JP4161570B2 - Methods for testing the ability to control neuronal plasticity - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for assaying the ability to control neuronal plasticity and the like.
[0002]
[Prior art]
The cranial nerve function is built on the neural circuit made by a wide variety of nerve cells. This complex and accurate network is formed by the nerve axon being accurately guided to the target cell and synapse with the correct target cell. In this induction / connection process, nerve axon elongation is controlled (promotion, inhibition, attraction, repulsion, etc.).
Recent studies have shown that the decline in cognitive ability associated with normal aging is due to nerve dysfunction rather than the loss of neurons and synapses. Such nerve dysfunction has some structural plasticity (remodeling: hereinafter also referred to as nerve cell plasticity) of two types of projections (neuron dendrites and nerve axons) possessed by neurons. This is thought to be caused by the failure to maintain the normal state due to the cause. Similarly, abnormalities in neuronal plasticity are considered to be one of the etiologies in mental retardation, cognitive dysfunction due to Alzheimer's disease, and the like.
Therefore, by properly controlling the plasticity of nerve cells, it becomes possible to effectively improve cognitive dysfunction due to aging, mental retardation, cognitive dysfunction due to Alzheimer's disease, and the like.
[0003]
[Problems to be solved by the invention]
Therefore, there is a demand for the development of a drug for controlling nerve cell plasticity in mammals. In order to search for a substance that can be used as an active ingredient of such a drug, a method for testing the ability to control nerve cell plasticity Development was desired.
[0004]
[Means for Solving the Problems]
As a result of intensive studies under such circumstances, the present inventors have expressed the expression of proteins that have been reported to be involved in the control of neuronal plasticity, such as Eph A receptor and Rho GDP dissociation inhibitor (or Rho GTPase inhibitor). The present inventors have found that a certain type of transcriptional regulatory factor controls and have reached the present invention.
That is, the present invention
1. A method for testing an ability to control neuronal plasticity dependent on a transcriptional regulatory factor having any of the following amino acid sequences (hereinafter sometimes referred to as the present amino acid sequence):
(1) a first step of bringing a test substance into contact with a mammalian cell expressing the transcription regulatory factor; and
(2) After the first step, the expression level of the marker protein gene present on the transcription regulatory factor-dependent neuronal plasticization pathway in the mammalian cell or an index value correlated with the amount is measured. Two steps, and
(3) a step of evaluating the ability of the test substance based on the expression level measured in the second step or the index value having a correlation with the amount,
(Hereinafter sometimes referred to as the present invention assay method),
<Amino acid sequence group>
(A) the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3,
(B) an amino acid sequence of a protein having an amino acid sequence showing 90% or more amino acid identity to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 and having transcriptional regulatory ability;
(C) It has an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence represented by base numbers 102 to 2507 of the base sequence represented by SEQ ID NO: 4, and transcriptional regulation An amino acid sequence of a protein having
(D) It has an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence represented by base numbers 51 to 2456 of the base sequence represented by SEQ ID NO: 5 and transcriptional regulation An amino acid sequence of a protein having
(E) It has an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence represented by base numbers 35 to 2440 of the base sequence represented by SEQ ID NO: 6, and transcriptional regulation An amino acid sequence of a protein having an ability;
2. A method for assaying the ability to control neuronal plasticity dependent on a transcriptional regulator having this amino acid sequence,
(1) a first step of bringing a test substance into contact with a transformed mammalian cell into which a gene having a base sequence encoding this amino acid sequence has been introduced; and
(2) After the first step, the expression level of the marker protein gene present on the transcription regulatory factor-dependent nerve cell plasticizing pathway or the index value correlated with the amount is measured in the transformed mammalian cell A second step, and
(3) a step of evaluating the ability of the test substance based on the expression level measured in the second step or the index value having a correlation with the amount,
An assay method characterized by comprising:
3. The assay method according to item 1 or 2, wherein the marker protein gene is an Eph A receptor gene or a Rho GDP dissociation inhibitor gene;
4). The assay method according to item 1 or 2, wherein the marker protein gene is an Eph A receptor gene and a Rho GDP dissociation inhibitor gene;
5. The test substance has the expression level of the marker protein gene or the difference obtained by comparing the index value correlated with the expression level in the group using two or more different substances as the test substance independently. 3. The test method according to item 1 or 2, wherein the ability is evaluated.
6). 6. The assay method according to item 5 above, wherein at least one of the two or more different substances is a substance not having the ability;
7). A method for searching for a substance having the ability to control neuronal plasticity dependent on a transcriptional regulatory factor having this amino acid sequence, wherein the substance has the ability based on the ability evaluated by the assay method according to item 1 or 2. A search method characterized by selecting (hereinafter also referred to as the present invention search method);
8). A nerve cell comprising a substance selected by the searching method according to the preceding item 7 or a pharmaceutically acceptable salt thereof as an active ingredient, wherein the active ingredient is formulated into a pharmaceutically acceptable carrier. A plasticity modifier (hereinafter sometimes referred to as the present nerve cell plasticity regulator);
9. Use of DNA having a base sequence encoding this amino acid sequence for controlling neuronal plasticity in mammalian cells;
10. Transcription regulator-dependent neuronal plasticization pathway having any of the following amino acid sequences in the cell by providing the foreign DNA to the mammalian cell so that the DNA is placed in a position where the DNA is expressed in the cell Use of DNA having a base sequence encoding any one of the following amino acid sequences as foreign DNA for controlling the expression of the marker protein gene present above;
Etc. are provided.
[0005]
DETAILED DESCRIPTION OF THE INVENTION
The present invention is described in detail below.
In the present invention, “a transcriptional regulatory factor having any of the following amino acid sequences” (hereinafter sometimes referred to as the present transcriptional regulatory factor) is a basic helix-loop-helix: (Hereinafter referred to as bHLH.) A protein having a motif and a PAS domain (Per-Arnt-Sim homology domain), which forms a heterodimer and binds to DNA having a specific base sequence to regulate transcription It is a protein that functions as a factor and is involved in the control of neuronal plasticity.
As used herein, “neural cell plasticity dependent on a transcriptional regulatory factor having any of the following amino acid sequences (ie, the present amino acid sequence)” refers to a transcriptional regulatory factor having the present amino acid sequence (ie, the present transcriptional regulatory factor). ) Is involved, and is subject to control when cascade reactions such as metabolic processes and signal transduction involving the transcription factor occur. Such a cascade reaction is described as “the transcription regulatory factor-dependent neuronal plasticization pathway (ie, having the present amino acid sequence)” in the present invention. In addition, a protein that is included in the pathway and is subject to expression regulation, and that can be used as a measure of neuronal plasticity in the present invention is referred to as “depending on the transcription regulator (that has this amino acid sequence). -"Marker protein present on the nerve cell plasticization pathway". Specifically, for example, Eph A receptor, Rho GDP dissociation inhibitor (or Rho GTPase inhibitor) and the like can be mentioned.
The Eph A receptor is a tyrosine kinase-type cell membrane receptor and is widely known to be involved in brain development. Recently, it has been shown that Eph A receptor is expressed in the adult brain, particularly the hippocampus, and observation of LTP (long-term enhancement of synaptic transmission efficiency) and behavioral changes when agonists and antagonists of the receptor are administered to the hippocampus. Several studies have led to evidence that the receptor is involved in synaptic plasticity, learning and memory. It has also been observed that the receptor is involved in memory enhancement.
Rho is one of intracellular signaling substances, and is a protein that is widely known to be involved in control of cell proliferation, regulation of cell adhesion formation, structure formation of the actin cytoskeleton, and the like. As a factor that controls Rho activation, there is a regulatory factor (inhibitory protein) called Rho GDP dissociation inhibitor (or Rho GTPase inhibitor). Rho GDP dissociation inhibitor forms a complex with Rho's GDP binding type and prevents Rho from becoming GTP binding type (Rho active type), and thus inhibits Rho activation (Rho GTPase activity). Rho GDP dissociation inhibitors are thought to regulate neuronal plasticity by controlling Rho GTPase activity as described above.
[0006]
The “transcriptional regulatory factor having any of the following amino acid sequences (ie, the present amino acid sequence) (ie, the present transcriptional regulatory factor)” used in the assay method of the present invention is represented by any one of SEQ ID NOs: 1 to 3. A protein having the amino acid sequence (that is, the present amino acid sequence) (here, the protein having the present amino acid sequence represented by SEQ ID NO: 1 is a human transcriptional regulatory factor, and the present amino acid sequence represented by SEQ ID NO: 2) And the protein having the amino acid sequence represented by SEQ ID NO: 3 is a rat-derived transcriptional regulatory factor derived from a rat). A protein having an amino acid sequence showing 90% or more amino acid identity with respect to the amino acid sequence shown in FIG. A protein having an amino acid sequence encoded by DNA that hybridizes under stringent conditions with a DNA comprising the base sequence represented by the numbers 102 to 2507, and having transcriptional regulatory ability, a base sequence represented by SEQ ID NO: 5 A protein having an amino acid sequence encoded by a DNA that hybridizes under stringent conditions with a DNA consisting of the base sequence represented by base numbers 51 to 2456 of FIG. A protein having an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence represented by base numbers 35 to 2440 of the base sequence and having transcriptional regulation ability is included. Such a protein preferably has a molecular weight of about 80,000 to 100,000 as a molecular weight in SDS-PAGE.
Examples of the difference from the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 found in the amino acid sequence of this transcriptional regulatory factor include amino acid deletion, substitution, modification, addition and other mutations. These include naturally occurring mutations such as differences in amino acid sequences due to differences in animal strains, individuals, organs, tissues, etc., in addition to mutations that can be artificially introduced by site-specific mutagenesis or mutation treatment. Type mutations are also included.
[0007]
In the present invention, “sequence identity” refers to the identity and homology of two base sequences or two amino acid sequences. The “sequence identity” is determined by comparing two sequences that are optimally aligned over the entire region of the sequence to be compared. Here, addition or deletion (for example, a gap) may be allowed in the optimal alignment of the base sequence or amino acid sequence to be compared. Such sequence identity can be determined, for example, by FASTA [Pearson & Lipman, Proc. Natl. Acad. Sci. USA, 4, 2444-2448 (1988)], BLAST [Altschul et al., Journal of Molecular Biology, 215, 403- 410 (1990)], CLUSTAL W [Thompson, Higgins & Gibson, Nucleic Acid Research, 22, 4673-4680 (1994a)] and the like. The above program is, for example, DNA Data Bank of Japan [International DNA Data Bank operated within the Center for Information Biology and DNA Data Bank of Japan (CIB / DDBJ)] Is generally available on the homepage (http://www.ddbj.nig.ac.jp). Sequence identity can also be determined using commercially available sequence analysis software such as Vector NTI, GENETYX-WIN Ver.5 (Software Development Co., Ltd.).
The amino acid identity in the present invention is preferably 90% or more, for example.
[0008]
In addition, as the above-mentioned “DNA that hybridizes under stringent conditions”, for example, a high ion concentration [for example, 6XSSC (900 mM sodium chloride, 90 mM sodium citrate) or the like is used. Are hybridized under a temperature condition of 65 ° C. to form a DNA-DNA hybrid, and a low ion concentration [for example, 0.1 × SSC (15 mM sodium chloride, 1.5 mM sodium citrate) or the like is used. The DNA that can maintain the hybrid even after washing for 30 minutes at a temperature of 65 ° C. can be mentioned. The transcriptional regulatory ability of the transcriptional regulatory factor can be evaluated based on, for example, an assay using a reporter gene described below.
[0009]
A gene having a base sequence encoding the amino acid sequence of the transcriptional regulatory factor (hereinafter referred to as the transcriptional regulatory factor gene) is obtained from, for example, J. Sambrook, EFFrisch, T from animal tissues such as humans, mice, and rats. By Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (published by Cold Spring Harbor Laboratory, 1989), etc.
Specifically, first, total RNA derived from animal tissues such as humans, mice, and rats is prepared. For example, brain tissue is pulverized in a solution containing a protein denaturant such as guanidine hydrochloride or guanidine thiocyanate, and the protein is denatured by adding phenol, chloroform or the like to the pulverized product. After removing the denatured protein by centrifugation or the like, total RNA is extracted from the collected supernatant fraction by a method such as guanidine hydrochloride / phenol method, SDS-phenol method, guanidine thiocyanate / CsCl method or the like. An example of a commercially available kit based on these methods is ISOGEN (manufactured by Nippon Gene). Using the obtained total RNA as a template, an oligo dT primer is annealed to the poly A sequence of RNA, and single-stranded cDNA is synthesized by reverse transcriptase. Next, double-stranded cDNA is synthesized using E. coli DNA polymerase I using the synthesized single-stranded cDNA as a template and RNA obtained by adding nicks and gaps to the RNA strand using E. coli RNaseH. To do. Furthermore, both ends of the synthesized double-stranded cDNA are smoothed with T4 DNA polymerase. The double-stranded cDNA with blunt ends is purified and recovered by conventional methods such as phenol-chloroform extraction and ethanol precipitation. Examples of commercially available kits based on these methods include cDNA synthesis system plus (Amersham Pharmacia Biotech) and TimeSaver cDNA synthesis kit (Amersham Pharmacia Biotech). Next, the obtained double-stranded cDNA is ligated with a vector such as plasmid pUC118 or phage λgt10 using a ligase to prepare a cDNA library. In addition, as a cDNA library, it is also possible to use a commercially available cDNA library (GIBCO-BRL, Clontech, etc.).
In addition, from tissue pieces of animals such as humans, mice, and rats, for example, by J. Sambrook, EFFrisch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (Cold Spring Harbor Laboratory published) 1989), Masamura Muramatsu, “Lab Manual Genetic Engineering” (Maruzen 1988), etc., to prepare genomic DNA. For example, in the case where the sample is hair, two to three hairs are washed with sterilized water and then with ethanol, and then cut into a length of 2 to 3 mm, and then BCL-Buffer [10 mM Tris-HCl (pH 7.5 ), 5 mM MgCl ₂ , 0.32M Sucrose, 1 Triton X-100], 200 μl, and proteinase K to a final concentration of 100 μl / ml and SDS to a final concentration of 0.5 (w / v) are added and mixed. This mixture is incubated at 70 ° C. for 1 hour, and then subjected to phenol / chloroform extraction to obtain genomic DNA. When the sample is peripheral blood, genomic DNA can be obtained by treating the sample with a DNA-Extraction kit (Stratagene) or the like. A genomic DNA library is obtained by ligating the obtained genomic DNA with a vector such as λgt10 using a ligase. As a genomic DNA library, a commercially available genomic DNA library (manufactured by Stratagene, etc.) can also be used.
[0010]
From the cDNA library or genomic DNA library as described above, for example, a partial base sequence of the base sequence represented by any of SEQ ID NOs: 4, 5, 6 or 33 (or 34) or complementary to the partial base sequence Polymerase chain reaction (hereinafter referred to as PCR) using an oligonucleotide having a base sequence as a primer, a base sequence represented by any of SEQ ID NOs: 4, 5, 6 or 33 (or 34) or a portion of the base sequence The transcriptional regulatory factor gene can be obtained by a hybridization method using DNA having a base sequence as a probe.
As a primer used for PCR, for example, an oligonucleotide having a length of about 10 bases to about 50 bases having a base sequence represented by any of SEQ ID NOs: 4, 5, 6 or 33 (or 34) An oligonucleotide having a base sequence selected from the 5 ′ untranslated region and a base sequence selected from the 3 ′ untranslated region of the base sequence represented by any of SEQ ID NOs: 4, 5, 6 or 33 (or 34) Examples include oligonucleotides having a complementary base sequence. Specifically, examples of the forward primer include an oligonucleotide having a base sequence represented by SEQ ID NO: 7 and an oligonucleotide having a base sequence represented by SEQ ID NO: 8. In addition, examples of the reverse primer include an oligonucleotide having a base sequence represented by SEQ ID NO: 9 and an oligonucleotide having a base sequence represented by SEQ ID NO: 10. The PCR conditions include, for example, 5 μl of 10-fold concentrated buffer for LA-Taq polymerase (Takara Shuzo), 2.5 mM dNTP mixture (each containing 2.5 mM dATP, dGTP, dCTP and dTTP in 50 μl of the reaction solution) .) 5 μl (final concentrations of dATP, dGTP, dCTP and dTTP are each 0.25 mM), 20 μM primer 0.25 to 1.25 μl each (final concentration is 0.1 to 0.5 μM), template cDNA 0.1 to 0.5 μg, LA-Taq polymerase (Takara Shuzo) For example, in a reaction solution having a composition containing 1.25 units, a temperature of 95 ° C. for 1 minute and then a temperature of 68 ° C. for 3 minutes is set as one cycle, and this is performed for 35 cycles.
[0011]
Examples of probes used in the hybridization method include DNA consisting of the base sequence represented by base numbers 102 to 2507 of the base sequence represented by SEQ ID NO: 4, and base numbers 51 to 2456 of the base sequence represented by SEQ ID NO: 5. DNA consisting of the base sequence represented by SEQ ID NO: 6, DNA comprising the base sequence represented by base numbers 35 to 2440 of the base sequence represented by SEQ ID NO: 6, and base numbers 1419 to 6164 of the base sequence represented by SEQ ID NO: 33 DNA having a base sequence to be prepared, or DNA having a partial base sequence of these DNAs. As hybridization conditions, for example, 6 × SSC (0.9 M sodium chloride, 0.09 M sodium citrate), 5 × Denhardt's solution (0.1 (w / v) Ficoll 400, 0.1 (w / v) polyvinylpyrrolidone, 0.1 ( w / v) BSA), 0.5 (w / v) SDS and 100 μg / ml denatured salmon sperm DNA, incubated at 65 ° C., then 1 × SSC (0.15 M sodium chloride, 0.015 M sodium citrate) and 0.5 (w / v) Incubate twice at room temperature for 15 minutes in the presence of SDS, and further in the presence of 0.1 × SSC (0.015M sodium chloride, 0.0015M sodium citrate) and 0.5 (w / v) SDS, 68 An example of the condition is that the temperature is kept at 30 ° C. for 30 minutes. Further, for example, 5x SSC, 50 mM HEPES pH 7.0, 10x Denhardt solution and 20 μg / ml denatured salmon sperm DNA were incubated at 65 ° C, then incubated in 2x SSC at room temperature for 30 minutes, and further 0.1x SSC Among them, the conditions for carrying out the heat insulation at 65 ° C. for 40 minutes twice may be mentioned.
The transcription regulatory factor gene can be obtained, for example, based on the nucleotide sequence represented by any of SEQ ID NOs: 4, 5, 6 or 33 (or 34), for example, the phosphite triester method (Hunkapiller, M. et al. , Nature, 310, 105, 1984) and the like, and can also be prepared by performing chemical synthesis of nucleic acids.
[0012]
The transcription factor gene thus obtained is, for example, published by J. Sambrook, EFFrisch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory And can be cloned into a vector according to the genetic engineering method described in 1989 and the like. Specifically, for example, cloning can be performed using a commercially available plasmid vector such as TA cloning kit (Invitrogen) or pBluescriptII (Stratagene).
The base sequence of the transcription factor gene thus obtained is determined by the Maxam Gilbert method (for example, described in Maxam, AM & W. Gilbert, Proc. Natl. Acad. Sci. USA, 74, 560, 1977) or the Sanger method ( (For example, it is described in Sanger, F. & ARCoulson, J. Mol. Biol., 94, 441, 1975, Sanger, F, & Nicklen and ARCoulson., Proc. Natl. Acad. Sci. USA, 74, 5463, 1977, etc.) Etc. can be confirmed.
Specific examples of the transcription regulatory factor gene include DNA having the base sequence represented by base numbers 102 to 2507 of the base sequence represented by SEQ ID NO: 4, and base numbers 51 to 2456 of the base sequence represented by SEQ ID NO: 5. DNA having the nucleotide sequence represented, nucleotide sequence represented by nucleotide numbers 35 to 2440 of the nucleotide sequence represented by SEQ ID NO: 6, nucleotide sequence represented by nucleotide numbers 1419 to 6164 of the nucleotide sequence represented by SEQ ID NO: 33 And the like having DNA.
This transcriptional regulator gene is provided as a foreign DNA to the cell so that it is placed at a position where it is expressed in the mammalian cell, whereby the transcriptional regulator-dependent neuronal plasticization pathway in the mammalian cell. It can also be used to regulate the expression of the marker protein gene present above.
[0013]
The transcriptional regulatory factor gene can be propagated autonomously, including a vector (hereinafter referred to as a basic vector) that can be used in a host cell into which the gene is introduced, for example, genetic information that can be replicated in the host cell, The transcription regulator gene vector can be constructed by incorporating it into a vector having a detectable marker, which can be isolated and purified from the host cell, in accordance with ordinary genetic engineering techniques.
Specific examples of basic vectors that can be used for the construction of this transcriptional regulator gene vector include plasmid pUC119 (Takara Shuzo), phagemid pBluescriptII (Stratagene), etc., when Escherichia coli is used as a host cell. be able to. When budding yeast is used as a host cell, plasmids pGBT9, pGAD424, pACT2 (manufactured by Clontech) and the like can be mentioned. When mammalian cells are used as host cells, plasmids such as pRc / RSV and pRc / CMV (Invitrogen), bovine papillomavirus plasmid pBPV (Amersham Pharmacia Biotech) or EB virus plasmid pCEP4 (Invitrogen) Examples include vectors containing autonomous origins of replication such as viruses, viruses such as vaccinia viruses, and the like, and insect viruses such as baculoviruses when insect animal cells are used as host cells. In order to incorporate this transcription regulatory factor gene into a virus such as baculovirus or vaccinia virus, a transfer vector containing a base sequence homologous to the genome of the virus to be used can be used. Specific examples of such transfer vectors include pVL1392, pVL1393 (Smith, GE, Summers MD et al .: Mol. Cell. Biol., 3, 2156-2165 (1983)), pSFB5 commercially available from Pharmingen. (Funahashi, S. et al .: J. Virol., 65, 5584-5588 (1991)). When the transcription regulator gene is inserted into the transfer vector as described above, and the transfer vector and the viral genome are simultaneously introduced into a host cell, homologous recombination occurs between the transfer vector and the viral genome, and the transcription is performed. It is possible to obtain a virus in which a regulator gene is embedded in the genome. As the viral genome, genomes such as Baculovirus, Adenovirus, and Vacciniavirus can be used.
More specifically, for example, when the transcriptional regulatory factor gene is incorporated into a baculovirus, the transcriptional regulatory factor gene is inserted into a multicloning site such as transfer vectors pVL1393 and pVL1392, and then the DNA of the transfer vector and Baculovirus genome DNA ( Baculogold (manufactured by Pharmingen) is introduced into insect cell strain Sf21 (available from ATCC) by the calcium phosphate method or the like, and the resulting cells are cultured. Virus containing the genome of the virus into which the transcription factor gene has been inserted is recovered from the culture solution by centrifugation or the like, and the virus particles containing the transcription factor gene are removed by deproteinization with phenol or the like. The genome of can be obtained. Furthermore, by introducing the genome of the virus into a host cell having the ability to form virus particles such as an insect cell strain Sf21 by the calcium phosphate method or the like, and culturing the resulting cells, virus particles containing the transcription regulator gene are obtained. Can be increased.
On the other hand, the transcription factor gene can be directly integrated into a relatively small genome such as a murine leukemia retrovirus without using a transfer vector. For example, the viral vector-DC (X) (Eli Gilboa et al., BioTechniques, 4, 504-512 (1986)) or the like incorporates the transcription regulatory gene into the cloning site on the vector. By introducing the obtained viral vector into which the transcription factor gene is incorporated into a packaging cell such as Ampli-GPE (J. Virol., 66, 3755 (1992)), Viral particles containing the inserted viral genome can be obtained.
[0014]
The transcription regulator gene is expressed in the host cell by binding a promoter capable of functioning in the host cell upstream of the transcription regulator gene and incorporating it into the basic vector as described above. The present transcription regulator gene vector can be constructed. Here, “to bind in a functional manner” means that the transcription factor gene and the transcription factor are expressed in the host cell into which the transcription factor gene is introduced so that the transcription factor gene is expressed under the control of the promoter. It means to bind a regulator gene. Examples of a promoter that can function in a host cell include DNA that exhibits promoter activity in the host cell into which it is introduced. For example, when the host cell is Escherichia coli, the E. coli lactose operon promoter (lacP), tryptophan operon promoter (trpP), arginine operon promoter (argP), galactose operon promoter (galP), tac promoter, T7 Promoters, T3 promoters, λ phage promoters (λ-pL, λ-pR), and the like. When the host cell is an animal cell or fission yeast, for example, the rous sarcoma virus (RSV) promoter, site Examples include megalovirus (CMV) promoter, simian virus (SV40) early or late promoter, mouse papilloma virus (MMTV) promoter, and the like. In the case where the host cell is budding yeast, ADH1 promoter) and the like can be mentioned.
In addition, when using a basic vector that already has a promoter that functions in the host cell, this transcription is downstream of the promoter so that the promoter possessed by the vector and the transcription factor gene are linked in a functional manner. What is necessary is just to insert a regulator gene. For example, the plasmids pRc / RSV, pRc / CMV, etc. described above have a cloning site downstream of a promoter that can function in animal cells, and the transcription regulator gene is inserted into the cloning site and introduced into animal cells. Thus, the transcription factor gene can be expressed. Since these plasmids have SV40 autonomous replication origin (ori) incorporated in advance, when the plasmid is introduced into cultured cells transformed with the SV40 genome lacking ori, such as COS cells, The number of copies of the plasmid is greatly increased, and as a result, the present transcription regulator gene incorporated into the plasmid can be expressed in a large amount. The yeast plasmid pACT2 has an ADH1 promoter, and if the transcription regulator gene is inserted downstream of the ADH1 promoter of the plasmid or its derivative, the transcription regulator gene can be expressed, for example, by CG1945 (manufactured by Clontech). This transcriptional regulatory factor gene vector that can be expressed in a large amount in budding yeast such as
[0015]
A transformant can be obtained by introducing the constructed transcription regulator gene vector into a host cell. As a method for introducing the present transcription regulatory factor gene vector into a host cell, a normal introduction method corresponding to the host cell can be applied. For example, when Escherichia coli is used as a host cell, J. Sambrook, EFFrisch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory, published in 1989, etc. Conventional methods such as the calcium chloride method and electroporation method described can be used. In addition, when a mammalian cell or insect animal cell is used as a host cell, the cell is introduced into the cell according to a general gene transfer method such as a calcium phosphate method, a DEAE dextran method, an electroporation method, or a lipofection method. can do. When yeast is used as a host cell, it can be introduced using, for example, a Yeast transformation kit (manufactured by Clontech) based on the lithium method.
When a virus is used as a vector, the virus genome can be introduced into a host cell by a general gene transfer method as described above, and a virus particle containing the virus genome into which the transcription factor gene is inserted. The virus genome can also be introduced into a host cell by infecting the host cell.
[0016]
In order to select the transformant, for example, a marker gene may be introduced into a host cell simultaneously with the transcription regulatory factor gene vector, and the cell may be cultured by a method according to the property of the marker gene. For example, when the marker gene is a gene that confers drug resistance to a selected drug exhibiting lethal activity in the host cell, the host cell into which the transcription factor gene vector is introduced using a medium to which the drug is added Can be cultured. Examples of combinations of a drug resistance-conferring gene and a selective drug include, for example, a combination of a neomycin resistance-conferring gene and neomycin, a combination of a hygromycin resistance-conferring gene and hygromycin, and a blasticidin S resistance-conferring gene and blasticidin S. Combinations can be given. In addition, when the marker gene is a gene that complements the auxotrophy of the host cell, the cells into which the transcription regulator gene vector has been introduced may be cultured using a minimal medium that does not contain the nutrient.
In order to obtain a transformant in which the transcription regulator gene is introduced into the host cell chromosome, for example, the transcription regulator gene vector and a vector having a marker gene are linearized by digestion with a restriction enzyme or the like. Then, these are introduced into a host cell by the above-described method, and the cell is usually cultured for several weeks, and the target transformant is selected and obtained using the expression of the introduced marker gene as an index. . In addition, for example, the transcription regulator gene vector having a gene for imparting resistance to a selective drug as described above as a marker gene is introduced into a host cell by the above-described method, and the cell is cultured in a medium to which the selective drug is added for several weeks or more. By subculturing and purifying the selected drug-resistant clones that have survived in a colony state, a transformant in which the transcription regulatory factor gene is introduced into the host cell chromosome can also be selected and obtained. In order to confirm that the introduced transcription factor gene has been integrated into the host cell chromosome, the genomic DNA of the cell is prepared according to the usual genetic engineering method, and introduced from the prepared genomic DNA. This transcription regulator gene can be obtained using a method such as PCR using primers having oligonucleotides having a partial base sequence of the present transcription regulator gene as primers and Southern hybridization using the introduced transcription regulator gene as a probe. What is necessary is just to detect presence of. Such a transformant can be cryopreserved and can be used after sleeping, if necessary, so that it is possible to save time and effort for preparing the transformant for each experiment. It becomes possible to carry out the test using the confirmed transformant.
[0017]
The transcriptional regulatory factor can be produced by culturing the transformant obtained as described above.
For example, when the transformant is a microorganism, the transformant is cultured using various media appropriately containing a carbon source, a nitrogen source, an organic or inorganic salt, etc., used for normal culture in general microorganisms. be able to. The culture is carried out according to the usual method for general microorganisms, and solid culture, liquid culture (swivel shaking culture, reciprocating shaking culture, JarFermenter culture, tank culture, etc.) are possible. The culture temperature and the pH of the medium can be appropriately selected from the range in which the microorganism grows. For example, the culture is generally performed at a culture temperature of about 15 ° C. to about 40 ° C. and a medium pH of about 6 to about 8. It is. The culture time varies depending on various culture conditions, but is usually about 1 day to about 5 days. Has an inducible promoter such as a temperature shift type or IPTG inductive type
When the expression vector is used, the induction time is preferably within one day, usually several hours.
In addition, when the transformant is an animal cell such as a mammal or an insect, the transformant can be cultured using a medium used for normal culture in general cultured cells. When the transformant is produced using a selective drug, it is desirable to culture in the presence of the selective drug. In the case of mammalian cells, for example, using a DMEM medium (Nissui, etc.) supplemented with FBS to a final concentration of 10% (w / v), 37 ° C, 5% CO ₂ Incubate while changing to a new culture solution every few days under the conditions such as the presence. Once the cells have grown to confluence, add PBS solution with trypsin so that the concentration is about 0.25% (w / v), for example, and disperse the cells in several times. Continue. Similarly, in the case of insect animal cells, for example, using a culture solution for insect cells such as Grace's medium containing 10% (v / v) FBS and 2% (w / v) Yeastlate at a culture temperature of 25 ° C to 35 ° C. What is necessary is just to culture. At this time, in the case of cells that are easily detached from the petri dish such as Sf21 cells, subculture can be performed by dispersing by pipetting without using a trypsin solution. In the case of a transformant containing a viral vector such as baculovirus, the culture time is preferably set to 72 hours after the virus infection, for example, before the cytoplasmic effect appears and the cells die.
The transcriptional regulatory factor produced by the transformant can be appropriately recovered by a combination of ordinary isolation and purification methods. For example, after completion of the culture, the transformant cells are centrifuged or the like. After the collected cells are suspended in a normal buffer, they are disrupted with polytron, sonication, Dounce homogenizer, etc., and the disrupted solution is centrifuged to collect the supernatant fraction. A fraction containing the factor can be obtained. Furthermore, the purified transcription factor can be recovered by subjecting the supernatant fraction to various types of chromatography such as ion exchange, hydrophobicity, gel filtration, and affinity. Further, when the polypeptide having the transcriptional regulatory factor or a partial amino acid sequence thereof is produced as a fusion protein with GST, it can be purified by affinity chromatography using glutathione sepharose (manufactured by Amersham Pharmacia). .
The transcriptional regulatory factor thus produced can be used, for example, as an immunizing antigen for producing an antibody that recognizes the transcriptional regulatory factor or a polypeptide having a partial amino acid sequence thereof. It can also be used in assays for screening substances that bind to factors.
[0018]
Using the transcriptional regulatory factor produced as described above as an immunizing antigen, for example, animals such as mice, rabbits, chickens, etc., described by Frederick M. Ausubel et al .; Short Protocols in Molecular Biology 3nd Edition, John Wiley & Sons, Inc. By immunizing according to an immunological method, an antibody that recognizes a polypeptide having the present transcription regulatory factor or a partial amino acid sequence thereof can be prepared. Specifically, for example, first, the transcriptional regulatory factor used as an antigen and complete Freunds adjuvant are mixed and emulsified. The resulting emulsion is administered subcutaneously to rabbits. About 4 weeks later, the antigen mixed with incomplete Freunds adjuvant and emulsified is administered. If necessary, administration is similarly performed every two weeks. Blood is collected to prepare a serum fraction, and the antibody titer against this transcriptional regulatory factor is confirmed. By fractionating a serum fraction having an antibody titer that recognizes the transcriptional regulatory factor or a polypeptide having a partial amino acid sequence obtained in this manner according to the usual ammonium sulfate precipitation method, the transcriptional regulatory factor is obtained. Alternatively, IgG that recognizes a polypeptide having the partial amino acid sequence can be obtained.
In addition, by chemically synthesizing a polypeptide having a partial amino acid sequence of the transcriptional regulatory factor and administering it to an animal as an immunizing antigen, an antibody that recognizes the transcriptional regulatory factor or a polypeptide having the partial amino acid sequence is produced. You can also Examples of the amino acid sequence of a polypeptide used as an immunizing antigen include, for example, animal species that have as low a homology as possible with amino acid sequences of other proteins from among the amino acid sequences represented by any of SEQ ID NOs: 1 to 3, and that are immunized. An amino acid sequence having a large difference from the amino acid sequence of the transcriptional regulatory factor of the present invention is selected. A polypeptide having a length of about 10 to 15 amino acids consisting of the selected amino acid sequence is chemically synthesized according to a conventional method, crosslinked with a carrier protein such as hemocyanin of Limulus plyhemus by MBS and the like, and then the same as above. Immunize animals such as rabbits.
In this way, an antibody that recognizes the transcription regulator or a polypeptide having a partial amino acid sequence thereof can be produced.
[0019]
To test the ability to control neuronal plasticity dependent on this transcriptional regulator (hereinafter also referred to as this neural cell plasticity regulating ability), (1) mammalian cells expressing this transcriptional regulator And (2) after the first step, the expression level or amount of the marker protein gene present on the transcription regulatory factor-dependent neuronal plasticization pathway in the mammalian cell after the first step; A second step of measuring an index value having a correlation, and an assay having a step of evaluating the ability of the test substance based on the expression level measured in the second step or the index value having a correlation with the amount. The method (ie, the assay method of the present invention) can be used. At this time, for example, the expression level of the marker protein gene in the section where two or more different substances as test substances are used independently, or an index value (corresponding to the quantity) The difference is examined by comparing the measured amount). As a result, based on the obtained difference (difference between the first measurement amount and the second measurement amount), the ability of the test substance to be tested is evaluated by evaluating the ability to control the nerve cell plasticity. Based on the neuronal plasticity control ability evaluated in this way, it can be confirmed that the substance has the neuronal plasticity control ability.
Thus, the present transcription regulatory factor is used in a method for effectively analyzing the expression level of a marker protein gene present on the transcription regulatory factor-dependent neuronal plasticization pathway or an index value correlated with the amount. Can be used to test the ability of the substance to control neuronal plasticity.
[0020]
The mammalian cell used in the assay method of the present invention may be a cell isolated from a tissue, a cell forming a population having the same function / morphology, or a cell in the body of a mammal. In some cases, the cell extraction system may be used. Examples of the origin of the cells include mammals, and more specifically humans, monkeys, cows, rabbits, mice, rats, hamsters and the like.
In the first step of the assay method of the present invention, the concentration of the test substance to be contacted with the mammalian cell expressing the transcription regulatory factor is usually about 0.1 μM to about 100 μM, and preferably 1 μM to 50 μM. The time for contacting the mammalian cells with the test substance is usually about 10 minutes to 2 days, preferably about several hours to 1 day.
The environment in which the test substance is brought into contact with the mammalian cell is preferably an environment in which the vital activity of the mammalian cell is maintained. For example, an environment in which the energy source of the mammalian cell coexists can be given. Specifically, it is convenient that the first step is performed in a medium.
[0021]
Furthermore, the first step of the assay method of the present invention is, for example, a transformed mammalian cell obtained by introducing a gene having a base sequence encoding the present amino acid sequence into a mammalian cell (hereinafter referred to as the present transformed mammalian cell). In some cases, the test substance may be contacted.
In this step, the concentration of the test substance to be contacted with the transformed mammalian cell is usually about 0.1 μM to about 100 μM, and preferably 1 μM to 50 μM. The time for which the transformed mammalian cell is brought into contact with the test substance is usually preferably about 10 minutes to 2 days, more preferably about several hours to 1 day.
[0022]
The transformed mammalian cell can be prepared as follows.
By inserting the transcription factor gene into a vector that can be used in a mammalian cell into which the transcription factor gene is introduced, in such a manner that it can be expressed in a form that can be expressed by a normal genetic engineering technique. A plasmid is prepared. The promoter used here may be any promoter that can function in a mammalian cell into which the present transcription regulatory gene is introduced. For example, the SV40 virus promoter, cytomegalovirus promoter (CMV promoter), Raus Sarcoma Virus promoter (RSV) Promoter), β-actin gene promoter and the like. A commercially available vector containing such a promoter upstream of the multicloning site may be used.
Next, the plasmid is introduced into mammalian cells together with a selection marker gene. Examples of the introduction method into mammalian cells include a calcium phosphate method, an electric introduction method, a DEAE dextran method, and a micelle formation method. As the calcium phosphate method, the method described in Grimm, S. et al., Proc. Natl. Acad. Sci. USA, 93, 10923-10927, etc., as the electrophoretic method and the DEAE dextran method, Ting, AT et al., The method described in EMBO J., 15, 6189-6196 etc., and the micelle formation method is the method described in Hawkins, CJ et al., Proc. Natl. Acad. Sci. USA, 93, 13786-13790 etc. Can be mentioned. When using the micelle formation method, commercially available reagents such as Lipofectamine (manufactured by Gibco) and Fugene (manufactured by Boehringer) may be used.
The transformed mammalian cell can be obtained by culturing a mammalian cell subjected to the plasmid introduction treatment, for example, using a selection marker gene introduced together with the plasmid in a medium under selection conditions according to the selection marker gene. Cells can be selected. Further, selection may be continued to obtain the present transformed mammalian cell that has become a stable transformant in which the present transcription regulatory factor gene has been introduced into the chromosome. In order to confirm that the introduced transcriptional regulatory factor gene has been incorporated into a chromosome present in a mammalian cell, the genomic DNA of the cell is prepared according to the usual genetic engineering method, and the transcriptional regulatory gene is prepared. This transcriptional regulation in genomic DNA using PCR, which uses DNA having a partial base sequence of the factor gene as a primer, or Southern hybridization using DNA having a partial base sequence of the transcriptional regulatory factor gene as a probe What is necessary is just to detect and confirm the presence of a factor gene.
Moreover, you may prepare this transformed mammalian cell according to a normal method from the transformed non-human animal mentioned later.
[0023]
In the assay method of the present invention, as a method for measuring the expression level of the marker protein gene present on the transcription regulatory factor-dependent neuronal cell plasticization pathway or the index value correlated with the amount, for example,
(1) A method for measuring the amount of the marker protein by a radioimmunoassay (RIA) method using an antibody that specifically recognizes the marker protein, an enzyme-linked immunosorbent assay (ELISA), Western blotting, or the like. ,
(2) A method for measuring the abundance of mRNA of the marker protein in the mammalian cell as an index value having a correlation with the amount of the marker protein,
(3) Measure the expression level of the marker protein using a DNA array or DNA chip that is hybridized with the marker protein and to which an oligonucleotide capable of measuring the amount of mRNA of the marker protein is immobilized. Method,
Etc.
For the quantification of mRNA in (2) above, conventional methods such as RT-PCR and Northern hybridization (eg, Sambrook J., Frisch EF, Maniatis T., Molecular Cloning 2nd edition) And the method described in Cold Spring Harbor Laboratory press).
[0024]
As described above, the expression value of the marker protein gene or the index value having a correlation with the amount in the group using two or more different substances as test substances independently, measured by the assay method of the present invention Based on the difference obtained by comparing the above, the ability of the substance to control plasticity of the nerve cell is evaluated. In this manner, the test ability of the present nerve cell plasticity control ability possessed by the test substance can be carried out.
In the assay method of the present invention, at least one of the two or more different substances may be a substance that does not have the ability to control neuronal plasticity (for example, a solvent, a test system solution that serves as a background, or the like). ) And as a reference, this nerve cell plasticity control ability of other test substances may be evaluated. Moreover, you may evaluate this nerve cell plasticity control capability which another test substance has on the basis of this nerve cell plasticity control capability which at least 1 substance has among two or more different said substances.
When the transcriptional regulatory factor and the test substance are brought into contact, the expression level of the marker protein or an index value (hereinafter referred to as measured value 1) having a correlation with the amount is expressed as the transcriptional regulatory factor and the test substance. By comparing the expression level of the marker protein when not in contact with an index value (hereinafter referred to as measurement value 2) having a correlation with the expression level of the marker protein, You may evaluate. For example, the present nerve cell plasticity control ability can be expressed as a control rate according to the following formula using the measured value.
Control rate (%) = [(measured value 1−measured value 2) / measured value 2] × 100
[0025]
In order to search for a substance having the present nerve cell plasticity control ability, a substance having this nerve cell plasticity control ability may be selected based on the present nerve cell plasticity control ability evaluated by the assay method of the present invention.
For example, a substance having a statistically significant control rate representing the ability of the test substance to control neuronal plasticity of the test substance shows a statistically significant value, specifically preferably a substance showing, for example, 30% or more, more preferably 50 % Or more is selected as a substance having the ability to control neuronal plasticity. Here, when the control rate is positive, the substance is selected as a substance having a promoting ability, and when it is negative, the substance is selected as a substance having a suppressing ability.
The substance may be any substance such as a low molecular weight compound, a protein (including an antibody) or a peptide as long as it has the ability to control neuronal plasticity.
Substances selected by the search method of the present invention have the ability to control neuronal plasticity, and are used as active ingredients of neuronal plasticity modifiers (for example, therapeutic agents such as cognitive dysfunction-improving agents and mental retardation improving agents). May be used.
[0026]
The nerve cell plasticity regulator (ie, the nerve cell plasticity regulator of the present invention) containing a substance selected by the search method of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient is effective orally or parenterally. In particular, it can be administered to mammals such as humans. For example, when administered orally, the nerve cell plasticity regulator of the present invention can be used in a usual form such as a tablet, capsule, syrup, suspension or the like. In addition, when administered parenterally, the nerve cell plasticity regulator of the present invention can be used in the form of a usual solution such as a solution, emulsion, suspension or the like. Examples of a method for parenteral administration of the above-mentioned form of the present neuronal plasticity-controlling agent include an injection method and a suppository method for rectal administration.
In the above-mentioned appropriate dosage form, a substance selected by the search method of the present invention or a pharmaceutically acceptable salt thereof is added to an acceptable normal carrier, excipient, binder, stabilizer, diluent, etc. Can be manufactured. When used in an injection form, acceptable buffering agents, solubilizing agents, isotonic agents and the like can be added.
The dosage varies depending on the age, sex, body weight, degree of disease, type of inhibitor of the exacerbation factor of arteriosclerosis, administration form, etc. of the mammal to be administered. About 1 mg to about 2 g as an active ingredient amount per dose, preferably about 5 mg to about 1 g as an active ingredient amount may be administered, and in the case of injection, about 0.1 mg to about 500 mg as an active ingredient amount may be administered as an adult. Good. In addition, the above-mentioned daily dose can be administered once or divided into several times.
Examples of the diseases considered to be applicable to the nerve cell plasticity regulator of the present invention include diseases such as cognitive dysfunction due to the elderly, mental retardation, and cognitive dysfunction due to Alzheimer's disease.
[0027]
This transcriptional regulator gene is provided as a foreign gene to a mammalian cell so that it is placed at a position where it is expressed in the cell, whereby the transcriptional regulator-dependent neuronal plasticization in the mammalian cell It can also be used to promote the expression of a marker protein gene present on the pathway.
Examples of mammalian cells include cells derived from mammals such as humans, monkeys, mice, rats, and hamsters. The cell may be a cell isolated from a tissue, a cell forming a group having the same function / morphology, or a cell in the body of the mammal. Therefore, when the mammal is a human, it means from a human cell subjected to general gene therapy to a cell line used for various experiments, and when the mammal is a non-human animal. The term means cells from non-human animals subjected to gene therapy in general to cells of model animals and cell lines used in various experiments. In the latter case, rats, mice and the like can be mentioned as preferred animal species. Furthermore, the case where a mammalian cell is a cell in the body of a mammal that can be diagnosed as suffering from a disease with mental retardation or Alzheimer's disease can be given as a more specific example.
[0028]
Preparation of the transcription regulatory factor gene may be performed according to a method equivalent to that described above. It can also be prepared by an in vivo transcription system that expresses the transcription factor gene under the control of a highly efficient promoter (eg, human cytomegalovirus promoter). By preparing a transformed cell as described below using the prepared gene, a transformed cell provided so that the gene is placed at a position where it is expressed in a mammalian cell can be obtained.
In the expression promoting method of the present invention, “placed at the position to be expressed” means that the DNA molecule directs transcription and translation from its base sequence (that is, RNA encoding the transcription regulatory factor and the transcription regulation, for example). It means that it is located adjacent to the base sequence (which promotes the production of factors).
The expression level of the transcriptional regulatory factor may be an amount that is sufficient to control the expression of the marker protein gene as compared to cells not provided with the transcriptional regulatory factor gene. In this case, the transcription regulatory factor gene may be DNA encoding the entire transcription regulatory factor or DNA encoding a part of the protein.
In the above expression promoting method, expression of the marker protein gene may be controlled by providing the transcription regulatory factor gene to a mammalian cell so that it is integrated into the genome.
[0029]
In the above expression promoting method, the gene is introduced into a gene construct (hereinafter also referred to as the present gene construct) used for introducing the transcription regulatory factor gene into a mammalian cell and the transfer delivery means. Retroviral vectors, adenoviral vectors, adeno-associated viral vectors or other viral vectors that have an affinity for mammalian cells to be used can be used. Specifically, see, for example, Miller, Human Gene Therapy 15-14, 1990; Friedman, Science 244: 1275-1281, 1989; Eglitis and Anderson, BioTechniques 6: 608-614. 1988; Tolstoshev and Anderson, current opinion in Biotechnology 1; 55-61, 1990; Sharp, The Lancet 337: 1277-1278, 1991; Cornetta et al., Nucleic Acid Research and Molecular Biology 36: 311-322, 1987; Anderson, Science 22-: 401-409, 1984; Moen, Blood Cells 17: 407-416, 1991; Miller et al., Biotechniques 7: 980 To 990, 1989; Le Gai La Salle et al., Science 259: 988-990, 1993; and Johnson, Chest 107: 77S-83S, 1995, and the like. Rosenberg et al. Engl. J. Med 323: 370, 1990; Anderson et al., US Pat. No. 5,399,346, etc., are particularly well developed and already used in clinical settings.
Further, as a means for transferring and delivering the DNA of the present invention, a non-viral technique can also be used. See, for example, Felgner et al., Proc. Natl. Acad. Sci. USA 84: 7413, 1987; Ono et al., Neurosci. Lett. 117: 259, 1990; Brigham et al., Am. J. Med. Sci. 298: 278, 1989; Staubinger et al., Meth. Enz. 101: 512, 1983), asialosonucoid polylysine conjugation (Wu et al., J. Biol. Chem. 263: 14621, 1988; Wu et al., J. Biol. Chem. 264: 16985, 1989, etc. And the microinjection, calcium phosphate method, DEAE dextran method, electroporation method and protoplast fusion method, and liposome method described in Wolff et al., Science 247: 1465, 1990.
[0030]
In the present gene construct, the present transcription regulatory factor gene may be placed under the control of a promoter that constitutively expresses the DNA. For example, SV40 virus promoter, cytomegalovirus promoter (CMV promoter), Raus Sarcoma Virus promoter (RSV promoter), β-actin gene promoter and the like can be mentioned.
Further, the transcription regulatory factor gene may be placed under the control of a promoter that regulates the expression of the gene by environmental stimulation. For example, the gene may be expressed using a chemical signal or an exogenous signal such as drug stimulation or a promoter activated by the introduction of a drug.
In addition, the transcription regulatory factor gene may be placed under the control of a tissue-specific or cell type-specific promoter. Such a promoter may include a promoter element (enhancer or the like) involved in transcriptional control specific to a tissue or cell in a mammal. For example, if necessary, an enhancer known to preferentially direct the expression of DNA in nerve cells may be used to express the present transcription regulatory factor gene. In addition, when a clone of genomic DNA (for example, DNA having the nucleotide sequence represented by SEQ ID NO: 33 or 34) encoding the transcription regulator is used as the gene construct, the original expression of the transcription regulator gene The transcription regulatory factor gene can be expressed via a cognate regulatory sequence contained in the regulatory region, and if necessary, expression can be controlled by adding any of the promoters or promoter elements described above.
A commercially available vector containing the above promoter or promoter element upstream of the multicloning site can also be used.
[0031]
When the above expression promoting method is applied as a gene therapy means, the present gene construct is preferably applied to a site where abnormal expression of the marker protein gene is expected (for example, by injection). Further, it may be applied to a tissue in the vicinity of a site where a phenomenon such as abnormal expression of the marker protein is expected or a blood vessel supplied to a cell where abnormal expression of the marker protein is expected to occur. In addition, the gene construct is introduced into a culturable cell exogenous or endogenous to the mammal to which the gene construct is to be applied, and then the introduced cell is serologically injected into the target tissue. You can also
Ideally, such gene therapy techniques should result in an expression level of the transcriptional regulator that is at least equivalent to the intracellular level of the normal transcriptional regulator in non-affected cells.
[0032]
Below, the said invention in case a mammal is a transformed non-human animal is demonstrated in detail as an example.
Examples of the method for introducing the transcription regulatory factor gene in the production of transformed non-human animals include a microinjection method, a method using a retrovirus, a method using embryonic undifferentiated cells (ES cells), and the like. Of these, the microinjection method is most widely used. The microinjection method is a method of injecting a solution containing the DNA into the pronucleus of a fertilized egg under a microscope using a micromanipulator.
First, this transcriptional regulator gene is injected into a fertilized egg. At that time, in order to integrate the gene into the chromosome with high probability, the vector region used for isolation of the gene should be removed as much as possible, the AU-rich region contributing to mRNA instability should be removed, It is preferable to use a straight chain. Moreover, it is preferable to insert an intron in advance with respect to the gene, and examples of the intron include a β-globin intron.
A fertilized egg is collected from a non-human animal of a line according to the purpose. For example, in the case of a mouse, an inbred C57BL / 6 mouse, a C3H mouse, or a cross between a C57BL / 6 mouse and another strain of mouse (for example, (C57BL / 6 × DBA / 2) F1, etc.), Examples include outbred ICR mice. A fertilized egg is usually collected from the female mouse after mating a female mouse and a male mouse in which superovulation has been induced by intraperitoneal administration of both pregnant horse serum gonadotropin and human chorionic gonadotropin. The collected fertilized eggs are placed in a culture drop, and CO ₂ By culturing and maintaining in a gas incubator, it can be stored until the gene injection operation.
The gene is injected under an inverted microscope equipped with a micromanipulator. As a fertilized egg to be used, a fertilized egg that is in a developmental stage from when the male pronucleus becomes larger than the female pronucleus to when both pronuclei are fused may be used. First, a fertilized egg is fixed, and a DNA solution containing the gene is injected into the male pronucleus of the fertilized egg. The DNA solution is prepared as a complex as necessary. Examples of the substance used for forming the complex include liposomes, calcium phosphate, and retroviruses. The injection of the DNA solution can be confirmed by the swelling of the male pronucleus. Examples of the gene injection amount include an amount containing about 200 to about 3,000 copies of the gene.
[0033]
In this way, the fertilized egg into which the transcription factor gene is injected is cultured in the same manner as described above until it becomes a blastocyst, and then transplanted to the uterus of the temporary parent. Preferably, it is transplanted to the oviduct of the temporary parent immediately after the DNA injection operation. As a temporary parent, a female mouse mated with a male mouse subjected to vasectomy surgery to be in a pseudopregnant state may be used. Specifically, first, the skin and muscle layer near the kidney on the dorsal side of the female mouse are incised to draw out the ovary, fallopian tube, and uterus, and the ovarian membrane is broken to find the fallopian tube mouth. Next, fertilized eggs that survived the DNA injection operation are transferred from the fallopian tube mouth, the ovaries, fallopian tubes, and uterus are returned to the abdominal cavity, the muscle layer is sutured, and the skin is clipped. A baby is born about 20 days later.
A part of the body structure of the obtained pup, for example, a part of the tail, is cut out and the presence or absence of the gene is confirmed by Southern blotting of DNA extracted from the site. In this way, it can be confirmed that the gene has been introduced into a non-human animal. Alternatively, other methods such as a confirmation method such as PCR may be used.
[0034]
The transcriptional regulatory factor gene, which is the active ingredient of the transcriptional regulatory factor gene therapeutic agent, may be prepared as described above. For example, it may be used in the form of a recombinant vector or a recombinant virus containing the gene. Such forms include, for example, retrovirus vectors, adenovirus vectors, adeno-related vectors, herpes simplex virus vectors, SV40 vectors, polyoma virus vectors, papilloma virus vectors, picornavirus vectors and vaccinia virus vectors. Viral vectors can be used. Further, when an adenovirus vector is used, for example, using AdEasy Kit manufactured by QUANTUM, this transcriptional regulatory factor gene is incorporated into the multi-cloning site of Transfer Vector, and the resulting recombinant vector is linearized, followed by pAdEasy A recombinant virus containing this transcriptional regulatory factor gene can be produced by transforming it into E. coli with a vector and incorporating the homologous recombinant DNA into human 293A cells, which can also be recovered and used. A non-viral vector such as plasmid DNA having a human cytomegavirus promoter region can also be used. In a system in which the transcription regulator gene is locally delivered using a non-viral vector, such as when the transcription regulator gene is directly injected into a site where abnormal expression of the marker protein is expected, Use is extremely beneficial. Any known introduction method can be used by introducing a recombinant vector containing the transcription regulatory factor gene into cells taken out of the body and returning it to the body, that is, using an ex vivo method. For example, a) direct injection, b) transduction via liposome, c) cell transfection by calcium phosphate method, electroporation method, DEAE-dextran method, d) delivery via polybrene, e) protoplast fusion, f) micro Non-viral vectors can be introduced by injection, g) transduction using polylysine, and the like.
[0035]
An effective amount of the gene therapy agent of the present invention can be parenterally administered to mammals such as humans. For example, the parenteral administration method can include, for example, the injection (subcutaneous, intravenous, etc.) as described above. The appropriate dosage form is pharmaceutically acceptable. For example, the transcription regulator gene (this can be used in a carrier such as a water-soluble solvent, a water-insoluble solvent, a buffer, a solubilizing agent, an isotonic agent, and a stabilizer). Including recombinant vectors, recombinant viruses, recombinant plasmids and the like in which the DNA of the invention is incorporated). You may add adjuvants, such as antiseptic | preservative, a suspending agent, and an emulsifier, as needed.
The dose varies depending on the age, sex, weight, disease level, type of the fat accumulation inhibitor, dosage form, etc. of the mammal to be administered, but the protein of the present invention is usually effective intracellularly in patient cells. It is sufficient to administer the amount of the active ingredient that brings about a concentration level that works. The daily dose can be administered once or divided into several times.
[0036]
【Example】
EXAMPLES The present invention will be described below with reference to examples, but the present invention is not limited to these examples.
Example 1 (Preparation of pGEM-mNXF, a vector containing this transcriptional regulatory factor gene)
An oligonucleotide consisting of the base sequence shown in SEQ ID NO: 7 (aagcacggag gaggaagccg ccggtgcgtc gggac) and an oligonucleotide consisting of the base sequence shown in SEQ ID NO: 8 (ggagagcggc tccacgtctt gatgacaata tgcca) were each made into a DNA synthesizer (Applied Biosystems model 394) ). PCR was performed using 10 ng of mouse Brain cDNA library (# 10655-017 Gibco BRL) as a template and the polynucleotide as a primer. In the PCR, 10 pmol of the above polynucleotide was added per 50 μl of the reaction solution, and LA-Taq polymerase (Takara Shuzo) and a buffer attached to the kit containing the enzyme were used. The PCR reaction solution was kept warm for 35 cycles using PCRsystem 9700 (Applied Biosystems), with 95 ° C. for 1 minute and then 68 ° C. for 3 minutes as one cycle.
Subsequently, the entire amount of the PCR reaction solution was subjected to low melting point agarose gel electrophoresis (Agarose L: Nippon Gene). After confirming a single band of about 2.5 kb DNA, the DNA was recovered. Prepare a sample for direct sequencing using a part of the recovered DNA and Dye Terminator Sequence Kit FS (Applied Biosystems), and use this with Auto Sequencer (Applied Biosystems, Model 3700) It was subjected to direct base sequence analysis.
Next, the DNA recovered as described above (about 1 μg) and pGEM T easy vector (Promega) (10 ng) are mixed, and T4 DNA ligase is added to this mixture and reacted, whereby pGEM is reacted. -Obtained mNXF. The base sequence of the obtained pGEM-mNXF was determined by the dye terminator method using an ABI model 3700 type auto sequencer. The determined base sequence was compared with the base sequence obtained by the direct sequence, and it was confirmed that the base sequence of the translation region was completely identical.
[0037]
Example 2 (Preparation of pRC / RSV-mNXFsense and pRC / RSV-mNXFantisense)
A plasmid for expressing the full length of the transcriptional regulatory factor in mammalian cells (hereinafter sometimes referred to as the present expression plasmid) was prepared as follows.
The pGEM-mNXF obtained in Example 1 was constructed so that the initiation codon of the translation region encoding the present transcription regulatory factor derived from the mouse was located downstream of the Sp6 promoter of the commercially available pGEM vector. Therefore, using this pGEM-mNXF (1 μg) as a template, the oligonucleotide shown in SEQ ID NO: 11 (forward primer 5′-gggcgctgcagcccagccaccatgtaccgatccaccaaggg-3 ′) and the oligonucleotide shown in SEQ ID NO: 12 (reverse primer 5′-aatctcggcgttgatctggt) -3 ') as a primer, a Kozak sequence (5'-CCAGCCACC-3') immediately before the start codon of the translation region encoding this transcriptional regulator by performing PCR using KODplus polymerase (manufactured by TOYOBO) Was obtained, and DNA encoding a part of this transcriptional regulatory factor into which a PstI restriction enzyme site was further introduced was obtained. The PCR reaction was carried out for 35 cycles with 95 ° C. for 1 minute, then 55 ° C. for 30 seconds and 72 ° C. for 1 minute as one cycle.
The DNA thus obtained was cleaved with PstI and BssHII, and then purified and recovered by subjecting to low melting point agarose gel electrophoresis (Nusieve GTG agarose; manufactured by FMCbio). The purified and recovered DNA was used as the insert DNA used below. Next, the DNA obtained by cleaving pGEM-mNXF with PstI and BssHII and then BAP-treated was subjected to low melting point agarose gel electrophoresis (AgaroseL; manufactured by Nippon Gene Co., Ltd.), and the DNA was recovered. By binding the insert DNA (0.5 μg) to the recovered DNA (0.1 μg) with T4 Ligase, a Kozak sequence (5 ′-(5′−) immediately before the start codon of the translation region encoding the transcription factor derived from mouse is obtained. Plasmid pGEM-mNXF Kozak into which CCAGCCACC-3 ′) was introduced was prepared.
Next, the plasmid pGEM-mNXF Kozak was simultaneously cleaved by PstI, NotI and ScaI, and then subjected to low melting point agarose electrophoresis to recover about 2.5 kbp DNA (mNXF Kozak PstI-NotI). The recovered DNA was blunt-ended with T4 polymerase, and the resulting DNA was used as insert DNA. The DNA obtained by cleaving the plasmid pRC / RSV (manufactured by Invitorgen) having the RSV promoter with HindIII and then blunting with T4 polymerase was BAP-treated, and this was used as vector DNA. By binding the insert DNA (0.5 μg) to this vector DNA (0.1 μg) with T4 Ligase, (a) DNA in which a translation region encoding the present transcription regulatory factor derived from mouse is connected downstream of the Kozak sequence. PRC / RSV-mNXFsense, which is a plasmid in which the sense strand is expressed under the control of the RSV promoter, and (b) an anti-DNA of a translation region encoding the transcriptional regulatory factor derived from mouse downstream of the Kozak sequence. PRC / RSV-mNXFantisense, a plasmid whose sense strand is expressed under the control of the RSV promoter, was prepared. In addition, it was confirmed by examining the base sequence of the boundary region between the vector DNA and the insert DNA that the prepared plasmid had a desired structure.
[0038]
Example 3 (EphA1 expression promotion by this transcription regulatory factor)
First, about 1x10 ⁷ SK-N-MC cells (ATCC No. HTB10; purchased from Dainippon Pharmaceutical Co., Ltd.) using DMEM medium (manufactured by Nissui Pharmaceutical) containing 10% FBS at 37 ° C. with 5% CO ₂ In the presence, the cells were cultured using a petri dish (Falcon) having a diameter of about 10 cm. The next day, the cultured cells were dispersed by trypsin treatment, washed twice with DMEM medium not containing FBS, and again 1 × 10 ⁷ The cells were dispersed in a DMEM medium containing no FBS so that the cell density was increased. After 0.4 μl of this cell dispersion was mixed with 10 μg of the present expression plasmid ((a) pRC / RSV-mNXFsense or (b) pRC / RSV-mNXFantisense) prepared in Example 2, this mixture was electroporated. The sample was transferred to a cuvette for transfection, and transfection was carried out under conditions of 200 V and 950 μF by electroporation using Gene Pulser (manufactured by BIORAD). After transfection, the medium was replaced with DMEM medium containing 10% FBS, and further cultured in a 10 cm petri dish for about 24 hours. After culturing, using 5 cells of petri dish, DNA is not included using Atlas Pure Total RNA Labeling system (K1038-1; manufactured by Clontech), a commercially available kit for RNA purification and radioactivity labeling. Total RNA was purified. The RNA yield is 23 μg when the plasmid (a) (ie, pRC / RSV-mNXFsense) is used, and 26 μg when the plasmid (b) (ie, pRC / RSV-mNXFantisense) is used. Met. Next, using the obtained RNA as a material, using the specific primer and reverse transcriptase included in the kit, [α-P ³² ] Each RNA was radiolabeled with -dATP (Amersham Pharmacia). Next, radiolabeled RNA (hereinafter referred to as probe) was purified using the above kit, and then 1.3 × 10 5 of the purified RNA. ^Five The DPM equivalent was used for the following hybridization. Hybridization was performed using (a) pRC / RSV using a kit containing a nylon membrane on which various genes were blotted (Atlas cDNA Expression array-Neurobiology; 7736-1 Clontech) and a hybridization buffer attached to the kit. -Probes derived from mNXFsense-introduced cells or (b) probes derived from pRC / RSV-mNXFantisense-introduced cells were added, and the reaction was performed under the same conditions for 18 hours. After hybridization, each nylon membrane was washed with 2XSSC, 1% SDS buffer (68 ° C., 30 minutes). This was repeated 4 times, and further washed with 0.1XSSC and 0.5% SDS buffer (68 ° C., 30 minutes). Each nylon membrane is wrapped in plastic wrap, exposed to an IP plate (Fuji Film) for 7 days, and then hybridization signals corresponding to various genes on the nylon membrane using an imaging analyzer (BASstation; Fujifilm) Quantification and comparison of the strength of were performed.
As a result, for the EphA1 gene, (a) the hybridization signal on the nylon membrane hybridized with the probe derived from pRC / RSV-mNXFsense-introduced cells hybridized with the probe derived from (b) pRC / RSV-mNXFantisense-introduced cells. It was detected significantly stronger than the hybridization signal on the nylon membrane. Thus, it was confirmed that this transcriptional regulatory factor has the ability to promote the expression of EphA1.
[0039]
Example 4 (Inhibition of Rho GDP dissociation inhibitor expression by this transcriptional regulatory factor)
First, about 1x10 ⁷ SK-N-MC cells (ATCC No. HTB10; purchased from Dainippon Pharmaceutical Co., Ltd.) using DMEM medium (manufactured by Nissui Pharmaceutical) containing 10% FBS at 37 ° C. with 5% CO ₂ In the presence, the cells were cultured using a petri dish (Falcon) having a diameter of about 10 cm. The next day, the cultured cells were dispersed by trypsin treatment, washed twice with DMEM medium not containing FBS, and again 1 × 10 ⁷ The cells were dispersed in a DMEM medium containing no FBS so that the cell density was increased. After 0.4 μl of this cell dispersion was mixed with 10 μg of the present expression plasmid ((a) pRC / RSV-mNXFsense or (b) pRC / RSV-mNXFantisense) prepared in Example 2, this mixture was electroporated. The sample was transferred to a cuvette for transfection, and transfection was carried out under conditions of 200 V and 950 μF by electroporation using Gene Pulser (manufactured by BIORAD). After transfection, the medium was replaced with a DMEM medium containing 10% FBS, and further cultured in a 10 cm petri dish for about 24 hours. After culturing, DNA free total RNA was purified using Atlas Pure Total RNA Labeling system (K1038-1; manufactured by Clontech), a commercially available RNA purification and radioactivity labeling kit, using 5 cells each as a material. did. The RNA yield is 23 μg when the plasmid (a) (ie, pRC / RSV-mNXFsense) is used, and 26 μg when the plasmid (b) (ie, pRC / RSV-mNXFantisense) is used. Met. Next, using the obtained RNA as a material, using the specific primer and reverse transcriptase included in the kit, [α-P ³² ] Each RNA was radiolabeled with -dATP (Amersham Pharmacia). Next, radiolabeled RNA (hereinafter referred to as probe) was purified using the above kit, and then 1.3 × 10 5 of the purified RNA. ^Five The DPM equivalent was used for the following hybridization. Hybridization was performed using (a) pRC / RSV using a kit containing a nylon membrane on which various genes were blotted (Atlas cDNA Expression array-Neurobiology; 7736-1 Clontech) and a hybridization buffer attached to the kit. -Probes derived from mNXFsense-introduced cells or (b) probes derived from pRC / RSV-mNXFantisense-introduced cells were added, and the reaction was performed under the same conditions for 18 hours. After hybridization, each nylon membrane was washed with 2XSSC, 1% SDS buffer (68 ° C., 30 minutes). This was repeated 4 times, and further washed with 0.1XSSC and 0.5% SDS buffer (68 ° C., 30 minutes). Each nylon membrane is wrapped in plastic wrap, exposed to an IP plate (Fuji Film) for 7 days, and then hybridization signals corresponding to various genes on the nylon membrane using an imaging analyzer (BASstation; Fujifilm) Quantification and comparison of the strength of were performed.
As a result, for the Rho GDP dissociation inhibitor gene, (a) the hybridization signal on the nylon membrane hybridized with the probe derived from the pRC / RSV-mNXFsense-introduced cell was: (b) the probe derived from the cell introduced with pRC / RSV-mNXFantisense Was significantly weaker than the hybridization signal on the nylon membrane hybridized with. Thus, it was confirmed that this transcriptional regulatory factor has the ability to suppress the expression of Rho GDP dissociation inhibitor.
[0040]
Example 5 (Test method of the present invention)
The pRC / RSV-mNXFsense-introduced cells prepared in Example 2 were treated with 5% CO at 37 ° C. using DMEM medium (Nissui Pharmaceutical) containing 10% FBS. ₂ In the presence, the cells were cultured using a petri dish (Falcon) having a diameter of about 10 cm. On the next day, the cultured cells are dispersed by trypsin treatment, washed twice with DMEM medium not containing FBS, and then dispersed again in DMEM medium.
Next, the dispersed cells are placed in a 6-well plate in which (a) a culture solution containing only DMSO or (b) a culture solution containing a test substance dissolved in various concentrations in DMSO is added in advance. 10 ⁶ Seed at a rate of individual / well. After culturing the cells in this plate at 37 ° C for about 24 hours, remove the culture medium from the plate, wash the cells with PBS (-), and extract total RNA from each well using Isogen (Nippon Gene). To do.
Using the total RNA thus extracted as a material, single-stranded cDNA is prepared using oligodT (Amersham Pharmacia) and Reverse transcriptase (Superscript II, Gibco, etc.). Next, using this single-stranded cDNA as a template, PCR was performed using LA-Taq (manufactured by Takara) and a combination of the following forward primer (SEQ ID NO: 13 to 17) and the following reverse primer (SEQ ID NO: 18 to 22). Do.
<Example of forward primer>
5'-agtgaacagcaacagatact-3 '(SEQ ID NO: 13)
5'-gcttcccgcgtccacgtgga-3 '(SEQ ID NO: 14)
5'-gatgtgggcattcagctccg-3 '(SEQ ID NO: 15)
5'-cagagcttcaccattcgaga-3 '(SEQ ID NO: 16)
5'-gtggccctggtgtctgtccg-3 '(SEQ ID NO: 17)
<Example of reverse primer>
5'-ctggcccgcgcgtggggcaa-3 '(SEQ ID NO: 18)
5'-acacatgcttcgccactgcc-3 '(SEQ ID NO: 19)
5'-taatggccgctctcacaggt-3 '(SEQ ID NO: 20)
5'-agctgagtccctgaggcaga-3 '(SEQ ID NO: 21)
5'-tgtgctgtgccctgacactg-3 '(SEQ ID NO: 22)
The condition of PCR is 30 cycles with 95 ° C for 1 minute and then 68 ° C for 1 minute for 1 cycle. Thus, the amount of EphA1 mRNA present in the total RNA was calculated, and various amounts of EphA1 mRNA in the total RNA extracted from the cells cultured using the culture solution added with DMSO alone, By comparing the amount of EphA1 mRNA in RNA extracted from cells cultured using the culture medium to which the test substance is added, and the test substance has neuronal cells dependent on this transcriptional regulatory factor Test and evaluate the ability to control plasticity. A desired substance is selected based on the result.
[0041]
Similarly, PCR is performed using a combination of the following forward primer (SEQ ID NO: 23 to 27) and the following reverse primer (SEQ ID NO: 28 to 32).
<Example of forward primer>
5'-ttgcagcggagaacgaggag-3 '(SEQ ID NO: 23)
5'-gatgagcactcggtcaacta-3 '(SEQ ID NO: 24)
5'-gcatccaggagatccaggag-3 '(SEQ ID NO: 25)
5'-ctggacaaggacgacgagag-3 '(SEQ ID NO: 26)
5'-cctgcgaaagtacaaggagg-3 '(SEQ ID NO: 27)
<Example of reverse primer>
5'-cctaccatgtagtcagtctt-3 '(SEQ ID NO: 28)
5'-ggtcaggaactcgtactcct-3 '(SEQ ID NO: 29)
5'-ttgggtgcctcctccacggg-3 '(SEQ ID NO: 30)
5'-gcgggacttgatgctgtagc-3 '(SEQ ID NO: 31)
5'-gtcttgtcgtcgtctgtgaa-3 '(SEQ ID NO: 32)
In this way, the amount of Rho GDP dissociation inhibitor mRNA in the total RNA was calculated, and the amount of Rho GDP dissociation inhibitor mRNA in the total RNA extracted from the cells cultured using a culture solution containing only DMSO. Is compared with the amount of Rho GDP dissociation inhibitor mRNA in the total RNA extracted from cells cultured using a culture solution to which various test substances are added. Test and evaluate the ability to control dependent neuronal plasticity. A desired substance is selected based on the result.
[0042]
【The invention's effect】
INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a method for testing the ability to control nerve cell plasticity, which is used to search for substances that can be used as active ingredients of drugs for controlling nerve cell plasticity in mammals. It was.
[Sequence Listing Free Text]
SEQ ID NO: 7
Oligonucleotide primers designed for PCR
SEQ ID NO: 8
Oligonucleotide primers designed for PCR
SEQ ID NO: 9
Oligonucleotide primers designed for PCR
SEQ ID NO: 10
Oligonucleotide primers designed for PCR
SEQ ID NO: 11
Oligonucleotide primers designed for PCR
SEQ ID NO: 12
Oligonucleotide primers designed for PCR
SEQ ID NO: 13
Oligonucleotide primers designed for PCR
SEQ ID NO: 14
Oligonucleotide primers designed for PCR
SEQ ID NO: 15
Oligonucleotide primers designed for PCR
SEQ ID NO: 16
Oligonucleotide primers designed for PCR
SEQ ID NO: 17
Oligonucleotide primers designed for PCR
SEQ ID NO: 18
Oligonucleotide primers designed for PCR
SEQ ID NO: 19
Oligonucleotide primers designed for PCR
SEQ ID NO: 20
Oligonucleotide primers designed for PCR
SEQ ID NO: 21
Oligonucleotide primers designed for PCR
SEQ ID NO: 22
Oligonucleotide primers designed for PCR
SEQ ID NO: 23
Oligonucleotide primers designed for PCR
SEQ ID NO: 24
Oligonucleotide primers designed for PCR
SEQ ID NO: 25
Oligonucleotide primers designed for PCR
SEQ ID NO: 26
Oligonucleotide primers designed for PCR
SEQ ID NO: 27
Oligonucleotide primers designed for PCR
SEQ ID NO: 28
Oligonucleotide primers designed for PCR
SEQ ID NO: 29
Oligonucleotide primers designed for PCR
SEQ ID NO: 30
Oligonucleotide primers designed for PCR
SEQ ID NO: 31
Oligonucleotide primers designed for PCR
SEQ ID NO: 32
Oligonucleotide primers designed for PCR
[0043]
[Sequence Listing]

Claims (7)

A method for assaying the ability to promote or inhibit the dependent neuronal plasticity in the transcriptional regulatory factor having the amino acid sequence of any of the following,
(1) a first step in which a test substance is contacted with a mammalian cell derived from human, monkey, cow, rabbit, mouse, rat or hamster expressing the transcription regulatory factor; and (2) after the first step, the feeding A second step of measuring an expression level of a marker protein gene present on the transcription regulatory factor-dependent nerve cell plasticization pathway in animal cells or an index value correlated with the amount, and (3) the second step A step of evaluating the ability of the test substance based on the measured expression level or an index value correlated with the expression level,
A test method characterized by comprising:
<Amino acid sequence group>
(A) the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3,
(B) an amino acid sequence of a protein having an amino acid sequence showing 90% or more amino acid identity to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 and having transcriptional regulatory ability;
(C) It has an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence represented by base numbers 102 to 2507 of the base sequence represented by SEQ ID NO: 4, and transcriptional regulation An amino acid sequence of a protein having
(D) It has an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence represented by base numbers 51 to 2456 of the base sequence represented by SEQ ID NO: 5 and transcriptional regulation An amino acid sequence of a protein having
(E) having the amino acid sequence encoded by the DNA that hybridizes under stringent conditions with the DNA consisting of the base sequence represented by base numbers 35 to 2440 of the base sequence represented by SEQ ID NO: 6 and transcriptional regulation Amino acid sequence of the protein A method for assaying the ability to promote or inhibit the dependent neuronal plasticity in the transcriptional regulatory factor having the amino acid sequence of any of the following,
(1) a first step of bringing a test substance into contact with a transformed mammalian cell derived from human, monkey, cow, rabbit, mouse, rat or hamster into which a gene having a base sequence encoding the amino acid sequence has been introduced; and (2) After the first step, the expression level of the marker protein gene present on the transcription regulatory factor-dependent nerve cell plasticizing pathway or the index value correlated with the amount is measured in the transformed mammalian cell And (3) evaluating the ability of the test substance based on the expression level measured in the second step or the index value having a correlation with the amount,
A test method characterized by comprising:
<Amino acid sequence group>
(A) the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3,
(B) an amino acid sequence of a protein having an amino acid sequence showing 90% or more amino acid identity to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 and having transcriptional regulatory ability;
(C) It has an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence represented by base numbers 102 to 2507 of the base sequence represented by SEQ ID NO: 4, and transcriptional regulation An amino acid sequence of a protein having
(D) It has an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence represented by base numbers 51 to 2456 of the base sequence represented by SEQ ID NO: 5 and transcriptional regulation An amino acid sequence of a protein having an ability.
(E) having the amino acid sequence encoded by the DNA that hybridizes under stringent conditions with the DNA consisting of the base sequence represented by base numbers 35 to 2440 of the base sequence represented by SEQ ID NO: 6 and transcriptional regulation Amino acid sequence of the protein   The assay method according to claim 1 or 2, wherein the marker protein gene is an Eph A receptor gene or a Rho GDP dissociation inhibitor gene.   The assay method according to claim 1 or 2, wherein the marker protein gene is an Eph A receptor gene and a Rho GDP dissociation inhibitor gene.   The test substance has the expression level of the marker protein gene or the difference obtained by comparing the index value having a correlation with the expression level in the group using two or more different substances as the test substance independently. The test method according to claim 1, wherein the ability is evaluated.   6. The assay method according to claim 5, wherein at least one of the two or more different substances is a substance not having the ability. A method for searching a substance having the ability to promote or suppress dependent neuronal plasticity in the transcriptional regulatory factor with any of the following amino acid sequences, is evaluated by the method of claim 1 or 2, wherein the said capability The search method characterized by selecting the substance which has the said capability based on this.
<Amino acid sequence group>
(A) the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3,
(B) an amino acid sequence of a protein having an amino acid sequence showing 90% or more amino acid identity to the amino acid sequence represented by any one of SEQ ID NOs: 1 to 3 and having transcriptional regulatory ability;
(C) It has an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence represented by base numbers 102 to 2507 of the base sequence represented by SEQ ID NO: 4, and transcriptional regulation An amino acid sequence of a protein having
(D) It has an amino acid sequence encoded by DNA that hybridizes under stringent conditions with DNA consisting of the base sequence represented by base numbers 51 to 2456 of the base sequence represented by SEQ ID NO: 5 and transcriptional regulation An amino acid sequence of a protein having an ability.
(E) having the amino acid sequence encoded by the DNA that hybridizes under stringent conditions with the DNA consisting of the base sequence represented by base numbers 35 to 2440 of the base sequence represented by SEQ ID NO: 6 and transcriptional regulation Amino acid sequence of the protein