WO2006001396A1 - Procede et composition pour transferer de l'acide nucleique fixe a une phase solide dans une cellule - Google Patents

Procede et composition pour transferer de l'acide nucleique fixe a une phase solide dans une cellule Download PDF

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Publication number
WO2006001396A1
WO2006001396A1 PCT/JP2005/011671 JP2005011671W WO2006001396A1 WO 2006001396 A1 WO2006001396 A1 WO 2006001396A1 JP 2005011671 W JP2005011671 W JP 2005011671W WO 2006001396 A1 WO2006001396 A1 WO 2006001396A1
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Prior art keywords
cell
nucleic acid
cell adhesion
cells
collagen
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PCT/JP2005/011671
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English (en)
Japanese (ja)
Inventor
Masato Miyake
Eiichiro Uchimura
Tomohiro Yoshikawa
Jun Miyake
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National Institute Of Advanced Industrial Science And Technology
Cytopathfinder, Inc.
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Publication of WO2006001396A1 publication Critical patent/WO2006001396A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0041—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being polymeric

Definitions

  • the present invention relates to a technique for introducing a nucleic acid such as DNA and cell sorting induction. More specifically, the present invention relates to a technique for increasing the efficiency of introducing a nucleic acid into a cell and a technique for inducing differentiation of a cell after introducing the nucleic acid.
  • Transfection is a technique used to transiently express genes in cells such as animal cells and observe their effects.
  • This technique is frequently used to elucidate the function of the gene to be encoded.
  • Adherent mammalian cells recognize a specific sequence in an extracellular matrix protein via an integrin receptor on the cell surface, and a signal transduction pathway linked to the receptor in a cell line-dependent manner. It is known to respond via In the case of cell culture, these responses appear to cause other changes involved in adhesion as well as causing changes in cell morphology (eg, elongation of actin filaments and cell plain).
  • Non-patent Document 2 it has been reported that the extracellular matrix affects the cell migration involved in wound healing.
  • fibronectin and collagen IV cause the activity of the plasminogen activator family (eg, serine proteases and related molecules), while collagen types I and III suppress activity.
  • Non-patent Document 3 found that smooth muscle cells have their cell migration rate determined depending on the surface density of fibronectin and collagen IV.
  • Adhesive Atsey (Non-Patent Document 4), the force that was found to spread when using PC12 cell force S laminin and collagen type IV was not observed to spread with fibronectin.
  • Non-patent literature l Ciancotti FG, et al. Science 285, 1028-1032, 1999
  • Non-patent literature 2 Jones JM etal. Exp. Cell. Res., 280, 244-254, 2002
  • Non-patent literature 3 DiMilla et al J. Cell. Biol. 122, 729-737, 1993
  • Non-Patent Document 4 Tomaselli KJ., J. Cell. Biol. 105, 2347-2358, 1987 Disclosure of the Invention
  • the present invention introduces such a substance into a cell in which it is difficult to introduce a nucleic acid such as DNA when it is fixed on a support (particularly, transfection. )
  • a nucleic acid such as DNA
  • Another object of the present invention is to develop a technique that can further induce differentiation after introducing a nucleic acid into a cell as described above.
  • the above problem is an unexpected result that a nucleic acid introduction efficiency higher than expected was achieved by combining a cell adhesion molecule and a gene introduction reagent (for example, Lipofectamine) to a cell.
  • a gene introduction reagent for example, Lipofectamine
  • the above problem was solved by finding that differentiation can be induced by appropriate stimulation (for example, addition of a cell growth factor such as nerve growth factor (NGF)).
  • a cell growth factor such as nerve growth factor (NGF)
  • the present inventors have found that cell adhesion molecules (including extracellular matrix proteins (for example, fibronectin, collagen type I, collagen type IV, laminin, etc.) have improved nucleic acid introduction efficiency including transfection efficiency. It was found that the cells had an unexpected effect, and the cells into which the nucleic acid had been introduced retained the ability to be induced. These increases in nucleic acid transfer efficiency and retention of cell sorting ability were unexpectedly found on the solid phase. In particular, in a system using a local transfection cell array, the present inventors have found that it is effective in any cells including neurons and primary cultured cells.
  • extracellular matrix proteins for example, fibronectin, collagen type I, collagen type IV, laminin, etc.
  • the present inventors have unexpectedly discovered that this achieves a technique for efficiently introducing a nucleic acid based on a cell adhesion molecule (for example, collagen type IV) and retention of cell differentiation inducing ability. I saw it.
  • a cell adhesion molecule for example, collagen type IV
  • the ability to introduce nucleic acid into neurons and retain the ability to induce cell differentiation enables various analyzes of neurons, including Parkinson's disease, Creutzfeldt-Jakob disease, nerve regeneration, neural stem cell differentiation, and nerve development. Analysis such as has become possible.
  • the ease of genetic manipulation of primary cultured cells has made it possible to analyze the effects of genetic manipulation in a more living environment.
  • the present invention provides the following.
  • composition for introducing a nucleic acid fixed to a solid phase into a cell (1) A composition for introducing a nucleic acid fixed to a solid phase into a cell
  • a composition comprising:
  • composition according to item 1 wherein the cell adhesion molecule comprises an extracellular matrix.
  • composition according to item 1 wherein the cell adhesion molecule comprises at least one extracellular matrix selected from the group consisting of collagen, laminin and fibronectin, and a mixture thereof.
  • composition according to item 1 wherein the cell adhesion molecule comprises fiber-forming collagen or basement membrane collagen.
  • composition according to item 1 wherein the cell adhesion molecule comprises fiber-forming collagen and basement membrane collagen.
  • cell adhesion molecule comprises collagen type I or type IV
  • composition according to item 1, wherein the cell adhesion molecule comprises collagen type I and type IV
  • composition according to item 1, wherein the gene introduction reagent contains at least one reagent selected from the group consisting of a cationic polymer, a cationic lipid, a polyamine reagent, a polyimine reagent, and a calcium phosphate strength.
  • composition according to item 1, wherein the gene introduction reagent contains a cationic lipid.
  • composition according to item 10 The composition according to item 1, further comprising a nucleic acid to be introduced.
  • composition according to item 1, wherein the nucleic acid is circular or linear, single-stranded or double-stranded DNA.
  • composition according to item 11 wherein the nucleic acid comprises a gene-encoding sequence.
  • composition according to item 1 wherein the composition is a liquid.
  • composition according to item 1 wherein the cell adhesion molecule is in a multimeric form.
  • composition according to item 1 wherein the cell adhesion molecule has a three-dimensional structure.
  • composition according to item 1 wherein the cell is a cell in which an increase in gene transfer effect by fibronectin is not observed, and the cell adhesion factor comprises collagen or laminin.
  • composition according to item 1 wherein the cell is a neuronal cell, and the cell adhesion molecule comprises collagen.
  • composition according to item 1 wherein the introduction of the nucleic acid is performed in a state where the cells are arranged on a solid phase.
  • composition according to item 1 wherein the cell includes a primary cultured cell.
  • composition according to item 1 wherein the cell retains the ability to induce differentiation even after introduction of a nucleic acid.
  • (23) The composition according to item 22, wherein the fraction is induced by a cell growth factor.
  • a device including a support for introducing nucleic acid fixed to a solid phase into a cell
  • a device wherein the composition is secured to the support.
  • cell adhesion molecule comprises at least one extracellular matrix selected from the group consisting of collagen, laminin and fibronectin, and a mixture thereof.
  • the gene introduction reagent contains at least one reagent selected from the group consisting of a cationic polymer, a cationic lipid, a polyamine reagent, a polyimine reagent, and a calcium phosphate.
  • nucleic acid is circular or linear, single-stranded or double-stranded DNA.
  • a device according to item 34, wherein the nucleic acid comprises a gene-encoding sequence.
  • a device according to item 24, wherein the device is a cell of the nervous system, and the cell adhesion molecule contains collagen.
  • a device according to item 24, wherein the cells include primary cultured cells.
  • a device according to item 49, wherein the differentiation-inducing factor comprises a cell growth factor.
  • the cells retain the ability to induce differentiation after introduction of the nucleic acid.
  • cell adhesion molecule comprises at least one extracellular matrix selected from the group consisting of collagen, laminin and fibronectin, and a mixture thereof.
  • the gene introduction reagent comprises at least one reagent selected from the group consisting of a cationic polymer, a cationic lipid, a polyamine reagent, a polyimine reagent, and a calcium phosphate.
  • the cell is a cell of the nervous system, and the cell adhesion molecule contains collagen.
  • Item 54 The method according to Item 53.
  • nucleic acid comprises circular or linear, single-stranded or double-stranded DNA.
  • a composition comprising:
  • a kit comprising:
  • composition comprising a) a cell adhesion molecule; and b) a gene transfer reagent for introducing a nucleic acid anchored to a solid phase into a cell.
  • a composition comprising:
  • the above-mentioned derivation-inducing reagents are activin, insulin-like growth factor (IGF) -1, fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), transforming growth factor
  • composition of item 87 selected from the group consisting of (TGF) - ⁇ , nerve growth factor (NGF) and bone morphogenetic protein (BMP) 2/4.
  • a remarkable increase in efficiency was observed in introducing a nucleic acid fixed to a solid phase into a cell, and in particular, an increase in the efficiency of transfection was achieved.
  • Such a transfection efficiency increasing reagent is in a place where the desired diffusion is surely ensured because the nucleic acid to be introduced is introduced into the cell while being fixed to the solid phase, particularly while fixing the cell in the solid phase. It can be introduced into cells, and coconut is also useful for increasing the efficiency of transfection.
  • the present invention also provides a technique that enables differentiation induction after introduction of a nucleic acid. According to the present invention, it is possible to freely set a test in which an arbitrary nucleic acid is introduced and an arbitrary differentiation induction is performed. Since this technique is possible on a solid phase, it has an effect that a considerable condition necessary for the cell array can be arbitrarily set.
  • FIG. 1A Morphological changes of PC 12 cells observed using a phase microscope (FIGS. 1A to D) and a confocal microscope (FIGS. 1E to F) in Examples.
  • Figure 1A shows the effect of fibronectin
  • FIG. 1B Morphological changes of PC 12 cells observed using a phase microscope (FIGS. 1A to D) and a confocal microscope (FIGS. 1E to F) in Examples.
  • FIG. 1B shows the effect of type I collagen.
  • FIG. 1C Morphological changes of PC 12 cells observed using a phase microscope (FIGS. 1A to D) and a confocal microscope (FIGS. 1E to F) in Examples.
  • FIG. 1C shows type IV collagen.
  • FIG. 1D Morphological changes of PC 12 cells observed using a phase microscope (FIGS. 1A to D) and a confocal microscope (FIGS. 1E to F) in Examples.
  • Figure 1D shows the effect of laminin.
  • FIG. 1E Morphological changes of PC 12 cells observed using a phase microscope (FIGS. 1A to D) and a confocal microscope (FIGS. 1E to F) in Examples.
  • Figure 1E shows the effect of fibronectin
  • FIG. 1F1 Morphological changes of PC 12 cells observed using a phase microscope (FIGS. 1A to D) and a confocal microscope (FIGS. 1E to F) in Examples.
  • FIG. 1F1 shows type IV collagen.
  • FIG. 1F2 Morphological changes of PC 12 cells observed using a phase microscope (FIGS. 1A to D) and a confocal microscope (FIGS. 1E to F) in Examples.
  • Figure 1F2 shows the results of observation over time (1 hour, 2 hours, 3 hours, 6 hours) using type IV collagen and using PLL. It is shown that the nucleus gradually expands and the nucleic acid is easily introduced
  • FIG. 1G shows the cell adhesion form when 300,000-6000 cells are seeded in 3 mm ⁇ 3 mm on a transfer chip (slide glass, PLL coat).
  • FIG. 1G shows the cell adhesion morphology of PC12 cells.
  • FIG. 1H shows Neuro2a cells in a PLL-coated slide coated with collagen type IV (0.005 mgZml).
  • FIG. 2A Effect of exemplary type IV collagen shown in Examples (PC 12 cells).
  • Figure 2
  • A shows adhesion characteristics on various extracellular matrices in PC12 cells.
  • FIG. 2B Effect of exemplary type IV collagen shown in Examples (PC12 cells).
  • FIG. 2C Effect of exemplary type IV collagen shown in the examples (PC12 cells).
  • FIG. 3A Effect of presence or absence of PLL coating on PC12 cells in Examples.
  • Figure 3A shows the phase microscope for various coating conditions and combinations with extracellular matrix.
  • FIG. 3B Effect of presence or absence of PLL coating on PC12 cells in Examples.
  • Fig. 3B is a photograph showing the transformation efficiency of various ECMs.
  • FIG. 3C Effect of presence or absence of PLL coating on PC12 cells in Examples.
  • Figure 3C shows the isolation effect on plain and PLL coated slides, respectively.
  • FIG. 3D Effect of presence or absence of PLL coating on PC12 cells in Examples.
  • Figure 3D shows the results of testing various ECM and various coating combinations.
  • FIG. 3E Effect of presence or absence of PLL coating on PC 12 cells in Examples.
  • Figure 3E shows the numerical values of the strengths of the results of testing various ECM and various coating combinations. The graph is shown.
  • FIG. 3F Effect of presence or absence of PLL coating on PC12 cells in Examples.
  • FIG. 3F shows the actual cell fixation of FIG. 3D.
  • FIG. 3G Effect of presence or absence of PLL coating on PC12 cells in Examples (fixed time course).
  • Figure 3G shows the time course of the effects of various ECMs depending on the type of slide surface treatment.
  • FIG. 3H Effect of presence or absence of PLL coating on PC12 cells in the example (fixed time course).
  • Figure 3H shows the changes over time in the effects of various ECMs depending on the type of slide surface treatment.
  • FIG. 4 shows the behavior of calcium ion release observed using Fura2 technology.
  • FIG. 6A shows an application example of the present invention to array technology.
  • FIG. 6A shows PC12 cells.
  • FIG. 6B shows an example of application of the present invention to array technology.
  • FIG. 6B shows Neuro2a cells.
  • FIG. 6C shows an application example of the present invention to array technology.
  • FIG. 6C shows HepG2.
  • FIG. 6D shows an application example of the present invention to array technology.
  • FIG. 6D shows hMSC.
  • FIG. 6E shows an application example of the present invention to array technology.
  • FIG. 6E is a photograph showing the state of PC12 cells in FIG. 6A.
  • Experimental conditions Reagent: Lipofectamin2000, Glass substrate: PL
  • FIG. 7 shows an outline of a solid phase transformation.
  • FIG. 8 shows that the nucleic acid introduction efficiency does not depend on the type of gene introduction reagent. It shows that the effect of various ECM proteins does not depend on the type of gene transfer reagent.
  • the z-axis is transfection efficiency
  • the X-axis shows gene introduction reagents from various manufacturers
  • the y-axis shows various ECM proteins or conditions in the absence thereof. It became clear that the trend of transfection efficiency of PC12 cells did not change even when the manufacturer's reagent was used.
  • FIG. 9 shows the effect of siRNA when solid phase transfection (PC12) is performed on a collagen IV coating.
  • FIG. 9A shows PC12 cells co-transfected with EGFP vector and anti-EGFP siRNA. As shown, only HcRed develops color It was found that the green signal derived from pEGFP-Nl was suppressed.
  • FIG. 9B shows an example using scrambled siRNA. As shown, green fluorescence was observed, confirming that the effect in FIG. 9A was that of RNAi.
  • FIG. 9C shows the relative intensity of the fluorescence in FIGS. 9A and 9B. The y-axis is indicated by relative luminance. It can be seen that the effect of EGFP is almost completely suppressed.
  • FIG. 10 shows that NGF induces PC12 cells even on the array.
  • FIG. 10A shows solid phase transfection on fibronectin, collagen type I, collagen type IV, and laminin coated arrays.
  • Figure 10A shows a scanned image of a glass slide.
  • Figure 10B shows a scan-enhanced image of differentiation in a transfection array (left).
  • the middle of Fig. 10B shows a photomicrograph 3 days after transfection of PC12 cells.
  • the right side of Fig. 10B shows a photograph one day after NGF induction.
  • SEQ ID NO: 1 Nucleic acid sequence of mouse FVBZN collagen pro-a-1 type I chain (GENBAN K accession number: U08020).
  • SEQ ID NO: 2 Sequence of the protein encoded by the nucleic acid of SEQ ID NO: 1.
  • SEQ ID NO: 3 Mouse procollagen type I a 2 (Colla2) (GENBANK registration number: NM
  • SEQ ID NO: 4 Sequence of the protein encoded by the nucleic acid of SEQ ID NO: 3.
  • SEQ ID NO: 5 Nucleic acid sequence of mouse at type IV collagen (GENBANK accession number: Ml
  • SEQ ID NO: 6 Sequence of the protein encoded by the nucleic acid of SEQ ID NO: 5.
  • SEQ ID NO: 7 Nucleic acid sequence of mouse collagen a-2 (IV) chain (GENBANK accession number: X04647).
  • SEQ ID NO: 8 Sequence of the protein encoded by the nucleic acid of SEQ ID NO: 7.
  • SEQ ID NO: 9 Mouse procollagen type IV a 3 (Col4a3) (GENBANK registration number: N
  • SEQ ID NO: 10 Sequence of the protein encoded by the nucleic acid of SEQ ID NO: 9.
  • SEQ ID NO: 11 Mouse collagen type IV a 4 chain nucleic acid sequence (GENBANK accession number: Z
  • SEQ ID NO: 12 Sequence of the protein encoded by the nucleic acid of SEQ ID NO: 11.
  • SEQ ID NO: 13 Nucleic acid sequence of mouse collagen type IV ⁇ 5 chain (GENBANK accession number: ⁇
  • SEQ ID NO: 14 Sequence of the protein encoded by the nucleic acid of SEQ ID NO: 13.
  • SEQ ID NO: 15 Nucleic acid sequence of mouse procollagen type IV a 6 (Col4a6) (GENBANK accession number: NM-053185).
  • SEQ ID NO: 16 Sequence of the protein encoded by the nucleic acid of SEQ ID NO: 15.
  • SEQ ID NO: 17 Laminin nucleic acid sequence (mouse ex strand)
  • SEQ ID NO: 18 amino acid sequence of laminin (mouse ⁇ chain)
  • SEQ ID NO: 19 Laminin nucleic acid sequence (mouse ⁇ chain)
  • SEQ ID NO: 20 amino acid sequence of laminin (mouse ⁇ chain)
  • SEQ ID NO: 21 Nucleic acid sequence of laminin (mouse ⁇ chain)
  • SEQ ID NO: 22 amino acid sequence of laminin (mouse ⁇ chain)
  • SEQ ID NO: 23 Nucleotide sequence of fibronectin (human)
  • SEQ ID NO: 24 Amino acid sequence of fibronectin (human)
  • SEQ ID NO: 25 siRNA sequence used in Example 9
  • SEQ ID NO: 26 Scrambled RNA sequence used in Example 9
  • the terms “protein”, “polypeptide”, “oligopeptide” and “peptide” are used interchangeably herein and refer to a polymer of amino acids of any length.
  • the polymer may be linear or branched or cyclic.
  • the amino acid may be a modified amino acid, which may be natural or non-natural.
  • the term may also include those assembled into a complex of multiple polypeptide chains.
  • the term also encompasses natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, daricosylation, 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 amino acids (eg, including non-natural amino acids, etc.), peptidomimetic compounds (eg, peptoids), and the art! Other modifications are included.
  • Gene products of extracellular matrix proteins such as fibronectin usually take the polypeptide form.
  • polynucleotide As used herein, the terms “polynucleotide”, “oligonucleotide” and “nucleic acid” are used interchangeably herein and refer to a nucleotide polymer of any length. The term also includes “derivative oligonucleotide” or “derivative polynucleotide”. “Derivative oligonucleotide” or “derivative polynucleotide” refers to an oligonucleotide or polynucleotide that includes a derivative of a nucleotide or that has unusual linkages between nucleotides, and is used interchangeably.
  • oligonucleotides include, for example, 2 ′ O-methyl-ribonucleotides, derivative oligonucleotides in which a phosphodiester bond in an oligonucleotide is converted to a phosphoroate bond, and phosphorous in an oligonucleotide.
  • Derivative oligonucleotides in which the acid diester bond is converted to N3, 1 P5, phosphoramidate bond, ribose in the oligonucleotide Derivative oligonucleotide in which phosphodiester bond is converted to peptide nucleic acid bond, Derivative oligonucleotide in which uracil in oligonucleotide is replaced with C5 propyruuracil, uracil in oligonucleotide is C-5 thiazoleuracil
  • nucleic acid sequence may also contain conservatively modified variants (e.g., degenerate codon substitutes) as well as explicitly indicated sequences. ) And complementary sequences are contemplated.
  • a degenerate codon substitute creates a sequence in which the third position of one or more selected (or all) codons is replaced with a mixed base and a Z or deoxyinosin residue.
  • nucleic acid molecule is also used herein interchangeably with nucleic acids, oligonucleotides, and polynucleotides and includes cDNA, mRNA, genomic DNA, and the like.
  • nucleic acids and nucleic acid molecules can be included in the concept of the term “gene”.
  • Nucleic acid molecules encoding certain gene sequences also include “splice variants”.
  • a particular protein encoded by a nucleic acid includes any protein encoded by a splice variant of that nucleic acid.
  • splice variants are the product of alternative splicing of genes.
  • the initial nucleic acid transcript can be spliced such that different (alternate) nucleic acid splice products encode different polypeptides.
  • the production mechanism of splice variants involves alternative splicing of changing 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 splice products) is included in this definition.
  • useful extracellular matrix proteins such as collagen, fibronectin, laminin, and the like, can also include the splice variants.
  • gene refers to a factor that defines a genetic trait. Usually arranged in a certain order on a chromosome. Those that define the primary structure of a protein are called structural genes, and those that affect their expression are called regulatory genes (for example, promoters). In the present specification, genes include structural genes and regulatory genes unless otherwise specified. Recent genome sequencing has attracted attention for parts that are neither structural genes nor regulatory genes, suggesting that they play a role in life. Therefore, it is understood that a gene includes a nucleic acid present in a living organism and factors that determine the genetic form defined by the nucleic acid. Therefore, a collagen gene usually includes both a structural gene of collagen and a promoter of collagen.
  • gene may refer to “polynucleotide”, “oligonucleotide” and “nucleic acid” as well as Z or “protein” “polypeptide”, “oligopeptide” and “peptide”.
  • gene product refers to “polynucleotide”, “oligonucleotide” and “nucleic acid” and Z or “protein” “polypeptide”, “oligopeptide” expressed by a gene. And “peptide”. A person skilled in the art can understand what a gene product is, depending on the situation.
  • sequences refers to the degree of identity of two or more gene sequences with each other. Therefore, the higher the homology between two genes, the higher the sequence identity or similarity. Whether two genes have homology can be determined by direct sequence comparison or, in the case of nucleic acids, hybridization methods under stringent conditions. When directly comparing two gene sequences, the DNA sequence between the gene sequences is typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90% If they are 95%, 96%, 97%, 98% or 99% identical, the genes have homology. As used herein, sequences (eg, nucleic acid sequences, amino acid sequences, etc.
  • Similarity refers to the degree of identity of two or more gene sequences to each other when conservative substitutions are considered positive (identical) in the above homology. Thus, if there is a conservative substitution, identity and similarity differ depending on the presence of the conservative substitution. When there is no conservative substitution, identity and similarity indicate the same numerical value.
  • amino acid may be natural or non-natural as long as the object of the present invention is satisfied.
  • amino acid derivative or “amino acid analog” refers to an amino acid that is different from a naturally occurring amino acid but has the same function as the original amino acid. Such amino acid derivatives and amino acid analogs are well known in the art.
  • natural amino acid means the L isomer of a natural amino acid. Natural amino acids are glycine, alanine, norin, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, ⁇ -carboxyglutamic acid Arginine, orthine, and lysine. Unless otherwise indicated, all amino acids in this specification are L-forms. Forms using D-form amino acids are also within the scope of the present invention.
  • unnatural amino acid means an amino acid that is not normally found in proteins.
  • unnatural amino acids include norleucine, para-nitrophenylalanine, homophenylalanine, para-fluorophenylalanine, 3-amino-2-benzylpropionic acid, homoarginine D or L and D-phenolanine .
  • amino acid analog refers to a molecule that is not an amino acid, but is similar to the physical properties and function or function of an amino acid.
  • amino acid analogs include ethionine, canavanine, 2-methylglutamine and the like.
  • Amino acid mimetics refers to chemical 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.
  • the “nucleotide” includes a nucleotide derivative or a nucleotide variant as long as the desired function can be exhibited, whether natural or non-natural.
  • Nucleotide derivatives or “nucleotide analogs” refer to those that are different from naturally occurring nucleotides but have the same function as the original nucleotide. Such nucleotide derivatives and nucleotide analogs are well known in the art! Examples of such nucleotide derivatives and nucleotide analogs include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, chiral methylphosphonates, 2-O-methylribonucleotides, peptide nucleic acids (PNA) .
  • PNA peptide nucleic acids
  • Amino acids may be referred to herein by either their commonly known three letter symbol power or by the one letter symbol recommended by the IUPAC — IUB Biochemica 1 Nomenclature Commission. Nucleotides can also be referred to by their generally recognized single letter codes.
  • Thymine Z uracil or cytosine pyrimidine
  • a "corresponding" amino acid or nucleic acid refers to a predetermined amino acid or nucleic acid in a polypeptide molecule or polynucleotide serving as a reference for comparison with a certain polypeptide molecule or polynucleotide molecule.
  • an antisense molecule can be a similar part in an ortholog corresponding to a particular part of the antisense molecule.
  • corresponding amino acids or nucleic acids can be identified using alignment techniques known in the art. Examples of such alignment techniques include, but are not limited to, techniques described in Needleman, SB and Wunsch, CD, J. Mol. Biol. 48, 443-453, 1970.
  • a "corresponding" gene refers to a given gene (eg, a polynucleotide or polypeptide) in a species that serves as a basis for comparison.
  • the corresponding gene of a gene can be an ortholog of that gene. Therefore, the gene corresponding to the mouse collagen gene can also be found in other animals (human, rat, pig, ushi, etc.).
  • Such corresponding genes can be identified using techniques well known in the art.
  • a corresponding gene in an animal is obtained by using the sequence (for example, SEQ ID NO: 1 to 24) of a gene (for example, mouse collagen) as a reference sequence of the corresponding gene as the query sequence. It can be found by searching a sequence database (eg, human, rat).
  • a sequence database eg, human, rat
  • fragment refers to a polypeptide or polynucleotide having a sequence length of 1 to n ⁇ 1 with respect to a full-length polypeptide or polynucleotide (length n).
  • length n the length of the fragment can be appropriately changed according to the purpose.
  • the lower limit of the length is 3, 4, 5, 6, 7, 8, 9, 10 in the case of a polypeptide. , 15, 2 Examples include 0, 25, 30, 40, 50 and more amino acids, and lengths expressed in integers not specifically listed here (eg, 11 etc.) may also be appropriate as lower limits.
  • examples include 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides.
  • the length of the polypeptide and the polynucleotide can be represented by the number of amino acids or nucleic acids, respectively, as described above. The above number as the upper limit or subtraction is intended to include those above and below that number (or 10% above and below for example). In order to express such intention, in this specification, “about” may be added before the number. However, it should be understood herein that the presence or absence of “about” does not affect the interpretation of the value.
  • the fragment preferably has a certain size (for example, 5 kDa) or more. Without being bound by theory, it seems that a certain size is necessary to function as a cell adhesion molecule.
  • region is meant a physically contiguous portion of the primary structure of a biomolecule.
  • a region is defined by a contiguous portion of the amino acid sequence of the protein.
  • domain is defined herein to refer to the structural portion of a biomolecule that contributes to a known or suspected function of the biomolecule.
  • a domain can have the same extent as a region or part thereof; a domain can also incorporate all or part of the region, as well as part of a biomolecule that is distinct from a particular region.
  • domains of cell adhesion molecules of the present invention include, but are not limited to, signal peptides, extracellular (ie, N-terminal) domains, leucine-rich repeat domains, RGD portions, and other conserved regions.
  • polynucleotide that hybridizes under stringent conditions refers to well-known conditions commonly used in the art.
  • the polynucleotide used in the present invention (for example, the one encoding collagen I) is selected as a probe using the colony hybridization method, plaque hybridization method, plaque hybridization method or By using the Southern blot hybridization method, etc. Renucleotides can be obtained. Specifically, using a filter immobilized with colony or plaque-derived DNA, hybridization was performed at 65 ° C in the presence of 0.7 to 1.OM of NaCl, and then 0.1 to 2 times.
  • the filter should be washed at 65 ° C using a concentration of SSC (saline-sodium citrate) solution (1x concentration of SSC solution is 150 mM sodium chloride and 15 mM sodium taenoate).
  • concentration of SSC solution is 150 mM sodium chloride and 15 mM sodium taenoate.
  • SSC saline-sodium citrate
  • Means a polynucleotide identifiable by Hybridization is an experimental document such as Molecular Cloning 2nd ed., Current Protocols m Molecular Biology, Supplements 1 to 38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). Can be carried out according to the method described in.
  • sequences that contain only the A sequence or only the T sequence are excluded from sequences that are hybridized under stringent conditions.
  • hybridizable polynucleotide refers to a polynucleotide that can be hybridized to another polynucleotide under the above hybridization conditions.
  • the polynucleotide capable of hybridizing is preferably a polynucleotide having at least 60% homology with the DNA base sequence encoding the polypeptide having the amino acid sequence specifically shown in the present invention.
  • “highly stringent conditions” allow for hybridization of DNA strands that have a high degree of complementarity in nucleic acid sequences, and DNA hybridization that has significant mismatches. Exclude conditions designed to exclude! The stringency of a hybridization is primarily determined by temperature, ionic strength, and the condition of the denaturing agent such as formamide. Examples of “highly stringent conditions” for such hybridization and washing are 0.0015M sodium chloride, 0.0015M sodium quenate, 65-68. C, or 0.005M sodium chloride, 0.0015M sodium citrate, and 50% formamide, 42 ° C.
  • hybridization buffer and wash buffer examples include
  • ushi serum albumin 0.1% polybulurpyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecyl sulfate (NaDodSO or SDS), Ficoll, Denhardt solution,
  • Sonicated salmon sperm DNA or another non-complementary DNA
  • dextran sulfate but other suitable agents can also be used.
  • concentration and type of these additives can be varied without substantially affecting the stringency of the hybridization conditions.
  • Hybridization experiments are usually carried out at pH 6.8 to 7.4; however, under typical ionic strength conditions, the speed of hybridization is almost exclusively at pH. See Anderson et al., Nucleic Acid Hybridization: a Practical Ap proach, Chapter 4, IRL Press Limited (Oxford, England).
  • Factors affecting the stability of DNA duplex include base composition, length, and degree of base pair mismatch. Hybridization conditions can be adjusted by those skilled in the art to apply these variables and allow different sequence related DNAs to form hybrids.
  • the melting temperature of a perfectly matched DNA duplex can be estimated by the following equation:
  • N is the length of the double chain formed
  • [Na +] is the molar concentration of sodium ions in the noble, hybridization solution or wash solution
  • % G + C is The percentage of (guanine + cytosine) bases in the.
  • the melting temperature decreases by about 1 ° C for each 1% mismatch.
  • moderately stringent conditions refers to the formation of DNA duplexes having a higher degree of base pair mismatch than can occur under "highly stringent conditions".
  • the conditions that can Typical examples of ⁇ moderately stringent conditions '' are 0.005M salt sodium silicate, 0.0015M sodium quenate, 50-65 ° C, or 0.005M sodium chloride, 0.005M salt sodium. 0015M sodium citrate, and 20% formamide, 37-50. C.
  • a “moderately stringent” condition of 50 ° C in 0.015M sodium ion will allow about 21% discrepancy.
  • Tm (2 ° C per A—T base) + (4 ° C per G—C base pair)
  • the sodium ion concentration in 6 X citrate sodium salt (SSC) is 1M (Suggs et al., Developmental Biology
  • the natural nucleic acid encoding the protein used in the present invention is represented by, for example, SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, etc. It is easily separated from a single cDNA library with PCR primers and hybridization probes containing part of the nucleic acid sequence.
  • Preferred nucleic acids of the invention are essentially 1% sushi serum albumin (BSA); 500 mM sodium phosphate (NaPO); ImM EDTA; at a temperature of 42 ° C.
  • Hybridization buffer containing% SDS and essentially 2 X SSC 600 mM NaCl; 60 mM sodium citrate); low as defined by wash buffer containing 0.1% SDS at 50 ° C
  • LX SSC of C 300 mM NaCl; 30 mM sodium citrate); 1% sushi serum under low stringent conditions defined by wash buffer containing 1% SDS, most preferably essentially at a temperature of 50 ° C Hybridization relaxation containing albumin (BSA); 200 mM sodium phosphate (Na PO); 15% formamide; ImM EDTA; 7% SDS
  • Immunity and essentially 65.
  • 0.5 X SSC 150 mM NaCl; 15 mM sodium taenate) in C; low stringency conditions defined by wash buffer containing 0.1% SDS. 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, etc. [Can be hybridized with one or a part of the self-indicating U shown.
  • search refers to other nuclei having a specific function and Z or property using a certain nucleobase sequence electronically or biologically or by other methods. This refers to finding an acid-base sequence.
  • Electronic searches include 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.
  • Biological searches include stringent hybridization, macroarrays with genomic DNA affixed to nylon membranes, or microarrays (microarray assay) affixed to glass plates, PCR and in situ noise. Examples include, but are not limited to redization. In this specification, it is intended that Fnl should include the corresponding genes identified by such electronic and biological searches.
  • introduction of a substance into a cell means that the substance enters into the cell membrane. Whether the substance has been introduced or not is caused by, for example, the force of detecting the label by labeling the substance itself (for example, using a fluorescent label, chemiluminescent label, phosphorescence, radioactivity, etc.) or the substance. Intracellular changes (eg gene expression, signal transduction, events due to binding to intracellular receptors, metabolic changes, etc.) physical (eg visual), chemical (measurement of secretions), biochemical, It can be determined by biological measurement. Thus, such “introduction” includes simple proteins, nucleic acids. In addition to the transfer of such substances into cells, operations such as transformation, transformation, and transduction, which are also commonly called genetic manipulations, are also included.
  • foreign substance refers to a substance that is intended to be introduced into cells.
  • the foreign substance contemplated by the present invention refers to a substance that is not introduced into cells under normal conditions.
  • substances that can be introduced into cells under normal conditions by diffusion or hydrophobic interactions are not subject to important aspects of the invention.
  • Target substances that are not introduced into cells under normal conditions include, for example, proteins (polypeptides), RNA, DNA, sugars (especially polysaccharides), and complex molecules (for example, glycoproteins, PNAs, etc.) These include, but are not limited to, complex molecules (eg, glycolipids) with other molecules, viral vectors, and other compounds.
  • interaction means that when two objects are referred to, the two objects exert a force on each other.
  • interactions include, but are not limited to, covalent bonds, hydrogen bonds, van der Waals forces, ionic interactions, nonionic interactions, hydrophobic interactions, electrostatic interactions, etc.
  • the interaction may be a normal interaction that occurs in vivo, such as a hydrogen bond, a hydrophobic interaction.
  • contact means that two substances (eg, a composition and a cell) are present at a sufficiently close distance to interact with each other.
  • the cell adhesion molecules used in the present invention often take the form of gene products.
  • the present invention can also use a substance produced by the following gene modification technique.
  • amino acids included in the sequence may be present in other protein structures, such as the cationic region or the binding site of the substrate molecule, without an apparent reduction or loss of interaction binding capacity.
  • amino acids It is the protein's ability to interact and the nature that defines the biological function of a protein.
  • substitutions of specific amino acids may occur in the amino acid sequence or in the DNA coding sequence. This can be done in the bell and can result in proteins that still retain their original properties after substitution. Accordingly, various modifications can be made in the peptide disclosed herein or in the corresponding DNA encoding this peptide without any apparent loss of biological utility.
  • hydrophobicity index of amino acids can be taken into account when designing such modifications as described above.
  • the importance of the hydrophobic amino acid index in conferring interactive biological functions in 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 protein produced, and then 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 their hydrophobicity and charge properties.
  • a certain amino acid can be replaced by another amino acid having a similar hydrophobicity index and still result in a protein having a similar biological function (eg, a protein 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. As described in US Pat. No. 4,554,101, the following hydrophilicity indices are assigned to amino acid residues!
  • an amino acid can be substituted with another that has a similar hydrophilicity index and can still provide a biological equivalent.
  • the hydrophilicity index is preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • conservative substitution means that the amino acid substitution is similar to the hydrophilicity index or Z and hydrophobicity index of the amino acid to be replaced with the original amino acid as described above. This refers to substitution. Examples of conservative substitutions include those having a hydrophilicity index or hydrophobicity index of 2 or less, preferably ⁇ 1 or less, more preferably ⁇ 0.5 or less. But not limited to them.
  • conservative substitutions are well known to those skilled in the art, for example, substitutions within the following groups: arginine and lysine; dartamic acid and aspartic acid; serine and threonine; glutamine and asparagine; Examples include, but are not limited to, leucine and isoleucine.
  • variant refers to a substance in which a part of the original substance such as a polypeptide or a polynucleotide has been changed. Such variants include substitutional variants, addition variants, deletion variants, truncated variants, allelic variants, and the like. Alleles are genetic variants that belong to the same locus and are distinguished from each other. Therefore, an “allelic variant” refers to a variant that has an allelic relationship with a 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 have different biological activities. May be included.
  • a “species homologue or homolog” is a homology (preferably 60% or more homology, more preferably 80% or more, 85% or more, 90% or more, 95% or more homology). The method for obtaining such species homologues will be apparent from the description herein.
  • “Ortholog” refers to a gene derived from speciation from a common ancestor with two genes, both orthologous gene and orthologous gene. For example, multigene Taking the structured hemoglobin gene family as an example, the human and mouse ⁇ -hemoglobin genes are orthologs, while the human a-hemoglobin gene and j8-hemoglobin gene are paralogs (genes generated by gene duplication). Orthologs are useful for estimating molecular phylogenetic trees. Since orthologs can usually perform the same function as the original species in another species, the orthologs of the present invention can also be useful in the present invention.
  • “Conservative (modified) variants” applies to both amino acid and nucleic acid sequences.
  • Conservatively modified variants with respect to a particular nucleic acid sequence refer to nucleic acids that encode the same or essentially the same amino acid sequence, and are essential if the nucleic acid does not encode an amino acid sequence. Refers to the same sequence.
  • the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
  • nucleic acid is a “silent modification (mutation),” which is one species of conservatively modified mutations.
  • Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of that nucleic acid.
  • each codon in a nucleic acid except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan, produces a functionally identical molecule. It is understood that it can be modified.
  • each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.
  • such modifications can be made to avoid substitution of cysteine, an amino acid that significantly affects the conformation of the polypeptide.
  • Such base sequence alteration methods include restriction enzyme digestion, DNA polymerase, Klenow fragment, DNA ligase treatment, and other ligation treatments, and site-specific base substitution using synthetic oligonucleotides ( Mark Zoller and Michael Smith, Methods in Enzymology, 100, 468-500 (1983)), but other modifications can be made by methods usually used in the field of molecular biology. It can also be done.
  • amino acid substitutions amino acid additions, deletions, or modifications can also be made in order to produce functionally equivalent polypeptides.
  • Amino acid substitution refers to substitution of the original peptide with one or more, for example, 1 to: LO, preferably 1 to 5, more preferably 1 to 3 amino acids.
  • Addition of amino acid means adding one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids to the original peptide chain.
  • Deletion of amino acids refers to deletion of one or more, for example, 1 to 10, preferably 1 to 5, more preferably 1 to 3 amino acids from the original peptide.
  • Amino acid modifications include, but are not limited to, amidation, carboxylation, sulfation, halogenation, alkylation, glycosylation, phosphorylation, hydroxylation, acylation (eg, acetylation) and the like.
  • the amino acid to be substituted or added may be a natural amino acid or a non-natural amino acid, or an amino acid analog. Natural amino acids are preferred.
  • peptide analog or "peptide derivative” is a compound that is a compound that is different from a peptide and is equivalent to 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 respect to the original peptide.
  • Peptide analogs have the same peptide function (for example, similar pKa values, similar functional groups, similar binding modes with other molecules, Such additions or substitutions are made so as to be substantially similar to (eg, similar water solubility).
  • Such peptide analogs can be prepared using techniques well known in the field.
  • a peptide analog can be a polymer containing an amino acid analog.
  • polynucleotide analog or “nucleic acid analog” is a compound that is different from a polynucleotide or nucleic acid and has at least one chemical or biological function. What is equivalent.
  • polynucleotide analogs or nucleic acid analogs include those in which one or more nucleotide analogs or nucleotide derivatives are added or substituted to the original peptide.
  • a nucleic acid molecule as used herein is a portion of the sequence of the nucleic acid as described above as long as the expressed polypeptide has substantially the same activity as the native polypeptide. May be deleted or replaced by other bases, or other nucleic acid sequences may be partially inserted. Alternatively, another nucleic acid may be bound to the 5 ′ end and the Z or 3 ′ end. Alternatively, it may be a nucleic acid molecule that encodes a polypeptide having substantially the same function as that of a polypeptide obtained by subjecting a gene encoding the polypeptide to a noble condition under stringent conditions. Such a gene is 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, or can be chemically synthesized.
  • a site-specific displacement induction method, a hybridization method, or the like may be combined with these methods.
  • substitution, addition or deletion of a polypeptide or polynucleotide refers to an amino acid or its substitute, or nucleotide, respectively, relative to the original polypeptide or polynucleotide. Or its substitute power is replaced, added or removed.
  • substitution, addition, or deletion techniques are well known in the art, and examples of such techniques include site-directed mutagenesis techniques. Any number of substitutions, additions or deletions may be used as long as it is one or more. Such numbers may be used in the variant having the substitutions, additions or deletions (eg, hormones, As long as the information transmission function is maintained). For example, such a number can be 1 or several and preferably can be within 20%, within 10%, or less than 100, less than 50, less than 25, etc. of the total length.
  • an "isolated" biological agent refers to other biological cells within the cells of the organism in which the biological agent is naturally present.
  • Factor e.g., if it is a nucleic acid, a nucleic acid containing a non-nucleic acid factor and a nucleic acid sequence other than the target nucleic acid; if it is a protein, it contains an amino acid sequence other than the non-protein factor and the target protein
  • a substance substantially separated or purified from a protein or the like A substance substantially separated or purified from a protein or the like.
  • isolated nucleic acids and proteins include nucleic acids and proteins purified by standard purification methods. Thus, isolated nucleic acids and proteins include chemically synthesized nucleic acids and proteins.
  • a "purified" biological agent eg, a nucleic acid or protein I.
  • a purified biological agent Means that at least part of the factors naturally associated with the biological factor has been removed.
  • the purity of a biological agent in a purified biological agent is usually higher (ie, enriched) than the state in which the biological agent is normally present.
  • purified and isolated as used herein are preferably at least 75% by weight, more preferably at least 85% by weight, and even more preferably at least 95% by weight. % And most preferably at least 98% by weight of the same type of biological agent is present.
  • vector refers to a vector capable of transferring a target polynucleotide sequence to a target cell.
  • vectors can be autonomously replicated in host cells such as prokaryotic cells, yeast, animal cells, plant cells, insect cells, animal individuals and plant individuals, or can be integrated into chromosomes. Examples include those containing a promoter at a position suitable for polynucleotide transcription.
  • a vector suitable for cloning is called a “cloning vector”.
  • Such cloning vectors usually contain multiple cloning sites that contain multiple restriction enzyme sites. Such restriction enzyme sites and multiple cloning sites are well known in the art, and those skilled in the art can select and use them appropriately according to the purpose. Such techniques are described in the literature described herein (eg, Sambrook et al., Supra).
  • expression vector refers to a nucleic acid sequence in which various regulatory elements are linked in a state in which they can operate in a host cell in addition to a structural gene and a promoter that regulates its expression.
  • the regulatory element may preferably include a terminator, a selectable marker such as a drug resistance gene, and an enhancer. 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 elements used can vary depending on the host cell.
  • Recombinant vectors for prokaryotic cells include pcDNA3 (+), pBluescript—SK (pcDNA3 (+), pBluescript—SK (pcDNA3 (+), pBluescript—SK (pcDNA3 (+), pBluescript—SK (pcDNA3 (+), pBluescript—SK (pcDNA3 (+), pBluescript—SK (pcDNA3 (+), pBluescript—SK (pcDNA3 (+), pBluescript—SK (
  • Recombinant vectors for animal cells include pcDNAlZAmp, pcDNAI, pCDM8 (all commercially available from Funakoshi), pAGE107 [JP-A-3-229 (Invitrogen), pAGEl 03 J. Biochem., 101, 1307 (1987) ], PAMo, ⁇ . Biol. Chem., 26 8, 22782—22787 (1993)], Murine Stem Cell Virus (MSCV) -based retroviral expression vectors, pEF—BOS, pEGFP, etc. Is exemplified.
  • MSCV Murine Stem Cell Virus
  • Recombinant vectors for plant cells include, but are not limited to, pPCVICEn4HPT, pCGN1548, pCGN1549, pBI221, pBI121 and the like.
  • the terminator 1 is usually located downstream of a region encoding a protein of a gene, and is the termination of transcription when DNA is transcribed into mRNA. This refers to the sequence involved in the addition. Terminators are known to affect gene expression by affecting mRNA stability.
  • promoter refers to a region on DNA that determines the transcription start site of a gene and directly regulates its frequency, and is usually a base that initiates transcription upon binding of RNA polymerase. Is an array. Therefore, in this specification, a portion having a promoter function of a gene is referred to as a “promoter portion”. Since the promoter region is usually 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 The region can be estimated.
  • the putative promoter region varies from structural gene to structural gene, but is usually upstream of the structural gene, but is not limited thereto, and may be downstream of the structural gene. Preferably, the putative promoter region is also present within about 2 kbp upstream of the first exon translation initiation force.
  • the term "announcer” refers to a sequence used to increase the expression efficiency of a target gene.
  • Such Jenno, sensors are well known in the art. You can use multiple Hansa, but you can use one! /, Or not! /.
  • silencer refers to a sequence having a function of suppressing and resting gene expression.
  • any silencer can be used as long as it has the function. It is not necessary to use a silencer.
  • operably linked means a transcriptional translational regulatory sequence (eg, promoter, enhancer, silencer, etc.) or translation that has the expression (operation) of a desired sequence. It is arranged under the control of a regulatory sequence.
  • a promoter In order for a promoter to be operably linked to a gene, the force with which the promoter is usually placed immediately upstream of the gene does not necessarily have to be placed adjacent.
  • any technique may be used for introducing a nucleic acid molecule into a cell. Examples thereof include transformation, transduction, and transformation. Such technology for introducing a nucleic acid molecule is well known and commonly used in the art, for example, Ausubel FA et al. (1988), Current Protocols in Molecular Biology, Wiley, New York; NY; Sambrook J et al. (1987) Molecular Cloning: A Laboratory Manual, 2nd Ed. And its third edition, Cold Spring
  • any of the methods described above for introducing DNA into a cell can be used.
  • transfection, transduction, transformation, etc. for example, Calcium phosphate method, ribosome method, DEAE dextran method, electro bolition method, method using particle gun (gene gun), lipofussion method, suwe mouth plast method [Proc. Natl. Acad. Sci. USA, 84, 1929 ( 1978)], lithium acetate method Q [. Bacteriol., 153, 163 (1983)], Proc. Natl. Acad. Sci. USA, 75, 1 929 (1978).
  • the term “foreign substance introduction reagent” refers to a substance used for promoting the introduction efficiency of a foreign substance.
  • reagents include, but are not limited to, cationic polymers, cationic lipids, polyamine reagents, polyimine reagents, calcium phosphate, and the like.
  • Such a reagent usually includes a “gene introduction reagent” because it increases the efficiency of nucleic acid introduction.
  • the term “gene introduction reagent” refers to a test used to promote the introduction efficiency of a nucleic acid (usually, but not limited to, a gene). Say medicine.
  • gene introduction reagents include, but are not limited to, cationic polymers, cationic lipids, polyamine reagents, polyimine reagents, calcium phosphate, and the like.
  • reagents used in the transfusion include those commercially available from various sources, such as Effectene Transfecti on Reagent (cat. No. 301425, Qiagen, C /, TransFast Transfection Reagent (E2431, Promega, Wl), Tfx TM — 20 Reagent (E2391, Promega, WI), SuperFect Transfection Reagent (301305, Qiagen, CA), PolyFect
  • Transfection Reagent (301105, Qiagen, CA), LipofectAMINE 2000 Reagent (11668— 019, Invitrogen corporation, CA), JetPEI (X 4) cone. (101-30, Polyplus-transfection, France) and ExGen 500 (R0511, Fer mentas Inc., MD) and the like.
  • cationic lipids as gene transfer reagents (particularly TransFast TM Transfection Reagent, LipofectAMINE 2000 Reagent, etc.).
  • Foreign substance introduction efficiency or gene introduction efficiency is determined by introducing (expression) the number of introduced foreign substances (transgene) (eg, fluorescent protein GFP) per unit area (eg, 1 mm 2 ), or It can be calculated by measuring the total signal (fluorescence for fluorescent proteins).
  • transgene eg, fluorescent protein GFP
  • transformant refers to all or part of a living organism such as a cell produced by transformation (tissue or the like).
  • transformants include all or part of living organisms such as cells of prokaryotes, yeasts, animals, plants, insects (tissues, etc.).
  • a transformant is also referred to as a transformed cell, transformed tissue, transformed host, etc., depending on the subject.
  • the cell used in the present invention may be a transformant.
  • the prokaryotic mitochondrion includes Escherichia, Serratia, Bacillus, Brevibacterium, Corynebacterium, ⁇ , Microbacterium ⁇ .
  • prokaryotic cells belonging to Pseudomonas® such as Escherichia coli XL1-Blue® Escherichia coli XL2-Blue, Escherichia coli DH1.
  • prokaryotic cells belonging to Pseudomonas® such as Escherichia coli XL1-Blue® Escherichia coli XL2-Blue, Escherichia coli DH1.
  • cells separated from natural products can also be used.
  • Animal cells that can be used in genetic engineering and the like in this specification include mouse.myeloma cells, rat 'myeloma cells, mouse' cells, hybridoma cells, Chinese hamster cells, CHO cells, BHK cells, Examples include African green monkey kidney cells, human leukemia cells, HBT5637 (Japanese Patent Laid-Open No. 63-299), and human colon cancer cell lines.
  • Mouse 'myeloma cells such as ps20, NSO, rat' myeloma cells such as YB2Z0, human fetal kidney cells such as HEK293 (ATCC: CRL-1573), human leukemia cells such as BALL-1 and African green monkey kidney
  • Examples of cells include COS-1 and COS-7
  • examples of human colon cancer cell lines include HCT-15, human neuroblastoma SK-N-SH, SK-N-SH-5Y, and mouse neuroblastoma Neuro2A. Is done.
  • primary cultured cells can also be used in the present invention.
  • plant cells that can be used for genetic manipulation and the like include callus or a part thereof and suspension cultured cells, solanaceae, gramineae, cruciferous, rosaceae, legumes. Examples include, but are not limited to, cells of plants such as family, cucurbitaceae, perilla family, lily family, akaza family, and cedar family.
  • detection or “quantification” of gene expression (eg, mRNA expression, polypeptide expression) refers to appropriate methods including, for example, mRNA measurement and immunological measurement methods. Can be achieved. Examples of molecular biological measurement methods include Northern blot method, dot plot method, PCR method and the like. Examples of the immunological measurement method include an ELISA method using a microtiter plate, an RIA method, a fluorescent antibody method, a Western plot method, and an immunohistochemical staining method. Examples of the quantification method include ELISA and RIA. It can also be performed by a gene analysis method using an array (for example, DNA array, protein array).
  • an array for example, DNA array, protein array.
  • DNA array (Shujunsha, edited by Cell Engineering, “DNA microarray and the latest PCR method”). Protein arrays are described in detail in Nat Genet. 2002 Dec; 32 Suppl: 526 —32.
  • gene expression analysis methods include, but are not limited to, RT-PCR, RACE method, SSCP method, immunoprecipitation method, two-hybrid system, and in vitro translation. Such further analysis methods are described in, for example, the genome analysis experiment method 'Yusuke Nakamura Lab' manual, edited 'Yusuke Nakamura Yodosha (2002), etc., all of which are referred to in this specification. Incorporated as.
  • expression of a gene product such as a gene, a polynucleotide, or a polypeptide means that the gene product or the like undergoes a certain action in vivo and takes another form.
  • it refers to force transcription and translation of genes, polynucleotides and the like to form a polypeptide, but transcription to produce mRNA can also be a form of expression. More preferably, such a polypeptide form may have undergone post-translational processing.
  • “Expression level” refers to the level at which a polypeptide or mRNA is expressed in a target cell or the like. Such expression level can be determined by any appropriate method including immunoassay methods such as ELISA, RIA, fluorescent antibody, western plot, and immunohistochemical staining using the antibody of the present invention.
  • the expression level of the peptide at the mRNA level can be mentioned. “Change in the expression level” means expression at the protein level or mRNA level of the polypeptide of the present invention evaluated by any appropriate method including the above immunological measurement method or molecular biological measurement method. It means that the amount increases or decreases.
  • expression or “reduction” in “expression amount” of a gene, polynucleotide, polypeptide, etc. means an action when the factor of the present invention is applied. This means that the amount of expression is significantly reduced compared to when not.
  • the decrease in expression includes a decrease in the expression level of the polypeptide.
  • expression or “increase” in “expression” of a gene, polynucleotide, polypeptide or the like refers to a factor related to gene expression in a cell ( For example, when the gene to be expressed or a factor that regulates it is introduced, the amount of expression is significantly increased compared to when it is not allowed to act.
  • the increase in expression includes an increase in the expression level of the polypeptide.
  • induction of “expression” of a gene means that a certain factor acts on a certain cell to increase the expression level of the gene. Therefore, the induction of expression means that the gene is expressed when the expression of the gene is not seen at all, and the expression of the gene has already been seen! / Including the increase in the expression of the gene when drowning.
  • a gene is "specifically expressed” means that the gene is different (preferably higher) from another site or time in a specific site or time of a plant. It is expressed. “Specific expression” may be expressed only at a certain site (specific site) or at other sites. The expression specifically preferably means that it is expressed only at a certain site.
  • biological activity refers to the activity that a certain factor (eg, polypeptide or protein) has in vivo, and has various functions (eg, transcription-promoting activity). ) Is exhibited.
  • a certain factor eg, polypeptide or protein
  • its biological activity includes the formation of conjugates or other biological changes.
  • such biological activity may be cell adhesion activity, heterogen 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 adhesion activity.
  • the heparin binding activity can be measured by performing affinity chromatography such as a heparin-fixed column and confirming that it binds thereto.
  • Collagen binding activity can be measured by performing affinity chromatography such as a collagen-fixed column and confirming that it binds to this. For example, if a factor is an enzyme, its biological activity includes that enzyme activity. In another example, when an agent is a ligand, the ligand includes binding to the corresponding receptor. Such biological activity can be measured by techniques well known in the art (see Molecular Cloning, Current Protocols (cited herein), etc.).
  • the term "kit” refers to a unit in which parts to be provided (eg, reagents, particles, etc.) are usually provided in two or more compartments. This kit form is preferred when it is intended to provide a composition that should preferably be mixed and used immediately prior to use. Such a kit is advantageously provided with instructions describing how to treat the provided parts (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 carrying a recombinant vector into which a DNA encoding the polypeptide is incorporated, according to a normal culture method.
  • the peptide can be produced by accumulating and collecting the polypeptide of the present invention from the culture.
  • the method of culturing the transformant in a medium can be performed according to a usual method used for culturing a host.
  • a carbon source that can be assimilated by the organism of the present invention for example, glucose, fructose, sucrose, and the like
  • Molasses carbohydrates such as starch or starch hydrolysates, organic acids such as acetic acid and propionic acid, alcohols such as ethanol and propanol), nitrogen sources (eg, ammonia, ammonium chloride, ammonium sulfate)
  • nitrogen sources eg, ammonia, ammonium chloride, ammonium sulfate
  • Ammonium salts of various inorganic or organic acids such as ammonia, ammonium acetate and ammonium phosphate, other nitrogenous substances, peptone, meat extract, fermented mother extract, corn steep liquor , Casein hydrolyzate, soybean meal and soybean meal hydrolyzate, various fermented cells and digested products thereof, etc.
  • inorganic salts for example, primary potassium phosphate)
  • Potassium phosphate for example, magnesium phosphate, magnesium sul
  • the 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.
  • the culture time is usually 5-7 days.
  • the pH is maintained at 3.0 to 9.0. The pH is adjusted using inorganic or organic acids, alkaline solutions, urea, calcium carbonate, ammonia, etc.
  • an antibiotic such as ampicillin or tetracycline may be added to the medium as needed during the cultivation.
  • E)-Sepharose (Pharmacia), DIAION HPA—75 (Mitsubishi Chemical) and other anion-exchange chromatography methods, S—Sepharose FF (Pharmacia) and other cation-exchange chromatography methods Hydrophobic chromatography method using butyl- Sepharose, Phenyl- Sepharose, etc., gel filtration method using molecular sieve, affinity chromatography method, chromatofocusing method, electrophoresis method such as isoelectric focusing etc. A purified sample can be obtained using the above method.
  • the polypeptide used in the present invention accumulates in a dissolved state in the cells of the transformant, the cells in the culture were collected by centrifuging the culture, and the cells were washed. Later, the cells are broken with an ultrasonic crusher, French press, Manton Gaurin homogenizer, Dynomill or the like to obtain a cell-free extract.
  • the solvent extraction method From the supernatant obtained by centrifuging the cell-free extract, the solvent extraction method, salting out method using ammonium sulfate, desalting method, precipitation method using organic solvent, jetylaminoethyl (DEAE)-Sepharose, DIAION Anion exchange chromatography using HPA-75 etc., cation exchange chromatography using S- Sepharose FF, etc., Butyl- Sepharose, Phenyl- Se By using techniques such as hydrophobic chromatography using pharose and other gels, gel filtration using molecular sieves, affinity chromatography, chromatofocusing, and electrophoresis such as isoelectric focusing A purified preparation can be obtained.
  • the polypeptide used in the present invention is expressed by forming an insoluble substance in the cell, similarly, the cell is recovered and then disrupted and centrifuged from the precipitate fraction obtained, After recovering the polypeptide of the present invention by an ordinary method, the insoluble substance of the polypeptide is dissolved with a polypeptide denaturant. This soluble solution is not contained in the polypeptide denaturing agent or the polypeptide denaturing agent does not denature the polypeptide! After constructing the peptide into a normal 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 [eg, J. Evan. Sadler et al .: Methods in Enzymology, 83, 458].
  • a conventional protein purification method eg, J. Evan. Sadler et al .: Methods in Enzymology, 83, 458.
  • affinity chromatography using a substance having an affinity for the fused protein is used. [Akio Yamakawa, Experimental Medicine, 13, 469-474 (1995)].
  • Lowe et al. Proc. Natl. Acad.
  • the polypeptide can be produced as a fusion protein with protein A 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 Dev elop., 4, 1288 (1990)].
  • polypeptide of the present invention can be transcribed in vitro according to a known method Q [. Biomolecular NMR, 6, 129-134, Science, 242, 1162-1164, Biochem., 110, 166-168 (1991)]. ⁇ Can be produced using a translation system.
  • the polypeptide of the present invention can also be obtained by chemical synthesis methods such as the Fmoc method (fluorenylmethylcarbol method) and the tBoc method (tbutyloxycarbon method). Peptides can be produced. Alternatively, chemical synthesis can be performed using peptide synthesizers such as Advanced ChemTech, Applied Biosystems ⁇ Pharmacia Biotech, Protein Technology Instrument, Synthecell-Vega, PerSeptive, Shimadzu Corporation.
  • differentiation generally refers to the separation of one system into two or more qualitatively different systems, function and Z when used on cells, tissues or organs. Or it means that the form is special. With differentiation, pluripotency usually decreases or disappears. For example, epidermal cells, splenic parenchymal cells, splenic duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, skeletal myoblasts, neurons, vascular endothelial cells, pigment cells They may differentiate into morphologically different cells such as smooth muscle cells, adipocytes, bone cells, and chondrocytes, or the ridges may expand or proliferate like nerve cells. When used in the present invention, differentiation-induced differentiated cells can take the form of a population or tissue.
  • differentiation induction includes an increase in the speed of separation and directing cells in which the differentiation is stopped or in the direction of separation into the direction of separation.
  • the ability to induce differentiation is retained, when used on a cell, the nucleic acid is introduced, and preferably the cell is differentiated in a state in which the nucleic acid is expressed. It has the ability to induce differentiation when exposed to. Such ability can be confirmed by observing various cell markers or morphologies to determine whether or not they are exposed to differentiation factors and differentiate. The ability to differentiate often disappears when the nucleic acid is introduced into the cell. However, in the present invention, it has been clarified that the ability to induce separation was retained even after the introduction of the nuclear acid.
  • minute key factor In the present specification, “minute key factor”, “minute key inducer” or “minute key promoter” are used interchangeably, and promote or induce minute key to minute cells.
  • Factors e.g. chemicals Quality, temperature, etc.). Examples of such factors include various environmental factors. Examples of such factors include temperature, humidity, pH, salt concentration, nutrition, metal, gas, organic solvent, pressure, chemical Examples include, but are not limited to, substances (eg, steroids, antibiotics, etc.) or any combination thereof.
  • Representative separation factors include, but are not limited to, cell bioactive substances.
  • DNA demethylating agents such as 5-azacytidine
  • histone deacetylating agents such as trichostatin
  • nuclear receptor ligands eg, retinoic acid (ATRA)
  • vitamins D, T3, etc.
  • IGF insulin-like growth factor
  • FGF fibroblast growth factor
  • PDGF platelet-derived growth factor
  • TGF transforming growth factor
  • ⁇ GF nerve growth factor
  • BMP 2Z4 bone formation Protein 2Z4, etc.
  • site force in LIF, IL 2, IL 6, etc.
  • hexamethylene bisacetamide dimethylacetamide, dibutyl cAMP, dimethylthiol sulfoxide, iodine deoxyuridine
  • examples include, but are not limited to, hydroxylurea, cytosine arabinoside, mitomycin C, sodium butyrate, affidicholine, fluorodeoxyuridine, polyprene, selenium and the like.
  • separation factor include the following. These separation factors can be used alone or in combination.
  • Skin keratinocytes: TGF- ⁇ , FGF-7 (KGF: keratinocyte growth factor), EGF
  • Vascular endothelium VEGF, FGF, angiopoietin
  • Kidney LIF, BMP, FGF, GDNF
  • HGF HGF, LIF, VEGF
  • HGF HGF, TGF- ⁇ , IL-6, EGF, VEGF
  • Intestinal epithelium EGF, IGF—I, HGF, KGF, TGF—j8, IL—11
  • Nerve nerve growth factor (NGF), BDNF (brain-derived neurotrophic factor), GDNF (glial cell-derived neurotrophic factor), neurotrophin, IL-6, TGF- ⁇ , ⁇ NF
  • Glial cells TGF— ⁇ , TNF-a, EGF, LIF, IL—6
  • K Peripheral neurons: bFGF, LIF, TGF- ⁇ , IL-6, VEGF
  • Placenta growth hormone (GH), IGF, prolatatin, LIF, IL-1, activin A, EG
  • FGF FGF
  • CNTF Chronic neurotrophic factor
  • Adipocytes insulin, IGF, LIF
  • Muscle cells LIF, TNF-a, FGF.
  • any culture solution can be used as long as the cells are maintained or differentiated into desired differentiated cells.
  • examples of such a culture solution include, but are not limited to, DMEM, P199, MEM, HBSS, Ham's F12, BME, RPMI1640, MCDB104, MCDB153 (KGM), and mixtures thereof.
  • Such cultures include corticosteroids such as dexamethasone, insulin, glucose, indomethacin, isobutyl-methylxanthine (IBMX), ascorbet 2-phosphate, ascorbic acid and its derivatives, glyce mouth phosphate, Estrogen and its derivatives, Progesterone and its derivatives, Androgen and its derivatives, Growth factors such as aFGF, bFGF, EGF, IGF, TGF ⁇ , ECGF, BMP, PDGF, pituitary extract, pineal extract, retinoic acid, vitamin D , Thyroid hormone, fetal bovine serum, horse serum, human serum, heparin, sodium bicarbonate, HEPES, albumin, transferrin, selenic acid (such as sodium selenite), linolenic acid, 3-isobutyl-1-methylxanthine, Five -Demethylating agents such as zanzanitidine, histone deacetylating agents such as trichost
  • the term “device” refers to a part that can constitute part or all of an apparatus, a support (preferably a solid support), and a standard to be supported on the support. Forces such as target materials are also constructed. Such devices include, but are not limited to, chips, arrays, microtiter plates, cell culture plates, petri dishes, films, beads, and the like.
  • support refers to a material capable of immobilizing a substance such as a biomolecule.
  • the material of the support is either a covalent bond or a non-covalent bond, and has a force having such a property as to bind to a substance such as a biomolecule used in the present invention, or such a property.
  • any material capable of forming a solid surface can be used.
  • the support may be formed from a plurality of layers of different materials.
  • inorganic insulating materials such as glass, quartz glass, alumina, sapphire, forsterite, silicon oxide, silicon carbide, and silicon nitride can be used.
  • Polyethylene ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polychlorinated butyl, polychlorinated vinylidene, polyacetic acid butyl, polybutyl alcohol, polybutylacetal, acrylic candy Fat, polyacrylonitrile, polystyrene, aceta Resin resin, polycarbonate, polyamide, phenol 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, and a PVDF membrane can also be used.
  • the material constituting the support is a solid phase, it is particularly referred to as “solid support” in the present specification.
  • solid support in the present specification, it may take the form of a plate, microwell plate, chip, slide glass, film, bead, metal (surface) and the like.
  • the support may or may not be coated.
  • liquid phase is used in the same meaning as that usually used in the art, and usually refers to a state in a solution.
  • solid phase is used in the same meaning as used in the art, and usually refers to a solid state.
  • liquid and solid may be collectively referred to as fluid.
  • salt is used in the same meaning as commonly used in the art, and includes both inorganic salts and organic salts.
  • the salt is usually produced by a neutralization reaction between an acid and a base.
  • NaCl, KSO, etc. produced by neutralization reaction
  • Salts include normal salts (those that do not contain acid H or base OH, such as NaCl, NH 4 Cl, CH COONa, Na 2 CO 3), acid salts (acid H remains in the salt).
  • Preferable salts include salts constituting the medium (for example, salt calcium carbonate, sodium hydrogen phosphate, sodium hydrogen carbonate, sodium pyruvate, HEPES, salt calcium carbonate, salt sodium chloride, rhodium chloride, sulfide).
  • salts constituting the medium for example, salt calcium carbonate, sodium hydrogen phosphate, sodium hydrogen carbonate, sodium pyruvate, HEPES, salt calcium carbonate, salt sodium chloride, rhodium chloride, sulfide.
  • Magnesium, iron nitrate, amino acids, vitamins, salts constituting buffer solutions for example, chloride chloride, magnesium chloride, sodium hydrogen phosphate, sodium chloride salt
  • These salts may be used alone or in combination. It is preferable to use a plurality. This is because the affinity for cells tends to increase.
  • salts eg, sodium chloride, magnesium chloride, sodium hydrogen phosphate, sodium chloride
  • the course may be customized.
  • this salt it may be advantageous to use this salt to fix the nucleic acid to the solid phase.
  • contact refers to the presence of two substances (eg, a composition and a cell) that are close enough to interact with each other.
  • interaction means that when two objects are referred to, the two objects exert a force on each other.
  • interactions include, but are not limited to, covalent bonds, hydrogen bonds, van der Waals forces, ionic interactions, nonionic interactions, hydrophobic interactions, electrostatic interactions, etc.
  • the interaction may be a normal interaction that occurs in vivo, such as a hydrogen bond, a hydrophobic interaction.
  • plate refers to a planar support on which molecules such as antibodies can be immobilized.
  • the plate is preferably based on a glass substrate having a metal thin film containing plastic, gold, silver, or aluminum on one side.
  • substrate refers to a material (preferably a solid) on which the chip or array of the present invention is constructed. Therefore, the substrate is included in the concept of a plate.
  • the material of the substrate can be either a covalent bond force or a non-covalent bond, a force having the property of binding to the biomolecule used in the present invention, or a derivative to have such a property. Any solid material may be mentioned.
  • Such materials for use as plates and substrates can be any material that can form a solid surface.
  • the substrate may be formed from a plurality of 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.
  • Preferred materials for the substrate vary depending on various parameters such as measuring equipment, and those skilled in the art can appropriately select various materials as described above.
  • a slide glass is preferred for the transformation array.
  • such a substrate can be coated.
  • coating refers to such a film that forms a film of a substance on the surface of the solid support or substrate when used on the solid support or substrate. Coating is done for a variety of purposes, such as improving the quality of the solid support and substrate (e.g., improving lifetime, improving environmental resistance such as acid resistance), materials to be bonded to the solid support or substrate. In many cases, the purpose is to improve the affinity.
  • the material for such a coating is referred to as a “coating agent”. As such a coating agent, various substances can be used.
  • biological substances such as DNA, RNA, protein, and lipid, polymers (for example, poly — L-lysine, MAS (available from Matsunami Glass, Kishiwada, Japan), hydrophobic fluorocarbon resin, silane (APS (eg y-aminominovir)), metal (eg gold) are used but you are not limited to them.
  • polymers for example, poly — L-lysine, MAS (available from Matsunami Glass, Kishiwada, Japan), hydrophobic fluorocarbon resin, silane (APS (eg y-aminominovir)), metal (eg gold) are used But you are not limited to them.
  • APS eg y-aminominovir
  • metal eg gold
  • such a coating comprises a poly L-lysine, a silane, such as an epoxy silane or mercapto silane, APS ( It may be advantageous to use a metal such as ⁇ -aminoprovirsilane)), MAS, hydrophobic fluorocarbon resin, gold.
  • a silane such as an epoxy silane or mercapto silane
  • APS It may be advantageous to use a metal such as ⁇ -aminoprovirsilane)
  • MAS acrylonitrile-butadiene triflate
  • hydrophobic fluorocarbon resin gold.
  • it is preferable to use a substance that is compatible with a cell or an object including the cell for example, a living body, a visceral organ, etc.
  • chip or “microchip” is used interchangeably and refers to a micro integrated circuit that has various functions and becomes a part of a system.
  • the chip include, but are not limited to, a DNA chip and a protein chip.
  • an "array” refers to a pattern in which a composition (eg, DNA, protein, transferate mixture) containing one or more (eg, 1000 or more) target substances is arranged and arranged. Or it refers to a substrate (for example, a chip) itself having a pattern. Among arrays, those patterned on a small substrate (eg, 10 ⁇ 10 mm) are called microarrays. In this specification, microarrays and arrays are used interchangeably. Therefore, even a pattern patterned on a larger substrate than the above substrate may be called a microarray. For example, an array is composed of a set of desired transferate 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 Uenore microtiter plate, or the size of 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 from 1 to the number of spots. For example, a composition containing about 10, about 100, about 500, and about 1000 target substances can be immobilized.
  • target substances for example, proteins such as antibodies
  • a solid surface or membrane such as a substrate as described above, but usually 10 8 per substrate.
  • up to 10 biomolecules in other embodiments up to 10 7 biomolecules, up to 10 6 biomolecules, up to 10 5 biomolecules, up to 10 4 biomolecules, up to 10 3 biomolecules.
  • up to 10 2 biomolecules can be deployed, although a composition containing more than 10 8 biomolecules of a target substance may be deployed. In these cases, the size of the substrate is 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 on the order of 1-2 nm) ).
  • the minimum substrate area is determined in some cases by the number of biomolecules on the substrate.
  • a composition containing a target substance intended to be introduced into a cell is usually immobilized in a spot form of 0.01 mm to 10 mm by covalent bonding or physical interaction.
  • spots of biomolecules can be placed on the array.
  • spot refers to a certain set of compositions containing a target substance.
  • spotting refers to producing a spot of a composition containing a certain target substance on a certain substrate or plate. Spotting can be done by any method, for example, it can be accomplished by pipetting or the like, or it can be done by automated equipment, such methods are well known in the art.
  • address refers to a unique position on a substrate, which may be distinguishable from other unique positions.
  • the address is suitable for associating with a spot with that address, and the entity at every address can take an arbitrary shape so that the entity forces at other addresses can also be identified (eg, optical). obtain.
  • the shape defining the address can be, for example, a force that can be circular, elliptical, square, rectangular, or an irregular shape. Therefore, “address” indicates an abstract concept, and “spot” can be used to indicate a specific concept, but when it is not necessary to distinguish between the two, And “spot” can be used interchangeably.
  • the size defining each address includes, among other things, the size of the substrate, the number of addresses on a particular substrate, the amount of the composition containing the target substance and Z or available reagents, the size of the microparticles and the It depends on the degree of resolution required for any method in which the array is used.
  • the size can be, for example, a force that can range from 1 to 2 nm force several centimeters, and can be any size consistent with the application of the array.
  • the spatial layout and shape that defines the address is designed to suit the particular application in which the microarray is used. Addresses can be densely distributed, can be widely distributed, or can be subgrouped into a desired pattern appropriate for a particular type of analyte.
  • Microarrays are widely outlined in the genome function research protocol (experimental medicine separate volume, post-genomic experimental course 1), genomic medical science and future genomic medicine (experimental medicine extra edition).
  • the database format includes, for example, a format called GATC (genetic analysis technology consortium) proposed by Affymetrix.
  • the present invention is useful when it has been found that a surprising effect (in addition to immobilization, the introduction efficiency increases dramatically) by the combination of a cell adhesion molecule and a gene introduction reagent. There is.
  • cell adhesion molecule or “adhesion molecule” is used interchangeably, and two or more cells approach each other (cell adhesion) or a substrate and a cell.
  • a molecule that mediates adhesion between In general, molecules related to cell-cell adhesion (cell-cell adhesion) and molecules involved in cell-extracellular matrix adhesion (cell-substrate adhesion molec) ule).
  • the cell adhesion molecule includes a protein on the substrate side during cell-substrate adhesion, but in the present specification, also includes a protein on the cell side (for example, integrin etc.). Even a molecule falls within the concept of a cell adhesion molecule or cell adhesion molecule herein as long as it mediates cell adhesion.
  • Biochemical quantification SDS—PAG method, labeled collagen method
  • immunological quantification enzyme antibody method, fluorescent antibody method, immunohistochemical examination
  • PD The determination can be made by determining the positive result in an assembly such as the R method or the hybridization method.
  • cell adhesion molecules include collagen (SEQ ID NO: 1-16), integrin, fibronectin (SEQ ID NO: 23-24), laminin (SEQ ID NO: 17-22), vitronectin, fibrinogen, immunoglobulin superfamily (eg, CD2, CD4, CD8, ICM1, ICAM2, VCAM1), selectin, cadherin and the like.
  • the adhesion factor used in the present invention is preferably one that transmits such an auxiliary signal of cell activity. This is because cell activity can promote the growth of cells and Z or cells in tissues or organs after application. Whether such auxiliary signals can be transmitted into cells is determined by biochemical quantification (SDS—PAG method, labeled collagen method), immunological quantification (enzyme antibody method, fluorescent antibody method, immunohistochemistry). Examination) Judgment can be made by determining positive in the PDR method or hybridization method.
  • extracellular matrix is also called “extracellular matrix” and refers to a substance existing between somatic cells regardless of whether they are epithelial cells or non-epithelial cells.
  • the extracellular matrix is involved in the organization of the internal environment that is necessary for the survival of all somatic cells, not just the tissue support.
  • Extracellular matrices are generally produced from connective tissue cells, but some are also secreted from the cells themselves that possess a basement membrane, such as epithelial cells and endothelial cells.
  • the fiber component and the matrix that fills it are roughly divided, and the fiber component includes collagen fiber and elastic fiber.
  • the basic component of the substrate is glycosaminodarlican (acid mucopolysaccharide).
  • the extracellular matrix used in the present invention include, but are not limited to, collagen, elastin, proteodarican, glycosaminodarican, fibronectin, laminin, elastic fiber, collagen fiber and the like.
  • the extracellular matrix preferably has the activity of attracting host autologous cells.
  • cell adhesion protein refers to a protein having a function of mediating cell adhesion as described above. Accordingly, in the present specification, the cell adhesion protein includes a protein on the substrate side during cell-substrate adhesion, but also includes a protein on the cell side (for example, an integrin). For example, when cultured cells are seeded under serum-free conditions on a substrate (glass or plastic) that has adsorbed the protein on the substrate side, the integrin, which is a receptor, recognizes the cell-adhesive protein, and the cell recognizes the substrate. Adhere to.
  • the proteins included in the present invention comprise RGD sequences such as RGD, YIGSR.
  • RGD sequences such as RGD, YIGSR.
  • Cell adhesion proteins are usually present in extracellular matrix, cultured cell surfaces, and plasma 'serum' body fluids. Its in vivo functions include not only cell adhesion to the extracellular matrix but also cell migration 'growth' morphology regulation and tissue construction. Apart from cell action, there are also proteins that exhibit the function of regulating blood coagulation 'complement action, and proteins having such functions may also be useful in the present invention. Examples of such cell adhesion protein include, but are not limited to, fibronectin, collagen, vitronectin, laminin and the like.
  • RGD molecule refers to a protein molecule comprising the amino acid sequence RGD (Arg-Gly-Asp) or a functionally identical sequence thereof.
  • RGD molecule is cell adhesive RGD, which is an amino acid sequence useful as an amino acid sequence of a cell adhesion active site of protein, or another functionally equivalent amino acid sequence is included.
  • the RGD sequence was found as a cell adhesion site for fibronectin, and was later found in many molecules with cell adhesion activity such as type I collagen, laminin, vitronectin, fibrinogen, von Willebrand factor, and enteractin.
  • the biomolecule in the present invention may be a chemically synthesized RGD molecule.
  • RGD molecules include, but are not limited to, the GRGDSP peptide in addition to the naturally occurring molecules described above. Since RGD sequences are recognized by integrins (eg, receptors for fibronectin), which are cell adhesion molecules (and receptors), RGD functionally equivalent molecules can be obtained using such integrins. It can be identified by examining the interaction.
  • collagen is a kind of protein, which is a fiber-forming collagen and is a general term for a region in which three polypeptide chains are wound in a triple helix, and is a scaffold for cell engraftment and proliferation. Yes, that forms a tissue skeleton.
  • Collagen is the main component of the extracellular matrix of animals.
  • Collagen also has an RGD sequence and is known to exhibit cell adhesion activity.
  • Collagen is known to be contained in about 20-30% of the total protein of animals and contained in large amounts in skin, tendons, cartilage and the like.
  • types I to XIII are known. Normally, each molecule has a triple helix structure with three polypeptide chains, and each chain is often called an ⁇ chain.
  • one molecule may have one type of ⁇ chain strength, or may have multiple types of ⁇ chain strength encoded by different genes.
  • the ⁇ chain is usually called ⁇ 1 (I) with a number after ⁇ , such as ⁇ ⁇ , ⁇ 2 and ⁇ 3, followed by a collagen type. Therefore, in the present invention, for example, in addition to naturally occurring collagen molecules such as [ ⁇ 1 (I) ⁇ 2 (I)] (type I collagen),
  • Such a combination of trimers can also be used privately.
  • Most of the primary structure of collagen is characterized by the amino acid alignment ability of [Gly—X—Pro (or hydroxyprolyl) ⁇ (X is any amino acid residue). This structure takes a left-handed helical structure with a three-residue period.
  • Collagen usually contains hydroxylysine as a special amino acid.
  • Collagen is a sugar protein. Sugar binds to the hydroxyl group of hydroxylysine! / [0141]
  • Collagen is classified into fibrillar collagen or interstitial collagen that exists in fibrous form and gathers to form collagen fibers.
  • fibrogenic collagens include type I, type II, type IV, type V, type XI collagen and are used in preferred embodiments of the present invention.
  • collagens include short-chain collagen (type VIII, type X, etc.), basement membrane collagen (type IV, etc.), FACIT collagen (type IX, type IV, XIV type, XVI type, XIX type, etc.) , Multiplexins collagen (XV type, XVIII type, etc.), microfibrillar collagen (VI type, etc.), long chain collagen (type VII, etc.), membrane-bound collagen (type IV, type XVII, etc.) All can be used in the present invention.
  • basement membrane collagen refers to the major collagen that constitutes the basement membrane.
  • type I collagen refers to [ ⁇ 1 (I) ⁇ 2 ( ⁇ )] t, a colloid having a coral structure.
  • Is composed of two a 1 (I) chains and three a 2 (I) polypeptide hetero-chains, a tissue skeleton present in every tissue in the living body and its functionally equivalent
  • amino acid sequences of such polypeptides include, but are not limited to, p02454 and p02464 in Genbank accession numbers (for example, as described in SEQ ID NOS: 1-4). Things).
  • the functionally equivalent molecule of type I collagen can be identified by, for example, an enzyme antibody method or an EIA method.
  • type IV collagen is basement membrane collagen, and its molecule is composed of four domains: 7S, NC2, TH2, and NCI. Polymerized and polymerized by NC1 at the C-terminal to form a network network, collagen or its functionally equivalent molecule, amino acid of such a polypeptide IJ , Typically Genbank accession numbers p02462, p08572, UO 2520, D17391, P29400, U04845 force ⁇ , but not limited to it! ⁇ (for example, sequence numbers 5-16 Etc.).
  • a functionally equivalent molecule of type IV collagen can be identified by, for example, an enzyme antibody method or an EIA method.
  • the term “cell” is defined in the same manner as the broadest meaning used in the field, and is a structural unit of tissue of a multicellular organism, and is a membrane that isolates the outside world. An organism that is wrapped in a structure and has self-regenerative ability inside and has genetic information and its expression mechanism.
  • the cells used in the present specification may be naturally occurring cells or artificially modified cells (eg, fusion cells, genetically modified cells).
  • the source of cells can be, for example, a single cell culture, or a cell from a normally grown transgenic animal embryo, blood, or body tissue, or a cell line that has grown normally. Cell mixtures such as, but not limited to.
  • the cells used in the present invention can be cells from any organism (eg, any type of unicellular organism (eg, bacteria, yeast) or multicellular organisms (eg, vertebrates, invertebrates). Animal), plant (for example, monocotyledonous plant, dicotyledonous plant, etc.))).
  • organism e.g., any type of unicellular organism (eg, bacteria, yeast) or multicellular organisms (eg, vertebrates, invertebrates). Animal
  • plant for example, monocotyledonous plant, dicotyledonous plant, etc.
  • cells derived from vertebrates e.g., metaraunagi, shark eels, cartilaginous fish, teleosts, amphibians, reptiles, birds, mammals, etc.
  • mammals e.g.
  • cells derived from primates are used, particularly but not limited to cells derived from humans.
  • stem cells have the ability to self-replicate and are pluripotent (ie pluripotent).
  • stem cells are usually able to regenerate the tissue when it is damaged.
  • stem cells can be, but are not limited to, embryonic stem (ES) cells or tissue stem cells (both tissue stem cells, tissue-specific stem cells or somatic stem cells).
  • an artificially produced cell for example, a fusion cell described herein, a reprogrammed cell, etc.
  • Embryonic stem cells are pluripotent stem cells derived from early embryos. Embryonic stem cells were first established in 1981 and have been applied since 1989 to the production of knockout mice. In 1998, human embryonic stem cells were established and are being used in regenerative medicine.
  • tissue stem cells are cells that have a limited direction of differentiation. They are located at specific locations in the tissue and have an undifferentiated intracellular structure. Therefore, tissue stem cells have a low level of pluripotency. Tissue stem cells have a nuclear Z cytoplasm ratio Insufficient intracellular organelles. Tissue stem cells generally have pluripotency and maintain proliferative capacity over the life of an individual with a slow cell cycle. As used herein, the 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, the skin system, digestive system, myeloid system, and nervous system.
  • skin tissue stem cells include epidermal stem cells and hair follicle stem cells.
  • tissue stem cells of the extinct system include knee (common) stem cells and liver stem cells.
  • myeloid thread and tissue stem cells include hematopoietic stem cells and mesenchymal stem cells.
  • Neural tissue stem cells include neural stem cells and retinal stem cells.
  • somatic cells refers to all cells that are not germ cells such as eggs and sperm and do not directly transfer the DNA to the next generation. Somatic cells usually have power or loss of limited pluripotency. As used herein, somatic cells may be naturally occurring or genetically modified.
  • Cells may be classified into stem cells derived from ectoderm, mesoderm and endoderm, depending on origin.
  • Cells derived from ectoderm are mainly present in the brain and include neural stem cells.
  • Cells derived from mesoderm are mainly present in 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 liver stem cells, spleen stem cells, and the like.
  • somatic cells may be derived from any germ layer.
  • lymphocytes, spleen cells or testis-derived cells can be used as the body cells.
  • isolated means that in a normal environment! Naturally occurring substances are at least reduced, preferably substantially free.
  • an isolated cell refers to a cell that is substantially free of other substances (eg, other cells, proteins, nucleic acids, etc.) that accompany it in its natural environment.
  • isolated is, for example, substantially free of cellular material or culture medium when made by recombinant DNA technology, and precursor when chemically synthesized.
  • An isolated nucleic acid is preferably a sequence that naturally flanks the nucleic acid in the organism from which it is derived (ie, located at the 5 'and 3' ends of the nucleic acid). Array) is not included.
  • established or “established” cells maintain certain properties (eg, pluripotency) and allow the cells to grow stably under culture conditions. It means a state in which people continue to do so. Thus, the established stem cells maintain pluripotency.
  • a "separated cell” refers to a cell (eg, muscle cell, nerve cell, etc.) that is specialized in function and morphology. There is little or no ability.
  • differentiated cells include epidermal cells, splenic parenchymal cells, splenic duct cells, hepatocytes, blood cells, cardiomyocytes, skeletal muscle cells, osteoblasts, skeletal myoblasts, nerve cells, vascular endothelial cells, dyes Examples thereof include cells, smooth muscle cells, fat cells, bone cells, and chondrocytes.
  • primary culture cell refers to a cell in a state of being cultured until a cell, tissue or organ separated from a living body is implanted and the first passage is performed. Therefore, it is strictly distinguished from the “passage” cells that have been passaged. Usually, primary cultured cells are considered to be quite difficult to introduce foreign substances such as genetic manipulation that is difficult to handle (for example, transfection).
  • nerve cell refers to a cell constituting a structural and functional unit of the nervous system, which is a combination of a cell body and a process that emerges therefrom, or a variant or functional equivalent thereof. Whether it is a nerve cell can be determined by checking whether nerve transmission occurs. The function of such a nerve cell can be confirmed by, for example, the notch clamp method.
  • PC12 cells can be used as a typical example of a neuron. PC12 cells are cells derived from adrenal medullary brown cells and are used as typical cultured cells as a model of the nervous system because their morphology is similar to neural cells.
  • the "instructions" of the present specification describe the substance introduction method of the present invention and the like to users (researchers, experimental assistants, doctors, patients, etc. who perform administration in the treatment). Is. This instruction describes a word indicating a method of using the composition of the present invention, for example. This instruction is prepared according to the format prescribed by the national supervisory authority in which the present invention is to be implemented, and it is clearly stated that it has been approved by the supervisory authority.
  • the document is usually in the form of a so-called package insert in the case of medicines or in the form of a manual in the case of laboratory reagents, usually provided in paper media, but is not limited thereto, for example, It can also be provided in the form of an electronic medium (for example, a home page or electronic mail provided on the Internet).
  • a so-called package insert in the case of medicines or in the form of a manual in the case of laboratory reagents, usually provided in paper media, but is not limited thereto, for example, It can also be provided in the form of an electronic medium (for example, a home page or electronic mail provided on the Internet).
  • the present invention provides a composition for improving cell nucleic acid introduction efficiency.
  • the composition is characterized by comprising A) a cell adhesion molecule; and B) a gene transfer reagent.
  • a gene introduction reagent such as a gene introduction reagent has been used.
  • the introduction efficiency will increase due to the addition of an external factor.
  • the effect of the cell adhesion molecule is surprising. In particular, it was not expected that the efficiency would change when the cells were fixed, but rather was thought to even decrease.
  • the present invention has an effect that it has been found that introduction of a nucleic acid unexpectedly improves when a gene introduction reagent and a cell adhesion molecule are combined and allowed to act on cells.
  • the present invention it has become possible to easily carry out transfection in a liquid phase or a solid phase in primary cultured cells, nerve cells and the like, which have been conventionally difficult.
  • the cell adhesion molecule and the gene introduction reagent only need to exist in a state where they can interact with each other. These substances may be configured to act simultaneously on cells or may be configured separately.
  • the cells retain the ability to induce differentiation even after introduction of nucleic acid. In this way, it can be said that it was a surprising effect that the ability to induce separation after gene transfer was maintained.
  • a device for improving nucleic acid introduction efficiency of a cell is provided.
  • a composition comprising A) a cell adhesion molecule; and B) a gene transfer reagent is fixed to a support.
  • Such a device may comprise instructions in a preferred embodiment.
  • the cells retain the ability to induce differentiation even after introduction of nucleic acid.
  • the cell adhesion molecule used in the present invention is preferably provided in a protein form, but is not limited thereto. This is because proteins are biomolecules and can be produced by genetic manipulation.
  • Preferred cell adhesion molecules include extracellular matrix. More preferably, the cell adhesion molecule comprises collagen, fibronectin or laminin.
  • Preferred collagens include, but are not limited to, fiber-forming collagen and basement membrane collagen. A plurality of these specific collagens may be present, including fiber-forming collagen and basement membrane collagen at the same time.
  • the cell adhesion molecule used in the present invention comprises collagen type I or type IV. More preferably, the cell adhesion molecule comprises collagen type I and type IV.
  • Collagen type I has traditionally been known only to act as a cell adhesion factor, and when used in combination with a gene transfer reagent, it was a special finding that the efficiency of nucleic acid transfer increased synergistically. .
  • collagen type IV increases the efficiency of nucleic acid introduction, which can be said to be a special effect.
  • combining these multiple collagens further increases the efficiency of nucleic acid introduction. This is because as the type of collagen increases, the points that facilitate the introduction of nucleic acid increase.
  • collagen and fibronectin are compatible with each other in cells, and fibronectin does not show an increase in gene transfer efficiency. Even in cells, collagen has shown an increase in gene transfer efficiency for the first time. It was revealed. Similar effects were also found for laminin in relation to collagen and fibronectin. Therefore, when the cells are cells where the increase in gene transfer effect due to fibronectin is not observed, the use of collagen or laminin as a cell adhesion factor can cause an unexpected increase in gene transfer effect. It became clear, and it became clear that there was a remarkable effect. Without wishing to be bound by theory, collagen appears to be suitable for increasing the gene transfer effect of neuronal cell systems. This was not obvious prior to the disclosure of the present invention o
  • the cell adhesion molecule used in the present invention preferably exists in a multimeric form. It is a force that stabilizes the cell fixation by being present in a multimeric form, and a force that has been found to dramatically increase the efficiency of nucleic acid introduction.
  • the cell adhesion molecule used in the present invention has a three-dimensional structure.
  • the “three-dimensional structure” means a structure that has at least two or more layers of force and has a molecular spread in a direction perpendicular to the plane in addition to the structure oriented in the plane.
  • the cell exists in a more natural state. Being present is a bit different.
  • the gene introduction reagent used in the present invention is at least selected from the group consisting of a cationic polymer, a cationic lipid, a polyamine reagent, a polyimine reagent, and a calcium phosphate force.
  • a cationic polymer e.g., a cationic polymer
  • a polyamine reagent e.g., a polyamine reagent
  • a polyimine reagent e.g., a calcium phosphate force
  • calcium phosphate force e.
  • one reagent may be used and a plurality of these reagents may be used.
  • cationic lipids are used.
  • TransFast TM Transfection Reagent e.g., LipofectA MINE 2000 Reagent.
  • composition for improving the nucleic acid introduction efficiency and fixation of the cell of the present invention may simultaneously contain a foreign molecule to be introduced.
  • the composition of the invention may comprise a nucleic acid molecule that encodes a gene intended to be introduced! / ⁇ !
  • composition of the present invention may be solid or liquid. Liquid is preferred U ⁇ . This is because the operation is easy and the arrangement on the solid phase is easy, and the effect in the liquid phase has also been confirmed.
  • the cell to which the present invention is directed is a cell into which normal gene transfer can be performed, as long as it is a cell to which nucleic acid introduction is intended.
  • Examples thereof include, but are not limited to, PC12, Neuro2a, SH-SY5Y, NG108-15, HCN-1A, and the like.
  • Examples of preferable cells include, but are not limited to, nerve cells and primary cultured cells.
  • the raw material of the primary cultured cells may be any material, and preferred examples include, but are not limited to, the mammalian brain (cerebral cortex, hippocampus, cerebellum).
  • the cell adhesion molecule used in the present invention is preferably present in a concentration range of 5 gZml to: LOO / z gZml. It can be understood that even if it exceeds the upper limit, the introduction efficiency can be improved without any problem.
  • Examples of the upper limit include, for example, lmg / ml, 750 ⁇ g / ml, 500 ⁇ g / ml, 400 ⁇ g / ml, 200 ⁇ g / ml, 250 ⁇ g / ml, 200 ⁇ g / ml, 150 ⁇ g / ml, 100 ⁇ g / ml, 90 ⁇ g / ml, 80 ⁇ g / ml, 70 ⁇ g / ml, 60 ⁇ g / ml, 50 ⁇ g / ml, 40 ⁇ g / m30 / z gZml, etc. But are not limited to these.
  • the lower limit for example, 75 ⁇ gm 50 ⁇ g / m 40 ⁇ gm 20 ⁇ gm 25 ⁇ gm 20 ⁇ g / ml, 15 ⁇ g, / ml, 10 ⁇ g, / ml, 9 ⁇ g, / ml, 8 ⁇ g, / ml, 7 ⁇ g, / ml, 6 ⁇ g, / ml, 5 ⁇ g / Forces, such as ml, 4 ⁇ g / ml, 3 ⁇ g / ml, 2 ⁇ g / ml, 1 ⁇ g / ml, etc.
  • a more preferable range is 20 gZml to 60 gZml.
  • the present invention takes advantage of the fact that nucleic acids can be introduced into cells on a solid phase. It has become possible to introduce nucleic acid into cells that have been difficult to transduce on the solid phase (eg, nervous system cells such as PC12 cells)! In this respect, the present invention should be noted.
  • the compositions of the invention can be used to introduce cells into nucleic acids on a collagen-coated solid support.
  • the present invention is used to induce differentiation after inducing nucleic acid. Therefore, preferably, the composition or kit of the present invention may contain a differentiation-inducing factor (eg, nerve growth factor). In addition, it is understood that the method of the present invention may further include a step of inducing a desired separation after introducing a nucleic acid.
  • a differentiation-inducing factor eg, nerve growth factor
  • the cell adhesion molecule of the present invention is preferably coated on the support. More preferably, the cell adhesion molecule is coated on the support in multiple layers.
  • the support is preferably coated with poly-L-lysine, APS (y-aminopropyl silane), or MAS.
  • nucleic acids are arranged on the support, and it is more preferable that nucleic acids (for example, DNA) are arranged on the support in an array. Yes.
  • nucleic acids for example, DNA
  • the present invention provides a method for introducing a nucleic acid into a cell.
  • the method comprises: A) a composition for improving cell gene transfer efficiency, comprising: a) a cell adhesion molecule; and b) a gene transfer reagent, wherein the composition encodes a gene intended for transfer.
  • the compositions used in the method of the invention are described herein. Methods for providing such a composition to cells can be accomplished using techniques well known in the art.
  • condition for introducing a nucleic acid include any condition under which the nucleic acid to be introduced is introduced and varies depending on the nucleic acid. It can be determined in consideration of the situation of nucleic acids and cells. For example, if the nucleic acid is DNA, such conditions are: pH: 6.0 to 8.0; temperature: 30 to 40; salt concentration: 0.1 to 0
  • the methods of the invention can be performed in solid or liquid phase.
  • a solid phase since a cell adhesion molecule fixes to a cell, a solid phase nucleic acid such as a solid phase transformation can be introduced.
  • nucleic acids such as nerve cells and primary cultured cells, which have been impossible to introduce nucleic acids such as conventional transfection, has made it possible to perform various analyses. Such an analysis has not been possible before.
  • the method of the present invention increases the nucleic acid introduction efficiency. Even in the liquid phase, it was not known that cell adhesion molecules would have an effect, and the effect was surprising.
  • the composition of the present invention and a nucleic acid are preferably immobilized on a support.
  • the support can be any material, but is preferably compatible with the cells. Such cell-compatible materials are as described above.
  • the nucleic acid does not elute even in an environment where the cells are exposed to cells after being fixed, so that the desired nucleic acid can be introduced into the cells at the desired site. This was not possible with the introduction of viral vectors using atelocollagen.
  • the method of the present invention also further comprises the step of immobilizing the composition of the present invention and a nucleic acid (eg, DNA) on a support.
  • a nucleic acid eg, DNA
  • examples of the method for immobilizing the composition and nucleic acid of the present invention on a support include an inkjet method, a bubble jet (registered trademark) method, and a manual method. It is not limited to them.
  • the present invention provides a step of inducing differentiation of a cell into which a nucleic acid has been introduced. May be included.
  • the present invention provides a kit for introducing a nucleic acid into a cell.
  • the kit comprises a composition comprising A) a) a cell adhesion molecule; and b) a gene transfer reagent; and B) the composition and a nucleic acid encoding the desired gene (e.g., DNA).
  • the cell adhesion molecule and the gene introduction reagent are as described above in the present specification and exemplified in the Examples.
  • the instructions describe how the nucleic acid and the composition of the invention can be treated to introduce the nucleic acid.
  • Such instructions may be in any form as described herein above.
  • the present invention may include a differentiation-inducing factor.
  • the present invention relates to the use of a composition
  • a composition comprising a) a cell adhesion molecule; and b) a gene transfer reagent for introducing a nucleic acid into a cell.
  • a composition comprising a) a cell adhesion molecule; and b) a gene transfer reagent for introducing a nucleic acid into a cell.
  • the composition of the present invention may comprise a protein inducer.
  • the present invention provides a composition for introducing a nucleic acid fixed to a solid phase into a cell, wherein the cell retains the ability to induce differentiation even after the introduction of the nucleic acid.
  • the composition of the present invention comprises A) a cell adhesion molecule; and B) a gene transfer reagent. Cells into which nucleic acid was introduced using such a composition retained the ability to induce differentiation even after introduction of the nucleic acid. Such a phenomenon has not been achieved so far. Therefore, it can be said that the present invention has powerful effects that cannot be predicted from the prior art.
  • the composition itself may comprise a separate inducer or may be provided separately. Where differentiation induction needs to be controlled, preferably the differentiation inducing factor is provided separately from the composition used to introduce the nucleic acid.
  • any factor can be used as long as the desired differentiation factor can be achieved, and examples thereof include a cell growth factor. If the cell is a nerve, the differentiation factor used may include nerve growth factor (NGF).
  • NGF nerve growth factor
  • the present invention provides a method for introducing a nucleic acid immobilized on a solid phase into a cell.
  • the method comprises: A) a) a cell adhesion molecule; and b) a gene transfer reagent, providing a composition with the nucleic acid intended to be introduced to the cell; and B) conditions under which the nucleic acid is introduced.
  • the composition used in the above-described method of the present invention may comprise a differentiation inducer or may be provided as a separate composition.
  • the wrinkle-inducing factor used may include, but is not limited to, a cell growth factor (eg, nerve growth factor (NGF)). It will be appreciated that such cell growth factors will vary depending on the cells used.
  • a cell growth factor eg, nerve growth factor (NGF)
  • NGF nerve growth factor
  • the method of the present invention further includes the step of inducing differentiation of the cell.
  • the present invention provides a composition for introducing a nucleic acid into a cell on a solid phase and for inducing the cell.
  • the composition comprises A) a cell adhesion molecule; B) a gene transfer reagent; and C) a differentiation inducing factor. Descriptions of such compositions, nuclear acids, cells, etc. have been given above, and it will be understood that any preferred embodiment may be employed.
  • the present invention provides a kit for introducing a nucleic acid into a cell on a solid phase and for inducing the cell.
  • the kit comprises A) a) a cell adhesion molecule; and b) a composition comprising a gene transfer reagent, and B) a wrinkle inducer.
  • Descriptions of such composition compositions, nucleic acids, cells, etc. have been given above, and it will be understood that any preferred embodiment may be employed.
  • the wrinkle-inducing factor is arranged separately from the yarn and the adult product.
  • kit of the present invention may be provided with instructions showing procedures such as nucleic acid introduction and differentiation induction.
  • the present invention provides a method for introducing a nucleic acid into a cell on a solid phase and inducing differentiation of the cell.
  • This method comprises the steps of A) a) a cell adhesion molecule; and b) a gene transfer reagent, together with the nucleic acid intended to be introduced, to the cell; and B) the conditions under which the nucleic acid is introduced. Exposing the cells; and C) Inducing differentiation of the vesicle. Descriptions of such compositions, nucleic acids, cells, etc. have been given above, and it will be understood that any preferred embodiment may be employed.
  • differentiation induction is performed independently of nucleic acid introduction.
  • the present invention when the composition comprises a nucleic acid in the present invention, the present invention is used to treat, inhibit or prevent a disease or disorder associated with abnormal expression and Z or activity of a polypeptide associated with the nucleic acid.
  • the composition of the invention is administered for gene therapy purposes.
  • Gene therapy refers to treatment performed by administration of a nucleic acid that is expressed or expressible to a subject.
  • the nucleic acids produce their encoded protein, which mediates a therapeutic effect.
  • the present invention when used as a medicament, such medicament can be administered orally or parenterally. Alternatively, such medicaments can be administered intravenously or subcutaneously.
  • the medicament used in the present invention may be in the form of a pharmaceutically acceptable aqueous solution that does not contain pyrogens. Preparation of such a pharmaceutically acceptable composition can be easily performed by those skilled in the art by considering pH, isotonicity, stability, and the like.
  • the administration method includes oral administration and parenteral administration (for example, intravenous administration). Administration, intramuscular administration, subcutaneous administration, intradermal administration, mucosal administration, rectal administration, intravaginal administration, topical administration to the affected area, dermal administration, and the like.
  • Formulations for such administration can be provided in any dosage form. Examples of such formulation forms include solutions, injections, and sustained release agents.
  • the medicament of the present invention may be a physiologically acceptable carrier, excipient or stabilizer as necessary (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 with the desired degree of purity and 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, target disease (type, severity, etc.), patient age, weight, sex, medical history, cell morphology or type, etc. In view of this, it can be easily determined by those skilled in the art.
  • the frequency with which the treatment method of the present invention is applied to a subject (or patient) also depends on the purpose of use, target disease (type, severity, etc.), patient age, weight, gender, medical history, treatment course, etc. In view of this, it can be easily determined by those skilled in the art. Examples of the frequency include administration once a few months every day (for example, once a week, once a month). It is preferable to administer once a week-once a month with the progress of the test.
  • Example [0200] Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to the following examples. Reagents, supports, etc. used in the following examples are commercially available, with the exception of Sigma (St. Louis, USA, Wako Pure Chemicals (Osaka, Japan), Matsunami Glass (Kishiwada, Japan). Using.
  • PC12 cells rat pheochromocytoma cells (ATCC, CRL-1721) at 37 ° C, 5% CO
  • the cells were cultured in DMEM (Dulbeccos modnied Eagle medium bath 14246-25, Nakalai Tesqu e, JPN) containing 10% sushi serum and antibiotics (penicillin and streptomycin).
  • Neuro2a cells are 10% FBS (fetal) at 37 ° C and 5% CO.
  • Each 9mm 2 grid of chips (Matsunami Glass Ind., LTD., JPN) is inoculated with 151 drops of culture medium containing the above cells and incubated at 37 ° C, 5% CO.
  • the cells were allowed to adhere to the substrate, after which culture medium was added and incubated for an additional 48 hours. Considering cell growth of PC12 cells and Neuro2a cells, 4500 and 3750 cells were seeded on each grid, respectively.
  • transfection reagent for each cell line was determined by several transfection assays.
  • a reagent containing Lipofectamine 2000 (11668-019, Invitrogen Corporation CA) was selected as a transfection reagent. 1. 5 1 pEGFP was diluted with 19 1 DMEM. After gently diluting the pDNA, 4.51 of lipofuctamine 2000 was added, and the resulting mixture was allowed to stand at room temperature to form a lipid DNA complex.
  • Jet—PEI NP5 101-30, Polyplus-transection, France
  • 1.51 pEGFP was diluted with 10.51 DMEM.
  • fibronectin 16 042- 41, Nakalai Tesque, JPN
  • lmgZml fibronectin (FN) (16 042- 41, Nakalai Tesque, JPN) (lmgZml) solution was diluted with water (final concentration 0. 1 ⁇ g / ml) 0 The solution then 400 1 And added to the glass slide to cover the area of the eight square sections. After 1 hour incubation at room temperature, the solution was removed and then the square compartments were washed once with ultrapure water.
  • type I collagen collagen l (TypeI-C, Nitta Gelatin Co., Ltd.
  • ECM extracellular matrix
  • fibronectin fibronectin, collagen type IV, collagen type I, laminin
  • phase microscopy Figures 1A-D
  • confocal microscopy Figures 1E, F
  • nucleic acid is increased by adopting those having a relative area of, for example, preferably more than 0.1.
  • the cell adhesion form is shown when 3000-6000 cells are seeded on X 3mm.
  • FIG. 1H shows Neuro2a cells on a PLL-coated slide coated with collagen type IV (0.005 mg / ml). As shown, there is a significant variation in morphology.
  • Transgenic gene expression of CMV promoter EGFP construction vector was measured by fluorescent cells using a confocal microscope.
  • the present inventors investigated the effect of coating plain glass slide with ECM protein with and without pre-coating with PLL (PC12 cells, Fig. 3; Neuro2a cells, Fig. 2C).
  • PLL PC12 cells, Fig. 3; Neuro2a cells, Fig. 2C.
  • type IV collagen increased the transfection up to about 1000 times compared to the PLL coating alone.
  • Laminin on the PLL coat slide showed similar adhesion to that of type IV collagen, but the transfer efficiency was lower (500 times). Adhesion to type I collagen was weaker than that of type IV collagen and laminin.
  • the increase in transfection efficiency was similar to that of type IV collagen. Since PC12 cell adhesion to fibronectin was weak, significant detachment was caused by damage from the transfection reagent during the transfection process. The cells that remained on fibronectin showed negligible force that showed more efficient transfection than when only PLL was used. The effect of ECM protein on uncoated glass slides showed a slightly different pattern than that observed when pre-coated with PLL. Adhesion and transfection efficiency to type I collagen was increased compared to PLL pre-coating. Fibronectin, laminin, and type IV collagen were less effective at transferring efficiency on plain slides than PLL pre-coating. Such an effect can be explained by lower adhesion for type IV collagen and higher adhesion for type I collagen on plain slides when compared to PLL precoated slides. A cooperative effect can also be envisaged.
  • FIGS. 3A, 3B and 3D Collagen (type I and type IV) and laminin showed increased transfer efficiency.
  • the effect of collagen is shown to be high in SPLL and plain.
  • Figure 3E shows a graph that numerically shows the intensity. It can be clearly seen that the effect of collagen is remarkable.
  • Fig. 3F shows the actual cell fixation of Fig. 3D. It can be seen that fibronectin has a low fixing effect, but collagen and laminin have a relatively fixing effect and an effect of increasing the efficiency of transfection.
  • Figure 3G shows the time course of the effects of various ECMs depending on the type of slide surface treatment. Thus, the effect of collagen is remarkable, but it is obvious that any ECM has a more or less adhesive effect.
  • the present inventors applied the knowledge obtained so far in the solution phase transfection to the LT transfection array system of the present inventors, and can perform gene expression screening based on a high-density chip for neuronal cell lines.
  • the transformation is localized to the area where the transformation mixture is spotted.
  • PC12 cells grown on different ECMs showed the same trend as observed in the solution phase method.
  • In grids coated with type I and type IV collagen up to 40 transfection bodies were observed in areas where 30 nl transfection complex (containing about lnl pEGFP) was spotted. .
  • adherence was maintained through more cell force transfection processes.
  • hMSC human mesenchymal stem cells
  • PT-2501 Cambrex Bioscience Walkersville, Inc.
  • MD human embryonic kidney cells
  • HEK293 human embryonic kidney cells
  • HEK293 cells NIH3T3-3 cells, HeLa cells and HepG2 cells
  • these cells are treated with 10% sushi fetal serum (FBS, 29-167-54, Lot No. 2025F, Dainippon Pharmaceutical CO., LTD., Maintained in Dulbecco's modified Eagle medium (DMEM, L-glutamine and sodium pyruvate (4.5 g / L); 14246-25, Nakalai Tesque, JPN). All cell lines were cultured in an incubator controlled at 37 ° C and 5% CO.
  • DMEM Dulbecco's modified Eagle medium
  • pEGFP—Nl and pDsRe d2—Nl vectors were used. In both cases gene expression was under the control of the cytomegalovirus (CMV) promoter. Transfected cells continuously expressed EGFP or DsRed2, respectively. Plasmid DNA was amplified using Escherichia coli, XL 1-blue strain (2002 49, Stratagene, TX) and purified by EndoFree Plasmid Kit (Endo Free Plasmid Maxi Kit 12362, QIAGEN, CA). In all cases, plasmid DNA was dissolved in DNase and RNase free water.
  • CMV cytomegalovirus
  • Transfusion reagents were obtained as follows: Effectene Transfection Reagent (Cat. No. 301425, Qiagen, CA), TransFastTM Transfection Reagent (E2431, Promega, Wl), TfxTM—20 Reagent (E2391, Promega, Wl), SuperFect Transfection Reagent (30 ⁇ 305, Qiagen, CA), PolyFect Transfection Reagent (301105, Qiagen, CA), LipofectAMINE 2000 Reag ent (11668— 019, Invitrogen corporation, CA), JetPEI (X 4) cone. (101 — 30, Polyp lus— transfection ⁇ France), and ExGen 500 (R0511, Ferme ntas Inc., MD).
  • the printing solution contains plasmid DNA and cell adhesion molecules (collagen type I, collagen type IV, laminin, ushi plasma fibronectin dissolved in ultrapure water at a concentration of 4 mgZmL (Cat. No. 16042— 4 1, Nakalai Tesque, JPN)
  • the above solution was applied using an inkjet printer (synQ UADTM, Cartesian Technologies, Inc., CA) or manually using a 0.5-10 / z L chip.
  • the printed slides were allowed to dry inside the safety cabinet for 15 minutes at room temperature, and the total Effectene reagent was placed on the DNA-printed slides before transfection.
  • Method B For other transfection reagents (TransFastTM, TfxTM-20, SuperFect TM PolyFect, LipofectAMINE 2000, JetPEI (X4) cone. Or ExGen), plasmid DNA; collagen type I, collagen type IV , Laminin or fibronec And the transfection reagent according to the ratio given in the manufacturer's instructions 1. Mix evenly in a 5 mL microtube and incubate for 15 minutes at room temperature before printing on the chip did. The printing solution was applied on the surface of the glass slide using an ink jet printer or 0.5-: L0 L chip. The printed slide was completely dried inside the safety cabinet at room temperature for 10 minutes.
  • the transfer array chip was constructed by microprinting a cell culture solution containing DNA Z transfer reagent and cell adhesion factors such as collagen type I, collagen type IV, laminin on a PLL-coated slide glass.
  • the present inventors used PC12 cells as a method of the present inventors (transfusion in a solid phase system) and conventional solutions. Both phase systems were used under a series of transformation conditions.
  • RFP red fluorescent protein
  • GFP green fluorescent protein
  • transfection reagents were evaluated: four liquid transfection reagents (Effectene, TransFastTM, TfxTM—20, LopofectAMI NE 2000), two polyamines (SuperFect, PolyFect), and two types of polyimines (jetPEI ( X 4) and ExGen 500).
  • Transfection efficiency was determined as total fluorescence intensity per unit area. Depending on the cell line used, optimal liquid phase results were obtained using different transfection reagents. These efficient transfection reagents were then used to optimize the solid phase protocol. Several trends were observed. By using conditions optimized for the SPTA methodology in the case of PC12 cells where it is difficult to transfect cells, we have achieved transfection efficiency of tens to hundreds while maintaining cell characteristics. It was observed that there was a double increase in calories. For PC12 cells, the best conditions included the use of Lipofectamine 2000 reagent.
  • N nitrogen atoms
  • P phosphate residues
  • a further cause of reciprocal contamination may be the mobility of the transfection cells on the solid support.
  • the present inventors successfully realized a transfusion array in a system using a cell adhesion molecule and a gene introduction reagent. This is a result of various studies using solid-phase transfection, such as the elucidation of the genetic mechanisms that control neuronal differentiation. It will enable high-throughput research. It became clear that the detailed mechanism of solid-phase transfection and the methodology for using this technology for high-throughput real-time gene expression monitoring can be applied for various purposes.
  • ECM protein used to coat the culture dish in the in vitro mouth retained significant effects on morphology, cell distribution, adhesion, and gene expression.
  • hMSC human mesenchymal stem cells
  • the ECM component appears to be a suitable substrate for chip-based cell arrays that are tested to be useful for the evaluation of unknown gene networks. This study revealed that fibronectin outweighs the role of functional adhesion.
  • Integrin-mediated cell dispersal force on fibronectin matrix causes nuclear plains, and this shape change has been shown to be accompanied by a transient increase in nuclear Ca2 + .
  • These studies showed the presence of activated Ca 2+ from permeation channels in the nuclear membrane responsible for the release of Ca 2+ from the perinuclear space and the penetration of the nucleus. The activity of transcription factors regulated by Ca 2+ was then enhanced in dispersed cells. In our recent report, we also showed a very relevant finding that transfection efficiency is dramatically increased by the presence of fibronectin. Similar cell adhesion profile force fibronectin (
  • fibronectin (+) conditions were observed, but the shape of adherent cells was significantly different. In conjunction with the time-dependent observation of the orientation of the actin filament, this plays an important role in increasing the efficiency of the actin filament induced by cell adhesion molecules such as fibronectin deposited on the surface. I suggest that. In the presence of fibronectin deposited on the surface, the actin filaments were rapidly rearranged and disappeared from the cytoplasmic space located below the nucleus in the process of cell expansion. Furthermore, the surface area of the nucleus was significantly increased in fibronectin (+) conditions. This is thought to be the force that facilitated the passage of DNA particles through possibly mechanical stress.
  • One exemplary purpose of the present inventors' research is to apply cells that have conventionally been difficult to introduce foreign substances, such as nerve cells, into a local transfection cell array.
  • This technique has proven to be an important high-throughput tool for the normalization of parallel cDNA gene expression studies.
  • the cell array can be used as a microscale overexpression array for morphological changes, cell proliferation, or alternative observations due to overexpression of a single gene.
  • nucleic acid introduction efficiency of the nucleic acid introduction according to the present invention depends on the type of gene introduction reagent. This was demonstrated in this example.
  • compositions mixed in the amounts shown in the following table were prepared.
  • Table 1 Amount of gene transfer reagent and plasmid DNA
  • DMEM serum-free
  • Plasmid DNA (1 mg / mL) 4 4 8 8 8 8 Gene transfer reagent ( ⁇ > 12 20 40 48 40 32 Completed 3 ⁇ 4 ⁇ amount ( ⁇ ') 500 500 500 500 500 500 500
  • the reagents used are as follows: Transfectin (170-3350; BIO-RAD, USA, CA), LipofectAMINE 2000 Reagent (11668-019, Invitrogen corporation, CA) C SureFECTOR ( EM- 101 -001, B—Bridg e, USA, CA), JetPEI (X 4) cone. (101— 30, Polyplus-transfection, Fran ce), SuperFect Transfection Reagent (301305, Qiagen, CA) and Tran sIT ( MIR2304; Mirus Co., USA, WI) G
  • FIG. 8 shows that the effects of various ECM proteins do not depend on the type of gene transfer reagent, but that the specific gene transfer reagent was more effective than other groups.
  • the z axis shows the efficiency of transfection
  • the X axis shows the gene introduction reagents from various manufacturers
  • the y axis shows the various ECM proteins (from the top, type IV collagen, type I collagen, laminin, fibronectin) or their Indicates a condition that does not exist.
  • Example 9 Application example using siRNA of PC12 cell microarray on collagen IV coated chip.
  • FIG. 9A in the case of PC 12 cells co-transfected with EGFP vector and anti-EGFP siRN A, it was found that only HcRed was colored and the green signal derived from pEGFP-Nl was suppressed. .
  • FIG. 9B in the case of scrambled siRNA, green fluorescence was observed, and the effect in FIG. 9A was confirmed to be the effect of RNAi.
  • FIG. 9C The relative intensity of the fluorescence in FIGS. 9A and 9B is shown in FIG. 9C.
  • the y axis is indicated by relative luminance. It can be seen that the effect of EGFP is almost completely suppressed.
  • FIG. 10B Figure 10B (left force magnification is 50x, 200x) shows a scan-enhanced image of the separation in the transfection array (left).
  • the middle of Fig. 10B shows a photomicrograph on the third day after transfection of PC12 cells.
  • the right side of FIG. 10B shows a photograph one day after NGF induction. As shown, it was revealed that PC 12 cells were neuronally differentiated.

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Abstract

L'invention concerne un procédé pour transférer un acide nucléique (par ex. un ADN) fixé sur un support dans une cellule, dans laquelle une telle substance peut être difficilement transférée (notamment transfectée) ou dans laquelle le transfert peut être amélioré. Selon l'invention, une combinaison d'une molécule adhésive cellulaire (par ex. un collagène de type IV) et d'un réactif de transfert génétique (par ex. la lipofectamine) est appliquée à une cellule, une efficacité de transfert d'acide nucléique étonnamment élevée pouvant ainsi être obtenue. En outre, après ce transfert d'acide nucléique, la cellule est apte à subir l'induction de différenciation.
PCT/JP2005/011671 2004-06-25 2005-06-24 Procede et composition pour transferer de l'acide nucleique fixe a une phase solide dans une cellule WO2006001396A1 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
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
JP2011079795A (ja) * 2009-10-09 2011-04-21 Osaka Univ Ｉ型−ｉｖ型コラーゲン混成ゲル
JP2014176308A (ja) * 2013-03-13 2014-09-25 Mitsubishi Rayon Co Ltd マイクロアレイ、その製造方法、及びその用途

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JP5789927B2 (ja) * 2010-07-30 2015-10-07 Jnc株式会社 放射線照射コラーゲン様ペプチドを用いた核酸導入法

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WO2003000297A1 (fr) * 2001-06-20 2003-01-03 Sumitomo Pharmaceuticals Company, Limited Procede facilitant le transfert d'acides nucleiques

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Cited By (3)

* Cited by examiner, † Cited by third party
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
JP2011079795A (ja) * 2009-10-09 2011-04-21 Osaka Univ Ｉ型−ｉｖ型コラーゲン混成ゲル
JP2014176308A (ja) * 2013-03-13 2014-09-25 Mitsubishi Rayon Co Ltd マイクロアレイ、その製造方法、及びその用途

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