic determinants” refer to sites in an antigen molecule to which an antibody or lymphocyte receptor binds. Methods for determining epitopes are well known in the art, and such epitopes can be determined by those skilled in the art using such well-known and conventional techniques given the primary sequence of a nucleic acid or amino acid.
- the partner of the "cell adhesion molecule” refers to a molecule that specifically interacts with the cell adhesion molecule.
- the term “specifically interacts" with a cell adhesion factor refers to an interaction with a higher affinity for a cell adhesion factor than for a substance other than the specific cell adhesion factor.
- the term "device” refers to a part that can constitute a part or the whole of an apparatus, and includes a support (preferably a solid support) and a support. It consists of a target substance to be carried. Such devices include, but are not limited to, chips, arrays, microtiter plates, cell culture plates, petri dishes, finolems, beads, and the like.
- support refers to a material to which a substance such as a biomolecule can be immobilized.
- the material of the support may be either covalently or non-covalently bonded, having the ability to bind to a substance such as a biomolecule used in the present invention, or derivatized to have such property. Any solid material that can be used.
- any material that can form a solid surface can be used, for example, glass, silica, silicon, ceramic, silicon dioxide, plastic, metal (also alloy). And natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon), and the like.
- the support may be formed from multiple layers of different materials. For example, glass, quartz glass, alumina, sapphire, forsterite, silicon oxide, silicon carbide And an inorganic insulating material such as silicon nitride.
- Polyethylene ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetate, acrylic resin, Polyacrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenolic resin, urea resin, epoxy resin, melamine resin, styrene 'acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, polysulfone, etc.
- Organic materials can be used.
- a membrane used for blotting such as a nitrocellulose membrane, a nylon membrane, or a PVDF membrane
- a membrane used for blotting such as a nitrocellulose membrane, a nylon membrane, or a PVDF membrane
- the material constituting the support is a solid phase, it is particularly referred to herein as "solid support".
- solid support In the present specification, it may take the form of a plate, a microwell plate, a chip, a slide glass, a finolem, a bead, a metal (surface), or the like.
- the support may be coated or uncoated.
- liquid phase is used in the same meaning as usually used in the art, and usually means a state in a solution.
- solid phase is used in the same meaning as used in the art, and usually means a solid state.
- a liquid and a solid may be collectively referred to as a fluid.
- contact means that two substances (eg, a composition and a cell) are present at a close enough distance to interact with each other.
- the term "interaction” means that when referring to two objects, the two objects exert a force on each other.
- Examples of such interactions include, but are not limited to, covalent bonds, hydrogen bonds, van der Waals forces, ionic interactions, nonionic interactions, hydrophobic interactions, electrostatic interactions, etc. Not done.
- the interaction can be a normal interaction that occurs in vivo, such as a hydrogen bond, a hydrophobic interaction.
- interacting substance refers to a substance that interacts with a certain subject.
- substances include, for example, antibodies to antigens, ligands to receptors Or vice versa, but is not limited thereto.
- the cell adhesion molecule used in the present invention often takes the form of a gene product, it is understood that such a gene product may be a variant thereof, as described above. Therefore, the present invention can also use substances produced by the following genetic modification techniques.
- certain amino acids included in the sequence may be present in the protein structure, such as, for example, a cationic region or a binding site of a substrate molecule, without any apparent loss or loss of interaction binding capacity. It can be replaced by another amino acid. It is the protein's ability to interact and its properties that define the biological function of a protein. Thus, certain amino acid substitutions may be made in the amino acid sequence, or at the level of its DNA coding sequence, resulting in a protein that retains its original properties after the substitution. Thus, various modifications may be made in the peptide disclosed herein or the corresponding DNA encoding the peptide without any apparent loss of biological utility.
- the hydropathic index of amino acids can be considered.
- the importance of the hydrophobic amino acid index in conferring interactive biological functions on proteins is generally recognized in the art (Kyte. J and Doolittle, RFJ Mol. Biol. 157 (1): 105-132, 1982).
- the hydrophobic nature of amino acids contributes to the secondary structure of the resulting protein, which in turn defines the interaction of the protein with other molecules (eg, enzymes, substrates, receptors, DNA, antibodies, antigens, etc.).
- Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge properties.
- an amino acid is replaced by another amino acid having a similar hydrophobicity index and It is well known in the art, however, that proteins having similar biological functions can be generated (eg, proteins that are equivalent in enzyme activity).
- the hydrophobicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5. It is understood in the art that such substitution of amino acids based on hydrophobicity is efficient.
- the hydrophilicity index may also be considered in modifying proteins. As described in US Pat. No. 4,554,101, the following hydrophilicity indices have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartic acid ( Glutamic acid (+ 3.0 ⁇ 1); Serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (— 0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cystine (-1.0); methionine (-1.3); valine ( —1.5); Leucine (—1.8); Isoloicin ( ⁇ 1.8); Tyrosine (—2.3); Phenylalanine (—2.5); and Tryptophan (—3.4).
- an amino acid can be substituted for another that has a similar hydrophilicity index and still provide a bioisostere.
- the hydrophilicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
- conservative substitution refers to a substitution in which the amino acid substitution and the amino acid to be substituted have similar hydrophilicity indexes and / or hydrophobicity indexes as described above.
- conservative substitution include, for example, those having a hydrophilicity index or a hydrophobicity index of two or less within soil 2, preferably within ⁇ 1 and more preferably within ⁇ 0.5. But not limited to them.
- conservative substitutions are well known to those skilled in the art and include, for example, substitutions within each of the following groups: arginine and lysine; daltamic and aspartic acid; serine and threonine; glutamine and asparagine; Leucine, isoleucine, and the like, but are not limited thereto.
- the "variant" refers to a substance in which a substance such as an original polypeptide or polynucleotide is partially changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, etc. And so on. Alleles refer to genetic variants that belong to the same locus and are distinct from each other. Therefore, “allelic variant” refers to a variant that has an allelic relationship to a certain gene. Such allelic variants usually have sequences that are identical or very similar to their corresponding alleles, and usually have nearly the same biological activity, but rarely different biological activities. It may also have.
- “Species homolog or homolog” refers to homology (preferably 60% or more homology, more preferably, homology at the amino acid level or nucleotide level) of a certain gene within a certain species. 80./o above, 85./ 0 or more, 90% or more, instances that have a homology) of 95% or more. Methods for obtaining such species homologs will be apparent from the description herein.
- the term "ortholog” refers to a gene derived from speciation from a common ancestor with two genes, both orthologous genes.
- Moguro bottles genes into range of the human and mouse force s, non-hemoglobin gene and the ⁇ -hemoglobin gene of human ortholog occurred at paralogs (genes duplicated gene ).
- Onoresologs are useful for estimating molecular phylogenetic trees.
- Orthologs of the present invention may also be useful in the present invention, since orthologs can usually perform the same function in another species as the original species.
- conservatively modified variants applies to both amino acid sequences and nucleic acid sequences.
- a conservatively modified variant is essentially a nucleic acid that encodes the same or essentially the same amino acid sequence, and if the nucleic acid does not encode an amino acid sequence, To the same sequence. Due to the degeneracy of the genetic code, many functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine.
- nucleic acid variation is a "silent modification (mutation)," which is one type of conservatively modified mutation.
- Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
- nuclear Each codon in the acid except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan, can be modified to produce functionally identical molecules It is understood that. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence.
- such modifications may be made to avoid substitution of the amino acid cysteine, which greatly affects the conformation of the polypeptide.
- base sequence modification methods include cleavage with a restriction enzyme or the like, ligation treatment with a DNA polymerase, Klenow fragment, DNA ligase, or the like, site-specific base substitution using a synthetic oligonucleotide, or the like. (Directed site-directed mutagenesis; Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)). The power S, and other methods usually used in the field of molecular biology. Can also be modified.
- amino acid addition, deletion, or modification can also be performed to produce a functionally equivalent polypeptide.
- Amino acid substitution refers to substitution of the original peptide with one or more amino acids, for example, 11 amino acids, preferably 115 amino acids, and more preferably 113 amino acids.
- the addition of amino acids refers to adding one or more, for example, 110, preferably 115, and more preferably 113 amino acids to the original peptide chain.
- Amino acid deletion refers to the deletion of one or more, for example, 110, preferably 115, more preferably 113 amino acids from the original peptide.
- Amino acid modifications include, but are not limited to, forces S including amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation), etc. .
- the amino acid to be substituted or added may be a natural amino acid or an unnatural amino acid or an amino acid analog. Natural amino acids are preferred.
- peptide analog or "peptide derivative” is a compound that is different from a peptide, but is equivalent to the peptide in at least one chemical or biological function.
- peptide analogs include those in which one or more amino acid analogs or amino acid derivatives have been added or replaced with the original peptide.
- Peptide analogs have a function similar to that of the original peptide (e.g., similar pKa values, similar functional groups, binding to other molecules). Such attachments or substitutions are made to be substantially similar (eg, similarity in similarity, similarity in water solubility).
- Such peptide analogs can be made using techniques well known in the field.
- a peptide analog can be a polymer comprising an amino acid analog.
- polynucleotide analog and “nucleic acid analog” are compounds that are different from polynucleotides or nucleic acids.
- a polynucleotide or nucleic acid and at least one chemical function or organism Are those whose biological functions are equivalent.
- a polynucleotide or nucleic acid analog includes one or more nucleotide analogs or nucleotide derivatives added to or substituted for the original peptide.
- the nucleic acid molecule used in the present specification has a part of the nucleic acid sequence deleted as long as the expressed polypeptide has substantially the same activity as the naturally-occurring polypeptide. Alternatively, it may be replaced by another base or another nucleic acid sequence may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 'end and / or the 3' end. Further, a nucleic acid molecule which hybridizes a gene encoding a polypeptide under stringent conditions and encodes a polypeptide having substantially the same function as the polypeptide may be used. Such genes are known in the art and can be used in the present invention.
- Such a nucleic acid can be obtained by a well-known PCR method, and can also be chemically synthesized. These methods may be combined with, for example, a site-specific displacement induction method, a hybridization method, or the like.
- substitution, addition or deletion of a polypeptide or polynucleotide refers to an amino acid or its substitute for the original polypeptide or polynucleotide, respectively. Or the substitution of nucleotides or their substitutes, additions or removals. Techniques for such substitution, addition or deletion are well known in the art, and examples of such techniques include site-directed mutagenesis techniques.
- the number of substitutions, additions, or deletions may be any number as long as it is one or more. Such a number may be a function having a desired function (for example, hormone, cytotoxicity, etc.) in a variant having the substitution, addition, or deletion. As long as the information transfer function is maintained) . For example, such a number may be one or several, and preferably may be within 20%, 10%, or 100 or less, 50 or less, 25 or less of the total length.
- an agent that specifically interacts with a polynucleotide or a polypeptide refers to an affinity for the polynucleotide or the polypeptide or other irrelevant (particularly, identity of 30./o). Typically less than or equal to the affinity for a polynucleotide or polypeptide (preferably significantly higher). Such an affinity can be measured by, for example, hybridization assay, binding assay, and the like.
- the "factor” may be any substance or other element (for example, energy) as long as the intended purpose can be achieved.
- substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (eg, DNAs such as cDNA, genomic DNA, mRNA, siRNA, RNAi Such as RNA), polysaccharides, oligosaccharides, lipids, small organic molecules (eg, hormones, ligands, signal transducers, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals) (Eg, small molecule ligands) and the like, but are not limited thereto.
- a polynucleotide typically, a polynucleotide having a certain degree of sequence homology (for example, 70% or more sequence identity) to the sequence of the polynucleotide, and And polypeptides such as transcription factors that bind to the promoter region, but are not limited thereto.
- an antibody specifically directed against the polypeptide, a derivative thereof, or an analog thereof eg, a single-chain antibody
- the polypeptide When is a receptor or a ligand, specific ligands or receptors, and when the polypeptide is an enzyme, include but are not limited to substrates.
- an "isolated" biological agent eg, a nucleic acid or protein
- a nucleic acid or protein is defined as another biological agent in a cell of an organism in which the biological agent naturally exists.
- Factors for example, in the case of nucleic acids, factors other than nucleic acids and nucleic acid sequences other than the target nucleic acid
- a nucleic acid containing stem IJ in the case of a protein, refers to a substance substantially separated or purified from a factor other than a protein and a protein containing an amino acid sequence other than a target protein.
- isolated nucleic acids and proteins include nucleic acids and proteins that have been purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.
- a “purified" biological agent eg, nucleic acid or protein
- a biological agent in which at least a part of a factor naturally associated with the biological agent has been removed. Say. Thus, typically, the purity of the biological agent in the purified biological agent is higher (ie, more concentrated) than in the state in which the biological agent is normally present.
- purified and isolated as used herein preferably refer to at least 75% by weight, more preferably at least 85% by weight, even more preferably at least 95% by weight. %, And most preferably at least 98% by weight, of the same type of biological agent.
- a "vector” or “recombinant vector” is a vector capable of transferring a target polynucleotide sequence into a target cell.
- Such vectors are capable of autonomous replication in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, animal individuals and plant individuals, or capable of integration into chromosomes, and Those containing a promoter at a position suitable for polynucleotide transcription are exemplified. Therefore, the plasmid is also one f of the vector. Of the vectors, those suitable for cloning are referred to as “cloning vectors”.
- Such cloning vectors usually contain a multiple cloning site that contains multiple restriction enzyme sites.
- restriction enzyme sites and multiple cloning sites are well known in the art, and those skilled in the art can appropriately select and use them according to the purpose. Such techniques are described in the literature described herein (eg, Sambrook et al., Supra).
- the term "expression vector” refers to a structural gene and a gene that regulates its expression.
- the regulatory elements may preferably include a terminator, a selectable marker such as a drug resistance gene, and a heterogeneous gene. It is well known to those skilled in the art that the type of expression vector of an organism (eg, an animal) and the type of regulatory element used can vary depending on the host cell.
- Examples of recombinant vectors for prokaryotic cells include pcDNA3 (+), pBluescript-SK (+/-), pGEM-T, pEF_B ⁇ S, pEGFP, pHAT, pUC18, pFT—DEST TM 42GA TEWAY (Invitrogen) and the like. Is exemplified.
- Recombinant vectors for animal cells include pcDNAl / Amp, pcDNAI, pCDM8 (also sold by Funakoshi Co., Ltd.), ⁇ 80 107 [Japanese Unexamined Patent Publication No. 3_229 (11 ⁇ 10 ⁇ 6, 103 [J. Biochem., 101, 1307 (1987)], pAMo, pAMoAU. Biol. Chem., 268, 22782--22787 (1993)], based on Murine Stem Cell Virus (MSCV). And retroviral expression vectors, pEF-BOS, pEGFP and the like.
- Examples of a recombinant vector for a plant cell include, but are not limited to, pPCVICEn4HPT, pCGN1548, pCGN1549, pBI221, pBI121, and the like.
- terminal 1 is usually located downstream of the protein-encoding region of a gene, and is involved in termination of transcription when DNA is transcribed into mRNA and addition of a poly-A sequence. Sequence. It is known that the terminator is involved in mRNA stability and affects the expression level of a gene.
- promoter refers to a region on DNA that determines the transcription initiation site of a gene and that directly regulates the frequency of transcription.
- the starting base sequence Therefore, in the present specification, a portion of a gene having a promoter function is referred to as a “promoter portion”. Since the promoter region is usually a region within about 2 kbp upstream of the first exon of the putative protein coding region, if the protein coding region in the genomic nucleotide sequence is predicted using DNA analysis software, the promoter region may be The area can be estimated. Putative promoter regions vary from structural gene to gene, but are usually upstream of, but limited to, structural genes. However, it may be downstream of the structural gene. Preferably, the putative promoter region is within about 2 kbp upstream of the first exon translation start site.
- the term “enhancer” refers to a sequence used to enhance the expression efficiency of a target gene. Such enhancers are well known in the art. A plurality of forces that can be used may be used or one may not be used.
- silencer refers to a sequence having a function of suppressing gene expression and arresting.
- any type of silencer may be used as long as the silencer has the function.
- operably linked refers to the expression (operation) of a desired sequence, a transcriptional / translational regulatory system (eg, a promoter, an enhancer, a silencer, etc.) or a translational regulatory system. It means to be placed under the control of the sequence.
- a transcriptional / translational regulatory system eg, a promoter, an enhancer, a silencer, etc.
- the promoter is usually, but not necessarily, positioned immediately upstream of the gene.
- any technique for introducing a nucleic acid molecule into cells may be used. Examples include transformation, transduction, and transfection. Techniques for introducing such nucleic acid molecules are well known and commonly used in the art, and are described, for example, in Ausubel FA et al. (1988), Current Protocols in Molecular Biology, Wiley, New York; NY; Sambrook J et al. "987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its two fragments, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. Co., 1997. Gene transfer can be confirmed using the methods described herein, such as Northern blot, Western blot analysis, or other well-known techniques.
- any of the above-described methods for introducing DNA into cells can be used, and examples thereof include transfection, transduction, and transformation (for example, calcium phosphate method, ribosome method, DEAE dextran method, elect port Pore Chillon method, a method using particle gun (gene gun)), Ribofuwekushiyon method, staple erotic plasto method [p ro Natl. Acad. Sci . USA, 84, 1929 (1978)] , Lithium acetate 153, 163 (1983)] and Proc. Natl. Acad. Sci. USA, 75, 1929 (1978).
- transformation for example, calcium phosphate method, ribosome method, DEAE dextran method, elect port Pore Chillon method, a method using particle gun (gene gun)
- Ribofuwekushiyon method for example, calcium phosphate method, ribosome method, DEAE dextran method, elect port Pore Chillon method, a method using particle gun (gene gun)
- gene introduction reagent refers to a reagent used for promoting the introduction efficiency in a gene introduction method.
- gene introduction reagents include, but are not limited to, cationic polymers, cationic lipids, polyamine-based reagents, polyimine-based reagents, calcium phosphate, and the like.
- reagents used in transfection include reagents commercially available from various sources, such as Effectene Transfection Reagent, cat.no.301425, Qiagen, CA), TransFastTM Transfection Reagent (E2431, Promega, WI), TfxTM-20 Reagent (E2391, Promega, WI), SuperFect Transfection Reagent (3 01305, Qiagen, CA), PolyFect Transfection Reagent (301105, Qiagen, CA), LipofectAMINE 2000 Reagent (11668) -019, Invitrogen corporation, CA), JetPEI (X4) cone. "01-30, Polyplus-transfection, France) and ExGen 500 (R0511, Fermentas Inc., MD). Not done.
- Effectene Transfection Reagent cat.no.301425, Qiagen, CA
- TransFastTM Transfection Reagent E2431, Promega, WI
- TfxTM-20 Reagent E2391,
- the "instructions" in the present specification describe the target substance introduction method and the like of the present invention to users (researchers, experimental assistants, or doctors and patients who administer patients in treatment, etc.). It was done.
- This instruction describes, for example, a word indicating a method of using the composition or the like of the present invention.
- the instructions shall be prepared in accordance with the format prescribed by the competent authority of the country in which the invention is to be implemented and shall specify that they have been approved by that competent authority.
- the instructions are usually in the form of a so-called package insert for pharmaceuticals or in the form of a manual for laboratory reagents and are usually provided in paper form, but are not limited thereto. For example, it may be provided in a form such as an electronic medium (eg, a home page provided on the Internet, an e-mail).
- the term "transformant” refers to all or a part (tissue or the like) of an organism such as a cell produced by transformation.
- the transformant include all or a part (tissue, etc.) of living organisms such as cells of prokaryotes, yeasts, animals, plants, insects and the like.
- Transformants may be transformed cells, transformed tissues, transformed It is also called the Lord.
- the cell used in the present invention may be a transformant
- prokaryotic cell when a prokaryotic cell is used in genetic engineering or the like, examples of the prokaryotic italium include Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium Noyu, Microbacterium, Pseudomonas, and the like. Examples include Noyu "5 J eukaryotic cells, for example, Escherichia coli XLl_Blue, Escherichia coli XL2-Blue, and Escherichia coli DH1. Alternatively, in the present invention, cells isolated from natural products can also be used.
- Animal cells that can be used in genetic manipulation and the like in the present specification include mouse myeloma cells, rat 'myeloma cells, mouse' nos, ibridoma cells, Chinese hamster cells CH ⁇ cells, and BHK cells. And African green monkey kidney cells, human leukemia cells, HBT5637 (JP-A-63-299), and human colon cancer cell lines.
- mouse 'myeloma cells include ps20 and NS ⁇
- rat myeloma cells include YB2 /
- human fetal kidney cells include HEK293 (ATCC: CRL_1573)
- human leukemia cells include BALL-1.
- C ⁇ S_1 and COS-7 as Zanore kidney cells
- HCT-15 as human colon cancer cell line
- SK—N_SH SK—N-SH-5Y
- human neuroblastoma Neuro2A etc. Is exemplified.
- primary cultured cells can also be used.
- the plant cells that can be used in genetic manipulation and the like in the present specification include callus or a part thereof and suspension cultured cells, Solanaceae, Grassicaceae, Brassicaceae, Rosaceae, Leguminosae, Laceae, Examples include, but are not limited to, cells of plants such as Labiatae, Lily, Acacia, Apiaceae and the like.
- the "detection" or “quantification” of gene expression is achieved using an appropriate method including, for example, mRNA measurement and immunological measurement methods.
- mRNA measurement and immunological measurement methods can be done.
- the molecular biological measurement method include a Northern blot method, a dot plot method, and a PCR method.
- the immunological measurement method include, for example, an ELISA method using a microtiter plate, an RIA method, a fluorescent antibody method, a Western blot method, and an immunohistochemical staining method.
- the quantification method and For example, the ELISA method or the RIA method is exemplified.
- DNA arrays are widely outlined in Shujunsha, edited by Cell Engineering, “DNA Microarrays and the Latest PCR Method”.
- the protein array is described in detail in Nat Genet. 2002 Dec; 32 Suppl: 526-32.
- Methods for analyzing gene expression include, but are not limited to, RT-PCR, RACE, SSCP, immunoprecipitation, two-hybrid systems, in vitro translation, and the like, in addition to those described above.
- expression of a gene product such as a gene, polynucleotide, or polypeptide means that the gene or the like undergoes a certain action in vivo to take on another form.
- it means that a gene, polynucleotide, etc. is transcribed and translated to form a polypeptide, but transcription and production of mRNA may also be a form of expression. More preferably, such forms of the polypeptide may have undergone post-translational processing.
- the term "expression level” refers to the level of expression of a polypeptide or mRNA in a target cell or the like. Such expression levels can be evaluated using the antibody of the present invention by any appropriate method including immunological measurement methods such as the ELISA method, RIA method, fluorescent antibody method, Western blot method, and immunohistological staining method.
- Expression level at the mRNA level “Change in expression level” refers to the expression level of the polypeptide of the present invention at the protein level or mRNA level, which is evaluated by any appropriate method including the above-described immunological measurement method or molecular biological measurement method. It means increasing or decreasing.
- expression or “reduction of expression level” of a gene, a polynucleotide, a polypeptide, or the like is defined as when the factor of the present invention is acted on or not. Means that the amount of expression is significantly reduced.
- the decrease in expression is a polypeptide Including a decrease in the expression level of
- “increase” of “expression” or “expression level” of a gene, polynucleotide, polypeptide, or the like refers to a factor related to gene expression in a cell (for example, a gene to be expressed or a gene that regulates the gene.
- the increase in expression includes an increase in the expression level of the polypeptide.
- induction of “expression” of a gene refers to increasing the expression level of a gene by causing a certain factor to act on a certain cell. Therefore, the induction of expression is to cause the gene to be expressed when no expression of the gene is found, and to increase the expression of the gene when the expression of the gene has already been seen It is included.
- the term "specifically expresses" a gene means that the gene is different (preferably higher) at a particular site or stage in a plant than at other sites or stages. It is expressed. To be specifically expressed may be expressed only at a certain site (specific site) or may be expressed at other sites. Preferably, specific expression means expression at a certain site only.
- biological activity refers to an activity that a certain factor (for example, a polypeptide or a protein) may have in a living body, and has various functions (for example, promotion of transcription).
- a cell adhesion-related factor interacts with a cell adhesion molecule
- its biological activity depends on the coexistence of the interacting substance when cells are seeded on a surface coated with an ECM substrate such as fibronectin. This confirms the adhesion inhibitory effect.
- a substrate having such an interaction activity is chemically or physically immobilized on the surface, an increase in the adhesion of cells to the surface is confirmed. Therefore, it can be measured by the technique based on such a principle.
- such biological activity can be cell adhesion activity, heparin binding activity, collagen binding activity, and the like.
- Cell adhesion activity can be measured by measuring the rate of cell adhesion to a solid phase after cell seeding and treating it as the adhesion activity.
- Heparin-binding activity can be measured by affinity chromatography using a heparin-immobilized column or the like, and can be confirmed by binding to this.
- Collagen binding activity is measured by affinity chromatography using a collagen-immobilized column. It can be measured by what can be identified as binding to it. For example, if a factor is an enzyme, its biological activity includes that enzymatic activity.
- an agent is a ligand, the ligand involves binding to the corresponding receptor.
- Such biological activity can be measured by techniques well known in the art (see, eg, Molecular Cloning, Current Protocols (cited herein) and the like).
- particle refers to a substance having a certain hardness and a size equal to or more than a certain size. In the present invention, it refers to a substance made of metal or the like. Examples of the particles used in the present invention include, but are not limited to, gold colloid, silver colloid, and latex colloid.
- kits refers to a unit which is usually divided into two or more sections and provided with a part to be provided (eg, a reagent, a particle, or the like). This kit form is preferred when it is intended to provide a composition such that it should preferably be mixed and used shortly before use, which should be provided as a mixture. Such kits will advantageously be provided with instructions that describe how to provide the provided components (eg, reagents, particles, etc.).
- the polypeptide used in the present invention is obtained by culturing a transformant derived from a microorganism, animal cell, or the like having a recombinant vector into which a DNA encoding the polypeptide has been incorporated, according to a conventional culture method. It can be produced by producing and accumulating a peptide, and collecting the polypeptide of the present invention from the culture.
- the method for culturing the transformant in a medium can be performed according to a usual method used for culturing a host.
- a medium for culturing a transformant obtained by using a prokaryote such as Escherichia coli or a eukaryote such as yeast as a host
- a carbon source for example, gnorecose, fructose, sucrose, or any of these containing assimilation
- Molasses Molasses, carbohydrates such as starch or starch hydrolysates, organic acids such as acetic acid and propionic acid, alcohols such as ethanol, propanol, etc.
- nitrogen sources eg, ammonia, ammonium chloride, ammonium sulfate
- Ammonium acetate ammonium phosphate, etc.
- inorganic salts for example,
- Culture is preferably performed under aerobic conditions such as shaking culture or deep aeration stirring culture, but is not limited thereto.
- the culture temperature is 15-40 ° C, and the culture time is usually 5 hours and 7 days.
- the culture maintain the pH between 3.0 and 9.0.
- the pH is adjusted using an inorganic or organic acid, an alkaline solution, urea, calcium carbonate, ammonia, or the like. If necessary, an antibiotic such as ampicillin or tetracycline may be added to the medium during the culture.
- a conventional polypeptide commonly used in the field is used.
- Peptide (eg, enzyme) isolation or purification techniques can be used.
- the culture is treated by a technique such as centrifugation to obtain a soluble fraction. Get minutes.
- solvents such as solvent extraction, salting-out desalting with ammonium sulfate, etc., precipitation with organic solvents, resins such as getylaminoethyl (DEA E) -Sepharose (Pharmacia), DIAION HPA-75 (Mitsubishi Chemical) Anion exchange chromatography method used, S_Sepharose FF (Pharmacia), etc. used resin Cation exchange chromatography method, Butyl-Sepharose, Phenyl-Sepharose, etc. used resin hydrophobic chromatography method, molecular sieve used Gel filtration, affinity chromatography, chromatofocusing, isoelectric focusing, etc.
- a purified sample can be obtained using a technique such as electrophoresis.
- the cells in the culture were collected by centrifuging the culture, and the cells were washed. Thereafter, the cells are crushed using an ultrasonic crusher, a French press, a Mantongaulin homogenizer, a Dynomill, or the like to obtain a cell-free extract.
- a purified sample can be obtained by using a gel filtration method using a molecular sieve, an affinity chromatography method, a chromatofocusing method, or an electrophoresis method such as isoelectric focusing.
- the polypeptide used in the present invention is expressed by forming an insoluble substance in the cells, the cells are similarly collected, crushed, and then separated from the precipitate fraction obtained by centrifugation. After recovering the polypeptide of the present invention by an ordinary method, the insoluble form of the polypeptide is solubilized with a polypeptide denaturing agent. The lysate is diluted or dialyzed into a solution containing no polypeptide denaturing agent or a solution in which the concentration of the polypeptide denaturing agent is so low that the polypeptide is not denatured. After having a three-dimensional structure, a purified sample can be obtained by the same isolation and purification method as described above.
- the protein can be purified according to a conventional protein purification method [for example, J. Evan. Sadler et al .: Methods in Enzymology, 83, 458].
- affinity chromatography using a substance having affinity for the fused protein may be used.
- Can also be purified [Akio Yamakawa, Experimental Medicine, 13, 469-474 (1995)].
- One such method is based on the method described by Lowe et al. [Pro Natl. Acad. Sci., USA, 86, 8227-8231 (1989), Genes Develop., 4, 1288 (1990)].
- polypeptide can be produced as a fusion protein with Protein II, and purified by affinity chromatography using immunoglobulin G.
- the polypeptide used in the present invention can be produced as a fusion protein with a FLAG peptide and purified by affinity chromatography using an anti-FLAG antibody [Proc. Natl. Acad. Sci., USA, 86, 8227 (1989), Genes Development, 4, 1288 (1990)].
- polypeptide of the present invention can be purified by affinity chromatography using an antibody against the polypeptide itself.
- the polypeptide of the present invention can be prepared in vitro according to a known method, Biomolecular NMR, 6, 129-134, Science, 242, 1162-1164, J. Biochem., 110, 166-168 (1991)]. Can be produced using a transcription'translation system.
- the polypeptide of the present invention can also be obtained by a chemical synthesis method such as the Fmoc method (fluorenylmethyloxycarbonyl method) or the tBoc method (t_butyloxycarbonyl method). Can be manufactured.
- peptide synthesis machines such as Advanced ChemTech, Applied Biosystems, Pharmacia Biotech ⁇ Protein Technology Instrument, Synthecell-Vega, PerSeptive, and Shimadzu Corporation can be used for peptide synthesis.
- plate refers to a flat support to which molecules such as antibodies can be immobilized.
- the plate is preferably made of a glass substrate having a metal thin film containing plastic, gold, silver or aluminum on one side as a base material.
- substrate refers to a material (preferably solid) on which the chip or array of the present invention is constructed.
- the substrate is included in the concept of a plate.
- the material of the substrate may have the property of binding to the biomolecule used in the present invention or be derivatized to have such property, either covalently or non-covalently. Any solid material is included.
- any material capable of forming a solid surface can be used.
- examples thereof include glass, silica, silicon, ceramic, silicon dioxide, plastic, and metal. (Including, but not limited to, alloys), natural and synthetic polymers (eg, polystyrene, cellulose, chitosan, dextran, and nylon).
- the substrate may be formed from multiple layers of different materials.
- inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon carbide, silicon oxide, and silicon nitride can be used.
- Organic materials can be used.
- a preferable material for the substrate varies depending on various meters such as a measuring instrument, and a person skilled in the art can appropriately select an appropriate material from the various materials described above.
- a slide glass is preferred for the transfection array.
- such a substrate can be coated.
- coating when used with respect to a solid support or substrate, refers to forming a film of a substance on the surface of the solid support or substrate and to such a film. Coating may be performed for various purposes, for example, to improve the quality of the solid support and the substrate (for example, to improve the service life and environmental resistance such as acid resistance), the substance to be bound to the solid support or the substrate. In many cases, the purpose is to improve the affinity of the compound.
- the substance for such a coating is called a “coating agent”. As such a coating agent, various substances can be used.
- biological substances such as DNA, RNA, proteins, and lipids, and polymers (for example, -L-lysine, MAS (available from Matsunami Glass, Kishiwada, Japan), hydrophobic fluoroplastics), silanes (APS (eg, ⁇ -aminopropyl silane)), and metals (eg, gold) It can be used but is not limited to them.
- -L-lysine available from Matsunami Glass, Kishiwada, Japan
- hydrophobic fluoroplastics for example, silanes (APS (eg, ⁇ -aminopropyl silane)), and metals (eg, gold) It can be used but is not limited to them.
- APS eg, ⁇ -aminopropyl silane
- metals eg, gold
- such a coating comprises poly-L-lysine, silane, (eg, epoxy silane or mercapto silane, APS (o / -aminopropyl silane)), MAS, hydrophobic fluoropolymer, It may be advantageous to use a metal such as gold.
- silane eg, epoxy silane or mercapto silane, APS (o / -aminopropyl silane)
- MAS hydrophobic fluoropolymer
- a metal such as gold.
- Such substances can be cells or objects containing cells (e.g., living organisms, It is preferable to use a substance that conforms to ()).
- chip or “microchip” is used interchangeably, has various functions, and refers to a microminiature integrated circuit that becomes a part of a system.
- the chip include, but are not limited to, a DNA chip and a protein chip.
- the term "array” refers to a pattern or pattern in which a composition (eg, a mixture of DNA, protein, and transfat) containing one or more (eg, 1000 or more) target substances is arranged. (E.g., a chip) itself having.
- Arrays that are patterned on a small substrate eg, 10 ⁇ 10 mm
- the terms microarray and array are used interchangeably herein. Therefore, what is patterned on a substrate larger than the above-mentioned substrate may be called a microarray.
- an array is composed of a set of desired transphosphate mixtures that are themselves immobilized on a solid surface or membrane.
- Array preferably comprises at least 10 two identical or different antibodies, at least 10 3 and more preferably, and more preferably at least 10 4, even more preferably at least 10 5 a. These antibodies are preferably placed on a surface of 125 ⁇ 80 mm, more preferably 10 ⁇ 10 mm.
- a microtiter plate such as a 96-well microtiter plate or a 384-well microtiter plate, or a size similar to a slide glass is contemplated.
- the composition containing the target substance to be immobilized may be one kind or plural kinds. The number of such types can be any number up to the number of individual spots. For example, a composition containing about 10, about 100, about 500, about 1000 target substances can be immobilized.
- the solid phase surface or film such as a substrate
- any number of target substances as described above e.g., proteins such as antibodies
- 10 8 to biomolecules child up to 10 7 biomolecules in other embodiments, 10 up to 6 biomolecules, 10 to 5 biological molecules, no more than 10 4 biomolecules, up to 10 3 biomolecules , or 10 although pieces of biological molecules up to two biological molecules can be arranged, 10 a composition containing target product quality more than eight biomolecules may be disposed.
- the size of the substrate is preferably smaller.
- the spot size of a composition containing a target substance can be as small as the size of a single biomolecule (this Can be of the order of 2 nm).
- the minimum substrate area is in some cases determined by the number of biomolecules on the substrate.
- a composition containing a target substance to be introduced into a cell is usually immobilized by covalent bond or physical interaction in the form of a 0.01 mm to 10 mm spot.
- spots of biomolecules may be arranged.
- spot refers to a certain set of compositions containing a target substance.
- spotting refers to producing spots of a composition containing a certain target substance on a certain substrate or plate. Spotting can be performed in any manner, for example, by pitting or the like, or can be performed by an automated device, and such methods are well known in the art.
- the term "address” refers to a unique location on a substrate, which may be distinguishable from other unique locations.
- the address takes any shape appropriate for association with the spot with that address, and the entity at every each address can be distinguished from the entity at the other address (eg, optically). obtain.
- the shape defining the address may be, for example, a circle, an ellipse, a square, a rectangle, or an irregular shape. Therefore, “address” indicates an abstract concept, and “spot” may be used to indicate a specific concept. However, when there is no need to distinguish between the two, “address” is used in this specification. And “spot” can be used interchangeably.
- the size defining each address depends, inter alia, on the size of the substrate, the number of addresses on a particular substrate, the amount and / or available reagents of the composition containing the target substance, the size of the microparticles and their size. Depends on the degree of resolution required for any method in which the array is used. The magnitude can be, for example, in the range of l_2 nm to a few cm. Any size is possible, consistent with the application of the array.
- the spatial arrangement and shape defining the address are designed to suit the particular application in which the microarray is used.
- the addresses can be densely distributed and widely dispersed, or sub-gnoled into a desired pattern appropriate for a particular type of analyte.
- microarrays refer to the Genome Function Research Protocol It has been extensively reviewed in experimental lectures in the medical era 1), genomic medical science and its powerful genomic medicine (Experimental Medical Research IJ).
- the term “cell” is defined in the same broadest sense as used in the art, and is a constituent unit of a tissue of a multicellular organism and isolates the outside world. An organism that is wrapped in a membrane structure that has self-renewal capability and has genetic information and its expression mechanism.
- the cell used in the present specification may be a naturally occurring cell or an artificially modified cell (for example, a fusion cell, a genetically modified cell).
- the source of cells can be, for example, a single cell culture, or cells from a normally grown transgenic animal embryo, blood, or body tissue, or a normally grown cell line. Cell mixtures such as, but not limited to.
- the cells used in the present invention include cells from any organism (eg, any kind of single cell organism (eg, bacteria, yeast) or multicellular organisms (eg, animals (eg, vertebrates, invertebrates) Animals), plants (eg, monocotyledonous plants, dicotyledonous plants, etc.))).
- any organism eg, any kind of single cell organism (eg, bacteria, yeast) or multicellular organisms (eg, animals (eg, vertebrates, invertebrates) Animals), plants (eg, monocotyledonous plants, dicotyledonous plants, etc.)).
- cells derived from vertebrates are used, and more specifically, mammals (eg, monopores) Species, marsupials, oligodonts, skin wings, winged fins, carnivores, insectivores, longnoses, equinoids, artiodactyls, tubulars, squamata, sponges, whales, Cells derived from primates, rodents, and egrets) are used.
- mammals eg, monopores
- mammals eg, marsupials, oligodonts, skin wings, winged fins, carnivores, insectivores, longnoses, equinoids, artiodactyls, tubulars, squamata, sponges, whales, Cells derived from primates, rodents, and egrets
- a primate eg, a chimpanzee
- Macaques, and humans in particular, but not limited to, human-derived cells.
- stem cell has a self-renewal ability and a pluripotency (ie, pluripotency).
- a stem cell can be, but is not limited to, an embryonic stem (ES) cell or a tissue stem cell (also referred to as a tissue stem cell, a tissue-specific stem cell, or a somatic stem cell).
- ES embryonic stem
- tissue stem cell also referred to as a tissue stem cell, a tissue-specific stem cell, or a somatic stem cell.
- artificially generated cells eg, the fused cells, reprogrammed cells, etc. described herein
- Embryonic stem cells are pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981 and have been applied to the production of knockout mice since 1989.
- Tissue stem cells unlike embryonic stem cells, are cells in which the direction of differentiation is limited, exist at specific positions in tissues, and have an undifferentiated intracellular structure. Thus, tissue stem cells have a low level of pluripotency. Tissue stem cells have poor intracellular organelles with a high nuclear / cytoplasmic ratio. Tissue stem cells are generally pluripotent and maintain their proliferative potential for more than one lifetime in individuals whose cell cycle is slow.
- a stem cell may be an embryonic stem cell or a tissue stem cell.
- tissue stem cells When classified according to the site of origin, tissue stem cells are classified into, for example, skin system, digestive system, bone marrow system, nervous system and the like.
- Skin tissue tissue stem cells include epidermal stem cells and hair follicle stem cells.
- Gastrointestinal tissue stem cells include knee (common) stem cells and hepatic stem cells.
- myeloid tissue stem cells include hematopoietic stem cells and mesenchymal stem cells.
- Tissue stem cells of the nervous system include neural stem cells and retinal stem cells.
- "somatic cells” are cells other than germ cells such as eggs and sperm, and refer to all cells that do not directly transfer their DNA to the next generation. Somatic cells usually have limited pluripotency or disappear.
- the somatic cell used in the present specification may be a naturally occurring one or a genetically modified one.
- Cells can be classified into ectoderm, mesoderm and endoderm-derived stem cells according to their origin.
- the cells derived from the ectoderm mainly exist in the brain, and include neural stem cells and the like.
- Mesodermal-derived cells mainly exist in the bone marrow, and include vascular stem cells, hematopoietic stem cells, mesenchymal stem cells, and the like.
- Endoderm-derived cells are mainly present in organs and include hepatic stem cells and ⁇ stem cells.
- somatic cells can be from any germ layer.
- lymphocytes, spleen cells or testis-derived cells can be used as body cells.
- isolated refers to a substance that naturally accompanies at least a reduced amount in a normal environment, and preferably is substantially free of the substance.
- an isolated cell refers to a cell that is substantially free from other attendant materials (eg, other cells, proteins, nucleic acids, etc.) in its natural environment.
- isolated refers to, for example, when substantially free of cellular material or culture medium when produced by recombinant DNA technology, and when chemically synthesized.
- the isolated nucleic acid is preferably a sequence that naturally flanks the nucleic acid in the organism from which it is derived (ie, the sequence IJ located at the 5 'and 3' ends of the nucleic acid). Not included.
- an "established” or “established” cell is a cell that maintains a specific property (eg, pluripotency) and A state in which stable growth is maintained. Therefore, the established stem cells maintain pluripotency.
- a specific property eg, pluripotency
- the term "differentiated (cell)” refers to a cell having a specialized function and morphology (eg, a muscle cell, a nerve cell, and the like). No or almost no gender.
- the differentiated cells include, for example, epidermal cells, parenchymal cells, duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, skeletal myoblasts, nerve cells, vascular endothelial cells, and pigments Cells, smooth muscle cells, adipocytes, bone cells, chondrocytes, etc.
- the present invention relates to a medicament for introducing an active ingredient into cells (for example, a medicament such as a vaccine, a health food, a medicament in which protein or lipid has reduced antigenicity), a cosmetic, a pesticide, a food, and the like.
- a medicament for introducing an active ingredient into cells
- a medicament for example, a medicament such as a vaccine, a health food, a medicament in which protein or lipid has reduced antigenicity
- a cosmetic for introducing an active ingredient into cells
- Such medicaments and cosmetics may further include pharmaceutically acceptable carriers and the like.
- the pharmaceutically acceptable carrier contained in the medicament of the present invention includes any substance known in the art.
- Such suitable formulation materials or pharmaceutically acceptable carriers include antioxidants, preservatives, colorants, flavors, and diluents, emulsifiers, suspending agents, solvents, fillers, Quantities, buffers, delivery vehicles, diluents, excipients and Z or pharmaceutical adjuvants include but are not limited to.
- the medicament of the invention will be administered in the form of a composition comprising the compound, or a variant or derivative thereof, together with one or more physiologically acceptable carriers, excipients or diluents.
- suitable vehicles may be water for injection, physiological solutions, or artificial cerebrospinal fluid, which may be supplemented with other materials common in compositions for parenteral delivery. is there.
- an acceptable carrier, excipient, or stabilizer is non-toxic to recipients, and is preferably inert at the dosages and concentrations employed.
- Exemplary suitable carriers include neutral buffered saline, or saline mixed with serum albumin.
- the product is a suitable excipient (eg, For example, sucrose is used as a lyophilization agent.
- suitable excipient eg, sucrose is used as a lyophilization agent.
- Other standard carriers, diluents and excipients may be included as desired.
- Other exemplary compositions include Tris buffer at pH 7.0-8.5 or acetate buffer at ⁇ 4.0-5.5, which further includes sorbitol or a suitable alternative thereof. obtain.
- the present invention when used as a medicament, such a medicament can be administered orally or parenterally. Alternatively, such a medicament may be administered intravenously or subcutaneously.
- the medicament used in the present invention may be in the form of a pyrogen-free, pharmaceutically acceptable aqueous solution. Preparation of such a pharmaceutically acceptable composition can be easily performed by those skilled in the art by considering ⁇ , isotonicity, stability, and the like.
- the administration method includes oral administration, parenteral administration (e.g., intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, vaginal administration, local administration to the affected area). , Dermal administration, etc.). Formulations for such administration may be provided in any form. Such preparations include, for example, solutions, injections, and sustained-release preparations.
- the medicament of the present invention may contain a physiologically acceptable carrier, excipient or stabilizing agent (Japanese Pharmacopoeia, 14th edition or its latest edition, Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company, 1990, etc.) and a composition having the desired degree of purity, prepared and stored in the form of a lyophilized cake or aqueous solution. Can be done.
- a physiologically acceptable carrier Japanese Pharmacopoeia, 14th edition or its latest edition, Remington's Pharmaceutical Sciences, 18th Edition, AR Gennaro, ed., Mack Publishing Company, 1990, etc.
- a composition having the desired degree of purity prepared and stored in the form of a lyophilized cake or aqueous solution. Can be done.
- the amount of the composition used in the treatment method of the present invention depends on the purpose of use, the target disease (type, severity, etc.), the age, weight, sex, medical history, cell morphology or type of the patient, etc. It can be easily determined by a person skilled in the art in consideration of the above.
- the frequency of applying the treatment method of the present invention to a subject (or patient) also depends on the purpose of use, the target disease (type, severity, etc.), the age, weight, sex, medical history, and course of treatment of the patient. A person of ordinary skill in the art can easily make a decision in consideration of the above. Frequency may include, for example, administration once every few months (eg, once a week—once a month). It is preferable to administer once a week to once a month while observing the progress.
- a composition for increasing the efficiency of introducing a target substance into cells including a cell adhesion-related factor.
- substances that are rarely introduced into cells under normal conditions e.g., DNA, RNA, polypeptides, sugar chains, or a complex thereof
- cell adhesion-related factors typically, integrin.
- the above-mentioned object was achieved by unexpectedly finding that the introduction of an antibody against the antibody) promotes the introduction.
- cell adhesion-related factors would have a remarkable effect on promoting the transduction efficiency during genetic manipulation using DNA such as transfection.
- the present invention should be noted as providing a significant breakthrough, especially in genetic research.
- the cell adhesion-related factor used in the present invention includes an interacting substance that interacts with a cell adhesion molecule (eg, extracellular matrix, integrin receptor, RGD molecule, etc.).
- a cell adhesion molecule eg, extracellular matrix, integrin receptor, RGD molecule, etc.
- the interacting substance used in the present invention reacts with an antigen-antibody reaction with a partner of a cell adhesion molecule. Therefore, the interacting substance of the present invention may be an antibody (for example, monoclonal antibody, polyclonal antibody, etc.) or a derivative thereof (chimeric antibody).
- an antibody for example, monoclonal antibody, polyclonal antibody, etc.
- a derivative thereof chimeric antibody
- the interacting substance includes an antibody selected from the group consisting of an anti-CD49a antibody, an anti-CD49b antibody, an anti-CD49c antibody, an anti-CD49e antibody and an anti-CD49f antibody.
- an antibody selected from the group consisting of an anti-CD49a antibody, an anti-CD49b antibody, an anti-CD49c antibody, an anti-CD49e antibody and an anti-CD49f antibody.
- Such antibodies are preferred because they unexpectedly increase the efficiency of cell transfer of genetic material (DNA).
- DNA genetic material
- a substrate such as an extracellular matrix to adhesion via integrins including CD49a-i3 ⁇ 4r.
- Adhesion through such cell adhesion-related factors has a favorable effect on cells, in this case, in particular, increases the efficiency of cell transfer of genetic material (DNA). Observed (prior patent or reference).
- an anti-CD49 antibody as an adhesion substrate is presumed to indicate a function that mimics this effect. Therefore, an antibody against a molecule called anti-integrin containing CD49a-fr3 ⁇ 4 related to CD49 can also be mentioned as a preferred embodiment of the present invention.
- the target substance targeted in the present invention includes genetic material (eg, a nucleic acid molecule such as DNA or RNA).
- genetic material eg, a nucleic acid molecule such as DNA or RNA.
- DNA include, but are not limited to, plasmid DNA, naked DNA, cDNA, and genomic DNA.
- the integrin receptor contemplated in the present invention is selected from the group consisting of CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, and CD29 force (eg, CD29, CD49a , CD49c, CD49d, CD49e and CD49f).
- CD29 force eg, CD29, CD49a , CD49c, CD49d, CD49e and CD49f.
- CD29 is also preferred because, by nature, attaching cells using a substrate such as extracellular matrix results in integrin-mediated attachment.
- CD29 is the beta subunit of the integrin receptor and a common unit of many integrin receptors.
- Adhesion via such cell adhesion-related factors has a favorable effect on cells, in which case, in particular, an increase in the efficiency of cell transfer of genetic material (DNA) is observed (prior patents or references). Therefore, the use of an anti-CD29 antibody as an adhesion substrate can be presumed to indicate a function that mimics this effect.
- the integrin receptor may interact with a molecule selected from the group consisting of collagen, fibronectin, vitronectin and laminin.
- the cells targeted by the present invention may include at least one cell selected from the group consisting of stem cells and differentiated cells.
- both stem cells and differentiated cells can be targeted.
- the cell adhesion molecule is one that is specifically expressed in the cell.
- Such cell-specific adhesion molecules can be those known in the art, or can be identified by those skilled in the art using any well-known techniques. The ability to do S.
- the target substance used in the present invention is a genetic substance, and it is advantageous to further include a gene transfer reagent.
- Genetic material is usually DNA, which can be genetic material suitable for a gene transfer reagent.
- the composition of the present invention preferably further comprises a gene transfer reagent. This is because by including such a gene transfer reagent, the effect of increasing the transfer efficiency of the present invention is synergistically exhibited.
- the gene introduction reagent used in the present invention can be selected from the group consisting of a cationic polymer, a cationic lipid, and calcium phosphate.
- the gene transfer reagent include, but are not limited to, Effectene, TransFast, TfxTM-20, SuperFect, PolyFect, LipofectAMINE 2000, JetPEI, and ExGen500. Li pofectAMINE 2000 force S preferred ,. This is because gene transfer efficiency is high.
- the composition of the present invention further comprises particles. This is because by including the particles, the substance can be efficiently introduced into the cell, particularly, the targeted introduction. Preferred examples of such particles include, but are not limited to, metal colloids such as gold colloid.
- the composition of the present invention further comprises a salt. While not wishing to be bound by theory, the inclusion of such a salt may enhance the anchoring effect when a support is used, or may allow the three-dimensional structure of the target material to be more appropriately shaped. It is considered that the effect retained by
- any salt can be used as long as it is an inorganic salt or an organic salt.
- the cell adhesion-related factor contained in the composition of the present invention is an antibody of an integrin receptor or a variant or fragment thereof.
- an antibody of the integrin receptor or a variant or a fragment thereof unexpectedly has a target substance-introducing effect.
- the extracellular matrix protein has the effect of increasing the efficiency of introducing a substance into cells.
- a polypeptide (for example, an integrin) considered in the present invention has a polypeptide having an amino acid sequence known in the art, or a modified sequence thereof, and has a biological activity. It may be a polypeptide.
- the number of substitutions, additions and deletions in the modified sequence is limited, for example, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, preferably 2 or less.
- the number of such substitutions, additions and Zs or deletions may be one or several. Lesser numbers of substitutions, additions and deletions are preferred, but higher numbers so long as they retain the biological activity (preferably have similar or substantially the same activity as the cell adhesion-related factor). May be.
- allelic variants of the polypeptide are used and may have at least 90% homology with the sequence on which the comparison is based. For example, in the same strain, it is preferable that such allelic variants have at least 99% homology.
- the species homologue is present in the gene sequence database of the species, the homologue of the species or the extracellular matrix protein of the present invention, the entire integrin receptor (eg, CD49 family) gene sequence or It can be identified by searching a part as a query sequence.
- the gene of the present invention can be identified by screening a gene library of the species using all or a part of the gene of the present invention as a probe or a primer. Such identification methods are well known in the art and have been described in the literature described herein.
- the species homolog has, for example, at least about 30% homology with the original sequence. Species homology preferably has at least about 50% homology with the original sequence.
- the species homolog preferably has at least about 30% homology with the original sequence.
- the species homolog preferably has at least about 50% homology with the original sequence.
- the concentration of the cell adhesion-related factor can be easily determined by those skilled in the art with reference to the description in the present specification.
- an example of such a concentration is at least about 0.1 / ig // L, preferably about 0.2 / ig / L, and more preferably 0.5 / ig // L. It is.
- OzgZxL may be a preferred concentration range, since concentrations above about 0.5 zg / zL reach a plateau.
- the present invention provides a kit for increasing gene transfer efficiency.
- a kit comprises (a) a composition comprising a cell adhesion-related factor; and (b) a gene transfer reagent.
- the cell adhesion-related factor the form described in detail in the above-described composition for increasing the efficiency of introduction of a target substance into a cell of the present invention can be applied.
- Such a form can be selected by a person skilled in the art based on the description in the present specification, and can be performed.
- such a kit may further comprise instructions. This instruction may be, but is not limited to, written in accordance with the format prescribed by the regulatory agency of the country in which the invention is implemented and approved by that regulatory agency.
- Cell adhesion-related factors those skilled in the art can arbitrarily select a form to be applied in a composition for increasing the efficiency of introducing a target substance into cells of the present invention, as described above.
- the invention can be implemented.
- the cell adhesion-related factor may be an antibody of the integrin receptor or a variant or fragment thereof.
- an antibody selected from the group consisting of an anti-CD49a antibody, an anti-CD49b antibody, an anti-CD49c antibody, an anti-CD49e antibody and an anti-CD49f antibody, a fragment thereof, or a variant thereof can be used.
- the present invention provides a composition for increasing the efficiency of introducing a target substance into cells.
- a target substance for example, DNA, RNA, polypeptide, sugar chain, or a complex substance thereof
- the present invention has been completed by unexpectedly finding that induction is promoted by the action of a cell adhesion-related factor (typically, an antibody of an integrin receptor). It is provided in the form of a composition containing a target substance and a cell adhesion-related factor.
- the cell adhesion-related factor may be an antibody to an integrin receptor or a variant thereof. More preferably, an antibody selected from the group consisting of an anti-CD49a antibody, an anti-CD49b antibody, an anti-CD49c antibody, an anti-CD49e antibody and an anti-CD49f antibody, or a fragment thereof or a variant thereof can be used.
- the target substance contained in the composition for introducing the target substance of the present invention into a cell preferably includes, for example, DNA, RNA, polypeptide, sugar, and a complex thereof. It is not limited to.
- DNA is selected as the target. When such DNA is used for gene expression, it is preferable to encode the gene of interest. Therefore, in an embodiment intended for transfection, the target substance comprises a DNA encoding the gene sequence to be transfected.
- RNA is selected as the target substance. When such RNA is intended for gene expression, it preferably encodes the gene of interest. In this case, it is preferable to use RNA encoding the gene sequence together with a gene transfer agent suitable for RNA.
- the composition for introducing a target substance of the present invention into cells may further contain a gene introduction reagent. While not being bound by theory, in one embodiment, such a gene transfer reagent and the cell adhesion-related factor found in the present invention act in unison to achieve an unprecedented efficient gene. Is thought to be introduced into cells.
- such a gene transfer reagent that can be contained in the composition of the present invention includes, for example, cationic polymers, cationic lipids, polyamine-based reagents, polyimine-based reagents, calcium phosphate, and the like.
- the composition for introducing the target substance of the present invention into cells may exist as a liquid phase. When present as a liquid phase, the invention is useful, for example, as a liquid phase transfer system.
- the composition for introducing the target substance of the present invention into cells may exist as a solid phase.
- the invention is useful, for example, as a solid phase transfection system.
- Preferred embodiments of the solid phase transfer extrusion include, but are not limited to, a transfer extrusion system using a microtiter plate, or a transfer extrusion system using an array (or chip). . These liquid and solid forms are also useful when introducing polypeptides.
- the present invention also provides a device for increasing the efficiency of introducing a target substance into cells.
- a composition comprising A) a target substance; and B) a cell adhesion-related factor is immobilized on a solid support.
- the device of the present invention is characterized in that a target substance that is hardly introduced into cells under normal conditions (for example, DNA, RNA, polypeptide, sugar chain, or a complex substance thereof) is a cell adhesion-related factor (typically, It has been completed by unexpectedly finding that the introduction is promoted by the action of an integrin receptor antibody or a variant thereof).
- the present invention provides a target substance and a cell adhesion-related factor.
- the cell adhesion-related factor may be an antibody to an integrin receptor or a variant thereof. More preferably, an antibody selected from the group consisting of an anti-CD49a antibody, an anti-CD49b antibody, an anti-CD49c antibody, an anti-CD49e antibody and an anti-CD49f antibody, or a fragment thereof or a variant thereof can be used.
- the target substance included in the device of the present invention preferably includes, but is not limited to, for example, DNA, RNA, polypeptide, sugar, and a complex thereof. What? In certain preferred embodiments, DNA is selected as the target substance. When such DNA is used for gene expression, it is preferable to encode the desired gene. Therefore, in an embodiment for transfection, the target substance includes DNA encoding a gene sequence to be transfused.
- the device of the present invention may further include a gene transfer reagent.
- a gene transfer reagent and the cell adhesion-related factor found in the present invention cooperate to produce an unprecedented efficient intracellular gene. It is thought that it will be introduced.
- the composition is immobilized on the solid support, it is preferable to use a gene transfer reagent that is compatible with the solid support.
- the solid support used in the device of the present invention is selected from the group consisting of a plate, a microplate, a chip, a slide glass, a finolem, a bead, and a metal. May be.
- a device of the invention when a device of the invention uses a chip as a solid support, such a device may also be referred to as an array.
- biomolecules to be introduced eg, DNA, protein, etc.
- transfer execution arrays those intended for transfer execution are also referred to herein as transfer execution arrays.
- the composition, device, and method using the cell adhesion-related factor of the present invention achieve an unprecedented effect of providing a transfection array that can be performed with any cell. Will be.
- the solid support used in the device of the present invention is preferably coated.
- the coating improves the quality of the solid support and the substrate (for example, increases the service life, improves the environmental resistance such as acid resistance), the affinity for the substance to be bound to the solid support or the substrate, and This is because the affinity for cells is increased.
- poly-L-lysine, silane eg, APS (—aminopropyl silane)
- MAS aminopropyl silane
- hydrophobic fluoropolymer epoxy silane
- a coating agent containing a silane such as mercaptosilane or a metal such as gold is used.
- the coating is poly L lysine.
- the present invention provides a method for increasing the efficiency of introducing a target substance into cells.
- the present invention presents a target substance (for example, DNA, RNA, polypeptide, sugar chain, or a complex substance thereof) which is hardly introduced into a cell under normal conditions to a cell together with a cell adhesion-related factor (preferably, This is an invention that has been completed by first finding an effect that the target substance is efficiently introduced into cells by contact. Therefore, the method of the present invention comprises, in any order, A) a step of providing a target substance; B) a step of providing a cell adhesion-related factor; and C) contacting the target substance and the cell adhesion-related factor with the cell. The method further includes a step.
- the target substance and the cell adhesion-related factor may be provided together or separately.
- the forms described in detail in the above-described composition for increasing the efficiency of introduction of the target substance into cells of the present invention can be applied.
- those skilled in the art can select and implement an appropriate form based on the description in this specification. Therefore, as such a cell adhesion-related factor, those skilled in the art can arbitrarily select a form to be applied in a composition for increasing the efficiency of introducing the target substance of the present invention into a cell, and determine the present invention.
- the cell adhesion-related factor can be an integrin receptor antibody or a variant thereof.
- an antibody selected from the group consisting of an anti-CD49a antibody, an anti-CD49b antibody, an anti-CD49c antibody, an anti-CD49e antibody and an anti-CD49f antibody, or a fragment thereof or a variant thereof can be used.
- the target substance used in the method of the present invention preferably includes, but is not limited to, for example, DNA, RNA, polypeptide, sugar, and complexes thereof.
- DNA is selected as the target substance.
- the target substance comprises DNA encoding the gene sequence to be transfected.
- the method of the present invention employs a gene transfer reagent. It may also be used. Without being bound by theory, in one embodiment, such a gene transfer reagent and the cell adhesion-related factor found in the present invention act in synergy to provide a cell with an unprecedented efficient gene. It is thought that it will be introduced inside.
- the provision of the gene transfer reagent may be together with or separate from the target substance and / or the cell adhesion-related factor.
- such a gene transfer reagent that can be used in the method of the present invention includes, for example, a cationic polymer, a cationic lipid, a polyamine-based reagent, a polyimine-based reagent, and calcium phosphate. But not limited to them.
- the target cells in the present invention may include any cells as long as the target substance is introduced, and examples include stem cells and somatic cells.
- a remarkable effect of the present invention is that the introduction of a target substance such as transfection is almost uniformly achieved in any cells regardless of the type of cells such as stem cells and somatic cells. This is an unexpected effect that the law did not have.
- the subject may include, but is not limited to, tissue stem cells, and embryonic stem cells may also be included as the subject. Without being bound by theory, among stem cells, tissue stem cells appear to have a higher transduction efficiency than embryonic stem cells.
- the target substance cell introduction method of the present invention can be partially or entirely performed in a liquid phase. In another specific embodiment, the target substance cell introduction method of the present invention can be performed partially or entirely on a solid phase. Therefore, the target substance cell introduction method of the present invention can be performed in a combination of a liquid phase and a solid phase.
- the present invention provides a method for increasing the efficiency of introducing a target substance into cells using a support.
- the present invention provides a method for immobilizing a target substance (e.g., DNA, RNA, polypeptide, sugar chain, or a complex substance thereof) that is hardly introduced into cells under normal conditions on a support, Present to (good More preferably, the present invention has been completed by first finding an effect that the target substance is efficiently introduced into cells by contact.
- the effect of increasing the introduction efficiency of the target substance (particularly DNA, preferably DNA containing the sequence encoding the gene to be transfected) on the support is not achievable with the prior art, and is not achieved.
- the method using the solid support of the present invention comprises a step of immobilizing a composition comprising I) A) a target substance; and B) a cell adhesion-related factor on a support; Contacting the cells with the composition.
- a cell adhesion-related factor the form described in detail in the composition for increasing the efficiency of introduction of the target substance of the present invention into cells can be applied.
- those skilled in the art can select and implement an appropriate form based on the description in this specification. Therefore, as such a cell adhesion-related factor, those skilled in the art can arbitrarily select a form to be applied in a composition for increasing the efficiency of introducing the target substance of the present invention into cells, and carry out the present invention. That can be S.
- the cell adhesion-related factor may be an integrin receptor antibody or a variant thereof. More preferably, an antibody selected from the group consisting of an anti-CD49a antibody, an anti-CD49b antibody, an anti-CD49c antibody, an anti-CD49e antibody and an anti-CD49f antibody, or a fragment thereof or a variant thereof can be used.
- the DNA When used as a target substance, the DNA may be provided naked (Naked DNA), but is preferably provided using a vector (plasmid) together with control sequences (promoter and the like). possible. In such cases, it is preferred that the DNA be operably linked to the control sequence.
- the method of the present invention also preferably further comprises the step of providing a gene transfer reagent, wherein the gene transfer reagent is provided to be contacted with a cell.
- a gene transfer reagent is preferred in the method of the present invention, since the transfer efficiency is further improved.
- the provision of a gene introduction reagent is well known in the art, and includes, but is not limited to, for example, adding a solution in which a gene introduction reagent is dissolved to an experimental system.
- the gene transfer reagent forms a complex with the target substance DNA, and then provides a cell adhesion-related factor. Without being bound by theory, taking this order This is because it has been found that the efficiency of introducing a target substance into cells on a solid support is dramatically increased.
- the gene transfer reagent (eg, cationic lipid) -target substance conjugate comprises a target substance (eg, DNA in an expression vector) and a gene transfer reagent, which may be water or deionized. Dissolved in a suitable solvent such as water. This solution is spotted on a surface such as a slide, thereby generating a surface to which the gene transfer reagent-target substance complex is attached at a specific position. Thereafter, a cell adhesion-related factor is appropriately added. The spotted transfection reagent-target substance conjugate is attached to the slide and dried sufficiently so that the spot remains in this attached position under the conditions used in subsequent steps of the method.
- a target substance eg, DNA in an expression vector
- a gene transfer reagent which may be water or deionized. Dissolved in a suitable solvent such as water. This solution is spotted on a surface such as a slide, thereby generating a surface to which the gene transfer reagent-target substance complex is attached at a
- the transfection reagent-target substance conjugate is coated on a slide or chip, such as a glass slide coated with poly-L-lysine (available from Sigma, Inc., Inc.), eg, manually or on a microarray. Spot using a manufacturing machine. The DNA spots can then be attached to the slide by drying the slide or chip at or above room temperature or under reduced pressure. The length of time required for sufficient drying to occur depends on several factors, such as the amount of mixture placed on the surface and the temperature and humidity conditions used. In the present invention, the cell adhesion-related factor is preferably provided after the complex is attached.
- the concentration of DNA present in the mixture is determined experimentally for each application, but generally ranges from about 0.01 / 1 ⁇ // 11 to about 0.2 ⁇ // ⁇ 1. In a particular embodiment, it is about 0.102 / 1 ⁇ // 11 to about 0.10 gZ / l.
- the concentration of DNA present in the gene transfer reagent-target substance complex is about 0.01 / 1 ⁇ / 1 to about 0.5 / 1 ⁇ // 11, about 0.1 Olugu 1 to about 0.4. ⁇ g / ⁇ l, and the stirrer may be 0.3 Olgu u 1 force, 0.3 ugu.
- the concentration of cell adhesion-related factors, or other carrier macromolecules, such as gene transfer reagents is determined empirically for each application, but typically ranges from 0.01% to 0.5%. In certain embodiments, about 0.05% force, about 0.5%, about 0.05% force, about 0.2%, or about 0.1% to about 0.2%.
- the final concentration of DNA in the cell adhesion-related factor-target substance complex is generally about 0.02 ⁇ gZ ⁇ 1 force, about 0.1 ⁇ g / ⁇ 1
- the DNA is It can be a final DNA concentration equal to about 0.05 / g // l.
- the vector can be of any type, such as a plasmid or a virus-based vector, in which the DNA of interest (to be expressed in cells) DNA) can be introduced and then expressed in the cell.
- a vector for example, a CMV-driven expression vector can be used.
- plasmid-based or virus-based vectors such as pEGFP (Clontech) or pcDNA3 (Invitrogen) may be used.
- the surface with the spot is coated with an appropriate amount of a lipid-based transfection reagent, and the resulting product Is maintained under conditions appropriate for complex formation between the DNA in the spot and the gene transfer reagent (eg, a transfection reagent such as a cationic lipid).
- the gene transfer reagent eg, a transfection reagent such as a cationic lipid
- the cell adhesion-related factor is provided thereafter, or at the same time the cell adhesion-related factor is provided.
- the resulting product is incubated at 25 ° C for about 20 minutes.
- the gene transfer reagent is removed, a surface with DNA (DNA in complex with the transfection reagent) is generated, and cells in a suitable culture are plated on the surface.
- the resulting product (the surface with the DNA and the plated cells) is maintained under conditions that cause entry of the DNA into the plated cells.
- transfection efficiency expression of the encoded product, effects on cells, and the like can be evaluated using known methods. For example, detection of immunofluorescence, or enzyme immunocytology, in situ hybridization, autoradiography, or other methods of detecting the expression of DNA or the effects of the encoded product or DNA itself on the introduced cells. By the means described above, the above parameter can be determined.
- antibodies that bind to the protein and are fluorescently labeled e.g., The slide is digested under conditions appropriate for binding of the body to the protein), and the location containing the protein (spot or area on the surface) is identified by detecting fluorescence.
- the presence of the fluorescent light indicates that transfection has occurred at defined locations that exhibit fluorescence and that the encoded protein has been expressed.
- the presence of a signal on the slide, detected by the method used indicates that expression of the transfection and encoded product or DNA transfer in the cell has occurred at the particular location where the signal was detected.
- the identity of the DNA present at each particular location may be known or unknown; thus, when expression occurs, the identity of the expressed protein may be known or unknown. Good. Preferably, such information is known. This is because it can be correlated with conventional information.
- Various cell adhesion-related factors were prepared as candidates for cell adhesion-related factors.
- the preparations in this example are as follows.
- Antibodies were prepared by using commercially available antibodies or by raising antibodies against the integrin receptor.
- As the polypeptide a commercially available product or a product produced by genetic manipulation was used.
- the CD29 and CD49 families can also be prepared using the amino acid sequence described in SEQ ID NOs: 3 to 14 for raising antibodies.
- Fibronectin SEQ ID NO: 1
- CD49a antibody IMMUN ⁇ TECH (a coulter company), france, monoclonal antibody CD49a—VLA alfal Cat. No. 1599 // COSMOBIO, Japan, mouse—ANTIratCD49a).
- CD49b antibody (IMMUNOTECH (a coulter company), france, monoclonal antibody CD49b Cat. No. 0717).
- CD49c antibody IMMUN ⁇ TECH (a coulter company), france, monoclonal antibody CD49c Cat. No2000).
- CD49d antibody IMMUNOTECH (a coulter company), france, monoclonal antibody CD49d Cat. No0764 // ENDOGEN, USA, mouse—ANTIratCD49d).
- CD49e antibody (IMMUNOTECH (a coulter company), france, monoclonal antibody CD49e Cat. No. 0771).
- CD49f antibody IMMUN ⁇ TECH (a coulter company), france, monoclonal antibody CD49f Cat. No. 0769 // AntigenixAmerica, USA, mouse—ANTIratCD49f, PRO. NO. MR49662C.
- CD29 antibody (IMMUNOTECH (a coulter company), france, monoclonal antibody CD29 Cat. Nol 5: 0.
- the solution obtained by reconstituting these antibodies and cell adhesion-related factors in the specified manner and dose was diluted 100 to 5000 times with PBS, and then seeded on a glass slide surface and dried. After drying, wash well with PBS and print the gene transfer reagent and gene complex on it. The target cells are seeded on these glass slides and the solid phase gene transfer method is performed.
- An anti-rat monoclonal antibody was used for PC12 cells and the like.
- FIG. 1 shows the relationship between the integrin receptor and the recognized extracellular matrix.
- a plasmid for transfection was prepared as DNA.
- plasmids pEGFP-Nl and pDsRed2-Nl (both BD Biosciences, Clontech, CA, USA) were used.
- gene expression is under the control of cytomegalovirus (CMV).
- Plasmid DNA was amplified and amplified in E. coli (XLl blue, Stratgene, TX, USA) and used as one of the complex partners. DNA was dissolved in distilled water containing neither DNase nor RNase.
- the transfection reagents used are as follows: Effectene Transfection Reagent (cat. No. 301425, Qiagen, CA), TransFastTM Transfection Reagent (E2431, Promega, WI), TfxTM-20 Reagent (E2391, Promega, WI), SuperFect Transfection Reagent (301305, Qiagen, CA), PolyFect Transfection Reagent (301 105, Qiagen, CA), LipofectAMINE 2000 Reagent (1 1668-019, Invitrogen corporation, CA), JetPEI (X4) cone. (101 — 30, Polyplus—transfection, France) and ExGen 500 (R0511, Ferment as Inc., MD). The power that seems to be preferable to use LipofectAMINE 2000 is not limited to that.
- the transfusion reagent was added to the above-mentioned DNA and the cell adhesion-related factor or used after forming a complex with DNA.
- PC12 rat pheochromocytoma cells: ATCC CRL-1721
- PC12 rat pheochromocytoma cells: ATCC CRL-1721
- DMEM / l 0% calf serum GIBCO
- Transfection reagents should be used according to the manufacturer's instructions.
- Plasmid DNA Glycerol stock was grown in lOOmL L_amp and purified using Qia prep Miniprep or Qiagen Plasmid Purification Maxi according to the standard protocol provided by the manufacturer.
- RIKEN Cell Bank JPN. These are DMEM containing L_glut and pen / strep
- the transfection reagent and DNA are mixed to form a DNA-transfection reagent complex. Since some time is required for complex formation, the mixture
- the cell adhesion-related factor may be used at the time of forming the complex, but in this example, it was physically coated on a solid support (for example, poly-L-lysine slide). Reconstitute these antibodies' Itoda cell adhesion-related factor with the specified dosage and administration in PBS at 100-5000. After dilution by a factor of 1, the cells were seeded on a glass slide surface and dried. After drying, wash well with PBS and print the gene transfer reagent and gene complex on it. The target cells were seeded on these glass slides and subjected to solid-phase gene transfer.
- a solid support for example, poly-L-lysine slide.
- Cells were distributed for transfatate. This distribution was usually performed by suctioning the reagent under reduced pressure in a hood. The slide was placed on the dish and the solution containing the cells was added for transfection.
- the distribution of cells is as follows.
- Proliferating cells were distributed such that the cell concentration was 10 7 cells in 25 mL. Cells were plated on slides in square 100 0 100 1 5111111 Petri dishes or 100111111 15 mm circular dishes. The transfection was allowed to proceed for about 40 hours. This is about two cell cycles. Slides were processed for immunofluorescence.
- Evaluation of gene transfer was achieved, for example, by immunofluorescence, fluorescence microscopy, laser scanning, radiolabeling and detection using sensitive films or emulsions.
- the expressed proteins to be visualized are fluorescent proteins, they can be viewed by fluorescence microscopy and photographed.
- the slide can be scanned with a laser scanner for data storage. If a fluorescent antibody can detect the expressed protein, an immunofluorescence protocol can be followed. If the detection is based on radioactivity, the slides can be attached as indicated above, and the radioactivity detected by autoradiography using film or emulsion.
- FIG. 2A shows the results of using various cell adhesion-related factors and fibronectin when using HepG2 cells as an example.
- FIG. 2B shows, as another example, the results of the transfusion efficiency using different cell adhesion-related factors (HLA, CD46 and CD54) in an experiment using HepG2 cells.
- FIG. 3A shows the relationship between various integrin receptors (CD49a, d, f) expressed on HepG2 cells and the recognized extracellular matrix, and the transfusion efficiency on each extracellular matrix-coated surface.
- FIGS. 3B-3E show changes in cells after transfection when various cell adhesion-related factors are coated.
- PC12 cells which are known as nerve-like cells
- nerve-like cells it was observed whether or not the effect of the cell adhesion-related factor of the present invention can be observed in differentiated cells.
- various reagents and cells were prepared. However, PC12 cells followed the following procedure.
- PC12 rat pheochromocytoma cells: ATCC CRL-1721
- DMEM / lO containing L_glut and pen / strep. / 0 calfserum GEBCO
- Growing cells were distributed such that the cell concentration was 10 7 cells in 25 mL.
- Cells were plated on slides in square 100 ⁇ 100 ⁇ 15 mm Petri dishes or circular dishes of radius 100 mm ⁇ 15 mm. The transfusion was allowed to proceed for about 48 hours. Slides were processed for immunofluorescence.
- FIGS. FIG. 4 shows a result similar to FIG. 3A in Example 3. As can be seen, it was revealed that the cell adhesion-related factor of the present invention also increases transfusion efficiency even in PC12 cells, which are less effective with fibronectin.
- Figs. 5A and 5B show the state of cell adhesion
- Fig. 6 shows photographs comparing the state of transfection.
- 5A and 5B show the state of cell adhesion of PC12 cells in Example 4. Inhibition of PC12 cell adhesion to TypelV collagen-coated surface using CD antibody and transfection. TypelV collagen coat on poly-L-lysine coated slide glass. Thereafter, PC12 cells which had been brought into contact with the antibody solution in advance were seeded, and transfection was performed using Lipofectamin2000 according to a normal (liquid phase system) protocol.
- Figure 5A shows that anti-CD49a markedly inhibited the adhesion of PC12 cells to the TypelV collagen-coated surface.
- the anti-CD49d does not inhibit the adhesion of PC12 cells and shows the same transfection efficiency as the control without antibody.
- PC12 cells do not express CD49d as is apparent from FIG.
- CD49d is a receptor for fibronectin, suggesting that the adhesion to the TypelV collagen-coated surface was not inhibited.
- the presence of the antibody itself does not affect the transfusion efficiency.
- FIG. 5B also shows the difference in transfusion efficiency in the presence of anti-CD49f. The number shown indicates the number of dilutions of the antibody stock solution and the force with which it was brought into contact with PC12 cells beforehand. As can be clearly seen from the figure, the transfusion efficiency decreases depending on the concentration of the antibody contacted.
- CD49f is a receptor for laminin.
- FIG. 7 shows the effect of coating the cell adhesion-related factor on the support. Coating with collagen type IV appears to increase transfer efficiency.
- FIG. 8 shows an outline of a solid-phase transfection using a cell adhesion-related factor.
- phase transfer array SPTA
- the present inventors used differentiated cells (PC12 cells) that were inefficient with HepG2 and fibronectin, by using the method of the present inventors ( Both the solid-phase transfer and the conventional liquid-phase transfer were studied under a series of transfusion conditions.
- Transfection efficiency The transfection efficiency was determined as the total fluorescence intensity per unit area. Optimum liquid phase results were obtained according to the cell line used. These efficient transfusion reagents were then used to optimize the solid phase protocol. Some trends were observed: easily transfatable cell lines (e.g., HEK293, For HeLa, NIH3T3), the transfer efficiency observed with the solid phase protocol was slightly better than the standard liquid phase protocol, but was achieved at essentially similar levels. I have.
- the present inventors have successfully realized PC12 cells and an hMSC transfection array in a system using cell adhesion-related factors. This is because of the pluripotent stem cell It will enable high-throughput research in various studies using solid-phase transfer exci- sion, such as elucidation of the genetic mechanism that controls the conversion. The detailed mechanism of solid phase transfer and the methodology related to the use of this technology for high-throughput real-time gene expression monitoring has been shown to be applicable to a variety of purposes.
- hMSCs human mesenchymal stem cells, PT_2501, Cambrex Bioscience Walkersville, Inc., MD
- human MSC cells these cells are converted to a commercially available human mesenchymal cell basal medium (MSCGM BulletKit PT-3001, Cambrex Bioscience Walkersville, MD). Inc., MD).
- Example 4 Similarly to the results of Example 4, the present inventors successfully implemented a transfusion array using human mesenchymal stem cells using a system using cell adhesion-related factors. This will enable high-throughput studies in various studies using solid-phase transfection, such as elucidation of the genetic mechanism that controls the differentiation of pluripotent stem cells. The detailed mechanism of solid-phase transfer and the methodology related to the use of this technique for high-throughput real-time gene expression monitoring has been shown to be applicable to a variety of purposes.
- SHSY5Y humanneuroblastoma: ATCC CRL-2266
- Example 4 Similarly to the results of Example 4, the present inventors successfully realized a transfection array for human neuroblastoma using a system using cell adhesion-related factors. This indicates that seeds using solid-phase transfection, such as elucidation of the genetic mechanism that controls the differentiation of cancer cells, have been developed. It will enable high-throughput research in various studies. The detailed mechanism of solid-phase transfer and the methodology related to the use of this technique for high-throughput real-time gene expression monitoring have been shown to be applicable to a variety of purposes.
- transfusion efficiency increasing reagent is particularly useful for carrying out a transfusion in a solid phase. Therefore, it is useful in any field that uses genetic engineering.
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Abstract
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP05709422A EP1715052A4 (en) | 2004-01-29 | 2005-01-27 | COMPOSITION AND METHOD FOR INCREASING GENTRANSFER EFFICIENCY |
JP2005517495A JPWO2005073385A1 (ja) | 2004-01-29 | 2005-01-27 | 遺伝子導入効率を上昇させるための組成物および方法 |
US10/587,776 US20080038824A1 (en) | 2004-01-29 | 2005-01-27 | Composition and Method for Elevating Gene Transfer Efficiency |
CA002554753A CA2554753A1 (en) | 2004-01-29 | 2005-01-27 | Composition and method for elevating gene transfer efficiency |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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JP2004-022315 | 2004-01-29 | ||
JP2004022315 | 2004-01-29 |
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WO2005073385A1 true WO2005073385A1 (ja) | 2005-08-11 |
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ID=34823826
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PCT/JP2005/001148 WO2005073385A1 (ja) | 2004-01-29 | 2005-01-27 | 遺伝子導入効率を上昇させるための組成物および方法 |
Country Status (5)
Country | Link |
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US (1) | US20080038824A1 (ja) |
EP (1) | EP1715052A4 (ja) |
JP (1) | JPWO2005073385A1 (ja) |
CA (1) | CA2554753A1 (ja) |
WO (1) | WO2005073385A1 (ja) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008049146A (ja) * | 2006-07-24 | 2008-03-06 | National Institute Of Advanced Industrial & Technology | 組織再生用スキャッフォールド及びその製造方法 |
WO2015005431A1 (ja) | 2013-07-12 | 2015-01-15 | 石原産業株式会社 | 細胞への遺伝子導入用組成物 |
Families Citing this family (1)
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DE10361932A1 (de) * | 2003-12-30 | 2005-07-28 | Celanese Ventures Gmbh | Protonenleitende Membran und deren Verwendung |
Citations (5)
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---|---|---|---|---|
WO1997011604A1 (en) * | 1995-09-29 | 1997-04-03 | Indiana University Foundation | Methods for enhanced virus-mediated dna transfer using molecules with virus- and cell-binding domains |
WO2000001836A1 (fr) * | 1998-07-01 | 2000-01-13 | Takara Shuzo Co., Ltd. | Procedes de transfert de genes |
WO2002018609A2 (en) * | 2000-08-31 | 2002-03-07 | Virxsys | Methods for stable transduction of cells with viral vectors |
WO2002083854A2 (en) * | 2001-04-13 | 2002-10-24 | Biogen, Inc. | Antibodies to vla-1 |
WO2003000297A1 (fr) * | 2001-06-20 | 2003-01-03 | Sumitomo Pharmaceuticals Company, Limited | Procede facilitant le transfert d'acides nucleiques |
Family Cites Families (3)
Publication number | Priority date | Publication date | Assignee | Title |
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AUPQ147799A0 (en) * | 1999-07-07 | 1999-07-29 | Medvet Science Pty. Ltd. | Mesenchymal precursor cell |
US6905878B2 (en) * | 2002-12-19 | 2005-06-14 | The Scripps Research Institute | DNA array for high throughput solid-phase transfection and method for producing the same |
SE0301087D0 (sv) * | 2003-04-14 | 2003-04-14 | Cartela Ab | New monoclonal antibody |
-
2005
- 2005-01-27 EP EP05709422A patent/EP1715052A4/en not_active Ceased
- 2005-01-27 WO PCT/JP2005/001148 patent/WO2005073385A1/ja active Application Filing
- 2005-01-27 US US10/587,776 patent/US20080038824A1/en not_active Abandoned
- 2005-01-27 CA CA002554753A patent/CA2554753A1/en not_active Abandoned
- 2005-01-27 JP JP2005517495A patent/JPWO2005073385A1/ja active Pending
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1997011604A1 (en) * | 1995-09-29 | 1997-04-03 | Indiana University Foundation | Methods for enhanced virus-mediated dna transfer using molecules with virus- and cell-binding domains |
WO2000001836A1 (fr) * | 1998-07-01 | 2000-01-13 | Takara Shuzo Co., Ltd. | Procedes de transfert de genes |
WO2002018609A2 (en) * | 2000-08-31 | 2002-03-07 | Virxsys | Methods for stable transduction of cells with viral vectors |
WO2002083854A2 (en) * | 2001-04-13 | 2002-10-24 | Biogen, Inc. | Antibodies to vla-1 |
WO2003000297A1 (fr) * | 2001-06-20 | 2003-01-03 | Sumitomo Pharmaceuticals Company, Limited | Procede facilitant le transfert d'acides nucleiques |
Non-Patent Citations (1)
Title |
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See also references of EP1715052A4 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2008049146A (ja) * | 2006-07-24 | 2008-03-06 | National Institute Of Advanced Industrial & Technology | 組織再生用スキャッフォールド及びその製造方法 |
WO2015005431A1 (ja) | 2013-07-12 | 2015-01-15 | 石原産業株式会社 | 細胞への遺伝子導入用組成物 |
Also Published As
Publication number | Publication date |
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JPWO2005073385A1 (ja) | 2008-01-10 |
EP1715052A1 (en) | 2006-10-25 |
EP1715052A4 (en) | 2007-10-03 |
US20080038824A1 (en) | 2008-02-14 |
CA2554753A1 (en) | 2005-08-11 |
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