JPH11276174A - Promotor - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new promotor which consists of a Dlx5 promotor having a specific base sequence, has a promotor activity against a transcription factor for a bone-forming protein, and is useful, for example, to produce a medicine having a function to promote differentiation of osteoblasts. SOLUTION: This is a new Dlx5 promotor which consists of a DNA fragment having a base sequence shown at least by the formula, has a promotor activity against a transcription control factor Dlx5 capable of promoting differentiation of osteoblasts, and is useful to use the function of Dlx5 for industries such as medical treatment. This promotor is obtained by the preparation of a genomic DNA library from a mouse genomic DNA by the conventional method, followed by the cloning by the polymerase chain eraction(PCR) using the genomic DNA library as a template, and using a mouse Dlx5-specific primer and an adapter primer.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a DNA fragment having a promoter activity for a transcription control factor and its application.

[0002]

2. Description of the Related Art In recent years, various proteins that regulate osteoblast differentiation have been identified.
ne Morphogenetic Protein (hereinafter abbreviated as BMP), which has a very low specificity that acts on various tissues (A. Yamaguchi et al., J. Cell Biol. 113:
681-687, 1991: M. Wozney et al., Science 242: 1528-1534,
1988) or distributed in the nucleus as in cbfa1 (F. Otto
Cell 89: 765-771, 1997: T. Komori et al., Cell 89:
755-764, 1997), a transcriptional regulator that acts directly on DNA.

For example, BMP is known to strongly stimulate osteoblast differentiation and induce bone, but it also ossifies soft tissues such as muscles. Difficult to use. Therefore, application to systemic bone metabolic diseases such as osteoporosis is extremely difficult, and its use is limited to fracture treatments and the like that can be expected to be effective by local use.

On the other hand, transcription factors that regulate the differentiation of osteoblasts include cbfa1 and Msx (A. Towler et al., Molecular Endocrinology 8, 1) which is a homeobox gene.
484-1493, 1994) and Dlx. Especially D
It is known that Dlx5 belonging to the lx family is specifically expressed in bone tissue during the developmental process of vertebrate individuals (Zhaol Developmental Biol. 164: 37-51, 199).
4: A. Simeone et al., Proc. Natl. Acad. Sci., USA 91:
2250-2254, 1994). Also, according to the maturation of osteoblasts, Dl
It has been reported that the expression level of the x5 gene increases, and that Dlx5 protein regulates the phenotype of osteoblasts at the gene level (H. Ryoo et al., Molecular Endocrinology).
11: 1681-1694, 1997).

[0005] Furthermore, the Dlx5 gene is specifically induced in osteoblasts by BMP stimulation.
It has been reported that osteoblasts overexpressed have significantly enhanced differentiation (Miyama et al., The 20th Annual Meeting of the Molecular Biology Society of Japan, abstract 4-B2-W30-3, 1997).

[0006]

However, secretory humoral factors including cytokines such as interferon and colony stimulating factor can be expected to have pharmacological effects by directly administering the protein molecule itself to a living body. On the other hand, a transcription factor such as a homeobox gene can regulate an increase or suppression of the transcription level of a target gene only when it is localized in a cell, especially in a nucleus. Therefore, it is difficult to use the transcription control factor as it is in a final use as a drug such as a medicine.

Accordingly, it is an object of the present invention to find a factor that regulates the expression of a transcriptional regulatory factor, instead of directly utilizing the transcriptional regulatory factor, and to develop a technique for applying the same.

[0008]

The present inventors have conducted various studies on the regulation of the expression of transcriptional regulators,
It has been found that a DNA fragment having the nucleotide sequence shown in SEQ ID NOs: 1 to 4 has a promoter activity for a transcription control factor. Further, if this DNA fragment is used, other various useful proteins are produced and the promoter is activated. The present inventors have found that screening of substances can be performed, and have completed the present invention.

That is, the present invention relates to at least SEQ ID NO: 4 (314 base pairs), SEQ ID NO: 3 (963 base pairs), SEQ ID NO: 2 (1484 base pairs) or SEQ ID NO: 1 (2257 base pairs).
Base pair). The present invention also relates to a DNA fragment which hybridizes with a DNA fragment having the nucleotide sequence of SEQ ID NO: 1, 2, 3, or 4, and which has a promoter activity.
For the NA fragment. Further, the present invention further provides a DN having a base sequence represented by SEQ ID NO: 1, 2, 3 or 4.
A fragment or D which hybridizes to these DNA fragments
The present invention relates to a promoter having an NA fragment. In addition, the present invention relates to a plasmid or a vector having a DNA fragment having the nucleotide sequence represented by SEQ ID NO: 1, 2, 3, or 4, or a DNA fragment that hybridizes to these DNA fragments. Further, the present invention relates to a DNA fragment having a nucleotide sequence represented by SEQ ID NO: 1, 2, 3 or 4, or a DNA fragment having a nucleotide sequence hybridizing to these DNA fragments.
The present invention relates to a vector or a plasmid containing a DNA fragment in which an NA fragment is bound upstream of a reporter gene. Further, the present invention relates to a DNA fragment comprising a DNA fragment having a nucleotide sequence represented by SEQ ID NO: 1, 2, 3 or 4, or a DNA fragment having a nucleotide sequence hybridizing to these DNA fragments bound upstream of a reporter gene. It relates to the introduced transformed cells. The present invention also relates to the above-mentioned transformed cells, wherein the reporter gene is a DNA encoding luciferase, and the transformed cells are MC3T3-E1 cells. The present invention also relates to a genetically modified animal having the above transformed cell. The present invention also relates to a method for evaluating a substance that promotes bone formation, comprising using the above-described transformed cell or gene-mutated animal. The present invention also relates to a method for evaluating a substance that promotes bone differentiation by adding a test substance to a cell culture solution of the above-mentioned transformed cells. The present invention also relates to a substance selected or evaluated by the above evaluation method. Further, the present invention relates to a bone formation promoting agent containing a substance selected by the above-described evaluation method or a substance evaluated as an active ingredient. The present invention also relates to a gene therapy method for the gene therapy method using the above-described vector or plasmid. Also, the present invention provides SEQ ID NO: 1,
DNA fragments having the nucleotide sequence represented by 2, 3 or 4, or DNAs hybridizing to these DNA fragments
The present invention relates to a protein expressed using a promoter having a fragment. Further, the present invention is characterized in that a gene encoding a useful substance protein is operably linked to the above-described vector or plasmid, and a eukaryotic cell transformed with a vector having the sequence of the recombinant DNA is used. Protein production method.

[0010]

BEST MODE FOR CARRYING OUT THE INVENTION The DNA fragment of the present invention is a DNA having the nucleotide sequence of SEQ ID NO: 1, 2, 3 or 4.
Fragments, DNA fragments containing their base sequences, or DNA fragments that hybridize with those DNA fragments.
Furthermore, the DNA fragment of the present invention may be a DNA fragment that hybridizes with a DNA fragment having the nucleotide sequence of SEQ ID NO: 1, 2, 3 or 4, or a DNA fragment having a nucleotide sequence highly homologous to these nucleotide sequences. Fragments shall also be included. The present invention provides a DN which hybridizes with a DNA fragment having the nucleotide sequence of SEQ ID NO: 1, 2, 3 or 4 under certain conditions.
An A fragment that has a promoter activity is also included. Hybridization in the present invention is preferably performed under the following conditions. . The plaque formed by genomic DNA or recombinant phage is transferred (blotted), and the immobilized nylon membrane is immersed in a hybridization buffer to which DNA labeled with ³² P or fluorescence is added as a probe under certain conditions to perform hybridization. Do. Here, under certain conditions, the hybridization temperature is preferably 50 ° C. to 60 ° C., particularly 55 ° C., and the hybridization time is preferably 1 hour to 12 hours, particularly preferably 1 hour to several hours. After performing the hybridization as described above, the hybridized nylon membrane is thereafter subjected to SDS (about 0.05
%) And a predetermined concentration of SSC solution is used for washing under predetermined conditions. Here, the predetermined concentration is 0.1 × SS
C to 4.0 x SSC concentration, especially 0.1 x SSC to 0.5
× SSC concentration is preferred. Further, as washing under predetermined conditions, after washing three times at room temperature for 15 minutes,
55% using 0.1 × SSC solution containing 0.1% SDS
Preferably, washing is performed at 20 ° C. for 20 minutes. Although the degree of cleaning has a certain standard, the D in the test at that time
The amount is appropriately adjusted depending on the amount of hybridization to the NA fragment. The degree of this adjustment is, for example,
The amount of NA labeling of P ³² and the like using as a label can be determined by examining by survey meter or the like.
After the washing after the hybridization is completed, the signal of the DNA fragment hybridized with the probe can be detected by exposing the membrane to an X-ray film.

The DNA fragment of the present invention is useful as a promoter. These promoters can be expressed regardless of animal species as long as they are mammalian cells. The most suitably expressed animal species, in view of their origin, include rodent cells, especially mouse cells.

The plasmid or vector having the DNA fragment of the present invention includes those capable of self-replication in prokaryotic cells such as bacteria and those capable of self-replication in eukaryotic cells such as mammalian cells, fungal cells, and insect cells. It is. Examples of the latter include, but are not limited to, for example, the pCRII vector.

[0013] SEQ ID NO: 1, 2, 3 or 4 of the present invention
A vector or plasmid containing a DNA fragment having the base sequence described above or a DNA fragment having a DNA fragment hybridized thereto bound upstream of a reporter gene contains the DNA fragment having the promoter activity of the present invention, A vector or a plasmid containing a DNA fragment in which a DNA fragment called a reporter gene is linked 3 ′ downstream of the portion is also included. Here, the reporter gene is a generic name of genes used for analyzing and detecting the functions of transcription regulatory regions such as promoters and enhancers of animal cells and viral genes, and the activity of the gene product is defined as the activity of other enzyme activities of cells. Must not be inhibited or compete with and be easily distinguished from similar activities inherent in cells. It is preferable that the activity of the gene product can be detected quickly and with high sensitivity.

The gene that can be used as a reporter gene in the present invention is not particularly limited as long as it can confirm expression, but it is desirable that the expression be easily confirmed. A specific example of such a reporter gene is firefly-derived luciferase which is excellent in sensitivity and convenience.
Luciferase is not normally expressed in mammals, but when luciferase is expressed in animal cells,
The cell extract contains luciferin and ATP as substrates.
Reacts with and chemiluminescence, and the promoter activity can be detected by measuring the luminescence intensity.

As the reporter gene, for example, any of luciferase, GFP (Green Fluorescent Protein), chloramphenicol acetyltransferase and the like may be used.
GFP can be used, and when it is desired to quantitatively detect a product reacted in an animal or in a cell in vitro, but a luciferase gene can be used as a reporter gene. Therefore, in the present invention,
Preferred examples of the reporter gene used include a luciferase gene. Further, a hybrid DNA in which a gene encoding luciferase is linked to the 3 ′ downstream side of the promoter of the present invention via ATG.
A fragment, a vector plasmid containing the DNA fragment,
Transformed cells produced from those vectors or plasmids, and genetically modified animals produced from the transformed cells, etc. can be mentioned as preferred examples of the present invention.

The transformed cell into which the DNA fragment of the present invention in which the DNA fragment is ligated upstream of a reporter gene has been introduced is not particularly limited as long as it is a mammalian cell. Animal cells are preferred because they are efficiently expressed, and among rodents, mouse cells are particularly efficiently expressed. A more preferred example is the MC3T3-E1 osteoblast cell line.

The transformed cell of the present invention can be obtained, for example, by transfecting the vector of the present invention and a vector having a drug resistance gene at the same time, selecting with a drug, and then expressing the reporter gene. The drug resistance gene is not particularly limited as long as its gene product does not inhibit or compete with the transcriptional regulatory activity of the promoter and other enzyme activities of the cell. At present, neomycin and zeocin resistance genes are commercially available. It is desirable that the genetically modified animal of the present invention has the DNA fragment of the present invention on a chromosome, and a transgenic mouse is preferable as such a genetically modified animal.

Next, the production of a transgenic mouse as a genetically modified animal will be described. (1) Pregnant horse serum gonadotropin (PMS) is intraperitoneally administered at about 4:00 pm to induce superovulation in the fertilized egg-collecting female mouse, and then coexist with the male mouse. (2) On the next morning, the oviduct is excised from the female whose mating has been confirmed, placed in a whole glass containing a culture solution, and a fertilized egg is collected from the magnum of the oviduct under a stereoscopic microscope. (3) Prepare a culture solution covered with liquid paraffin in a Petri dish and a drop of a previously prepared DNA solution to be introduced,
Put the collected fertilized eggs in the culture solution drop. (4) Two sets of micromanipulators on an inverted microscope,
Equipped with an injector, put a petri dish containing fertilized eggs on the stage. (5) Attach micropipette for holding and injection to the tip of the injector, and fill the injection pipette with DNA solution. (6) The fertilized egg is fixed with the holding pipette, and the DNA solution is injected into the male pronucleus of the fertilized egg with the injection pipette (microinjection). (7) A pseudopregnant mouse is anesthetized, the back is incised, and a fertilized egg into which DNA has been injected is transplanted into a fallopian tube. (8) A baby is born on the 19th to 20th days with the transplantation day as 0 day, and usually weaned 3 weeks after birth. (9) Extract genomic DNA from the tail of weaned offspring
A transgenic mouse (founder) is determined by performing an assay by the CR method or the Southern blot method. (10) A plurality of heterozygous individuals are obtained by crossing a founder with a normal individual, and a homozygous individual is obtained by crossing between heterozygotes to complete a transgenic mouse line.

The DNA solution used in the step (3) refers to a hybrid DNA in which a DNA encoding a protein to be expressed is ligated downstream of the promoter of the present invention via an initiation codon in an expressible state. Examples of proteins desired to be expressed include reporter genes such as β-galactosidase and luciferase. Hormones such as growth hormone or insulin may be used. The former can be used to evaluate a substance that activates the promoter of the present invention, and the latter has utility as a model for human gene therapy.

By using the above-mentioned transformed cell or gene-mutated animal of the present invention, a substance that regulates the promoter activity can be evaluated. In this case, the reporter gene is preferably ligated downstream of the DNA fragment having the promoter activity of the present invention. Substances selected using such an evaluation method have the property of controlling the promoter activity of the present invention. Controlling includes promoting and inhibiting promoter activity, but is particularly useful in evaluating substances that have promoting properties. Such a substance that promotes the promoter activity promotes osteoblast differentiation, promotes bone differentiation, and promotes bone formation, so that a substance that promotes these actions can be evaluated.

[0021] A specific method of the evaluation can be performed by adding a test substance to the prepared cell culture medium of the transformed cells and measuring the production amount of the expressed protein. Therefore, for the purpose of enabling the function of the homeobox gene that can regulate osteoblast differentiation to be used for industrial purposes such as medical treatment,
Drugs that increase the level of the homeobox gene can be evaluated. That is, a hybrid gene of a promoter region that controls the expression of a homeobox gene and a reporter gene is prepared, and expression of this gene in mammalian cells enables screening for a homeobox gene expression regulator.

As described above, by using the promoter of the present invention, the effect of the test substance on the promoter activity of the expression vector can be measured. That is, the evaluation can be performed by adding a test substance to the medium of cells into which the expression vector has been transfected, and measuring the luciferase activity of the cell extract after culturing for a certain period. The animal culture cells used here are not particularly limited as long as the expression vector can be introduced. Specific examples thereof include a mouse osteoblast-like cell line (MC3T3-E1) and mouse undifferentiated mesenchymal cells (C3H10T1 / 2). It should be noted that by correcting the luminescence intensity of firefly luciferase at the same time with the luminescence intensity of the transgenic Seapan luciferase, highly accurate measurement is possible.

Accordingly, the substances selected or evaluated by performing the method for evaluating a substance that activates a promoter using the transformed cells or the genetically modified animals of the present invention, and those substances as active ingredients Can be provided. A substance that promotes promoter activity selected using such an evaluation method is useful as a substance that promotes bone differentiation as described above. The gene therapy method of the present invention will be described. If the whole or a part of the gene having the promoter region of the homeobox gene obtained according to the present invention is incorporated as a promoter upstream of a specific gene, and used for gene therapy, a system capable of controlling the expression of a bone-specific protein can be obtained. It is possible to use. Gene therapy is broadly classified into a method of replenishing cells having defective genes with normal genes and a method of enhancing specific cell functions by introducing genes into certain cells for the purpose of treatment. For example, since osteoporosis is caused by a decrease in osteogenic ability, one of the factors is that it is presumed that gene therapy that enhances osteogenic ability, that is, enhances the function of osteoblasts, is effective for the treatment. . At this time, in order to enable gene therapy, the gene must be introduced into target cells, for example, osteoblasts, osteoblast precursor cells, cells constituting bone tissue, etc., and expressed efficiently. More effective if possible. However, such a method having no specificity for a target cell or tissue, such as a hybrid gene of the present invention (a gene in which a DNA fragment encoding a protein desired to be expressed in a living body is linked to the promoter of the present invention) is used for whole body. Even if introduced by administration and the hybrid gene of the present invention is randomly incorporated into the whole body, expression of the promoter of the present invention is limited to bone tissue. Therefore, even in such a systemic administration without specificity for the target tissue, since the expression occurs specifically, a useful effect can be expected by expressing the protein. In addition, the expression of the promoter of the present invention is presumed to be in an ON state in bone tissue and OFF in non-bone tissue,
According to the present invention, it is clear from the result of examining the tissue distribution of Dlx5 mRNA. Such gene transfer can be roughly classified into physical methods and biological methods.However, since physical methods have low transfer efficiency, they are applied to gene therapy using biological methods, that is, transfer efficiency. It is common to use a viral vector with a high level. Currently, viral vectors are retrovirus vectors, adenovirus vectors,
Adeno-associated vectors have been developed and used. The combination of these viral vectors and the homeobox gene promoter makes it possible to enhance the function of osteoblasts through regulation of bone-specific protein expression.

A method for producing a protein desired to be produced using the vector of the present invention and a protein produced by the method will be described. Using as a promoter having the DNA fragment of the present invention, a vector having a hybrid DNA in which a DNA fragment encoding a useful protein desired to be produced is operably linked downstream of the promoter via an initiation codon is prepared. . Next, the vector is introduced into the cells by a commonly known method such as a microinjection method and an electric stimulation method to prepare transformed cells. By culturing the transformed cells, a desired protein is produced in the cells. Therefore,
A protein produced by using such a transformed cell, which is read and expressed by a base sequence that can be expressed downstream of the promoter of the present invention, is the protein of the present invention. The type of the protein in this case is not particularly limited, but may be any as long as it can be expressed by the promoter of the present invention.

A method for cloning the promoter of the present invention will be described. (1) A gene having a promoter region of a homeobox gene of the present invention is a commercially available or self-made mammalian genomic DNA.
It can be prepared by cloning from a library by plaque hybridization or colony hybridization or by polymerase chain extension (PCR) using genomic DNA as a template.

(2) Hereinafter, an example in which the promoter region of the Dlx5 gene is cloned by the PCR method will be described. In this method, a library is prepared by digesting mouse genomic DNA with an appropriate restriction enzyme and adding an optional adapter. This adapter has a structure that suppresses nonspecific amplification by adapter primers,
Combination of primers designed in this adapter with primers specific to the target gene and PCR
By applying the method, it becomes possible to amplify the target gene specifically and efficiently. More specifically, to achieve this step, for example, the Universal Genome Walker Kit
Can be used.

(3) The heat resistance D used in the PCR method
As the NA polymerase, a Taq DNA polymerase having proofreading activity can be used to obtain a highly accurate long chain PCR product. The PCR product thus amplified can be subcloned using a commercially available PCR product cloning system such as a TA cloning vector (Invitrogen Inc., San Diego, CA). In some cases, PC
By repeating R twice, the yield can be increased. By the method as described above, the target promoter
The gene can be cloned.

(4-1) For the preparation of transformed cells, all or a part of the promoter gene sequence obtained by cloning as described above is introduced into cells. As a method for introducing a DNA fragment into a cell or into the nucleus of a cell, a commonly used method for introducing a DNA fragment into a cell or into the nucleus of a cell can be adopted. For example, the methods include microinjection, electrical stimulation, FuGENE 6 tr
ansfection reagent (Boehringer Mannheim), Tfx
Examples thereof include a method using a gene introduction reagent such as -50 (Promega) and a calcium phosphate coprecipitation method. The method is not particularly limited, but it is recommended that the introduction efficiency be higher. In the preparation of transient expression cells, the cells are useful as cells capable of expression even before the introduced gene is immobilized on the chromosome. When such a transient gene-transfected cell is prepared and used, it is sufficient for the gene transfection method to have a uniform transfection efficiency such that luciferase activity can be detected.

Hereinafter, the present invention will be described specifically with reference to examples.

Example 1 5 'of mouse Dlx5 gene
(1) Synthesis of oligonucleotide The oligonucleotide primer is DNA of Applied Biosystems (Foster City, CA)
Synthesis was performed in a synthesizer according to the manufacturer's instructions. An antisense primer specific for the mouse Dlx5 gene and located about 30 bp 3 'to its start codon;
5'-CGG AGC TTG GAA GTC GCC GGA TCGGAT GCT-3 '
(DSP1, 30-mer) and antisense primer containing around the start codon 5'-GAC TCT TCT GTC AAA CAC TCC TGT CAT AGC-3 '(DSP2
, 30-mer). In addition, a sense primer, which is an adapter primer designed in the DL adapter, 5′-GTA ATA CGA CTC ACT ATA GGG C-3 ′ (AP1, 22-me
r) and 5'-ACT ATA GGG CAC GCG TGG T-3 '(AP2, 19-mer) are the Universal Genome Walker Kit (Clontech Laborator
ies, Palo Alto, CA).

(2) Preparation of Genomic DNA Library In order to amplify the untranslated region 5 ′ from the start codon of mouse Dlx5 gene, a library using a Universal Genome Walker Kit as a template according to the manufacturer's instructions ( DL). That is, mouse genomic DNA (Clontech Laboratories, PaloAlto, CA) 25
0 ng of restriction enzymes (DraI, EcoRV, PvuII, ScaI and St
digested overnight at 37 ° C with uI). After digestion, TNE buffer (10 mM Tris-HCl, pH 8.0 / 150 mM NaCl / 1 mM ED)
TA) Add an equal volume of phenol saturated with
After centrifugation at 8,000 rpm for 5 minutes, the upper aqueous layer separated into two layers was removed, and the restriction enzyme was removed. Then, an equal amount of chloroform was added to the aqueous layer, and the mixture was gently suspended, and
The mixture was centrifuged at rpm for 5 minutes, and the upper aqueous layer separated into two layers was separated. Further, 1/10 volume of a 3M sodium acetate solution (pH 4.5) and 2 volumes of ethanol were added to perform ethanol precipitation. The precipitated DNA was washed with 70% ethanol, dried, and then 20 μl of TE buffer. (Ten
mM Tris-HCl, pH 8.0 / 1 mM EDTA) was added and dissolved. Each D
Add DL adapter to 4 μl of NA solution, T4 DNA
After the ligation reaction with ligase, the mixture was heated at 70 ° C. for 5 minutes to inactivate the ligase, and TE buffer was added to bring the total volume to 80 μl.
And DL-1 and DL-
2, DL-3, DL-4 and DL-5.

The DL adapter, 5'-GTA ATA CGA C
TC ACT ATA GGG CAC GCG TGG TCG ACG GCC CGG GCT GGT
-3'3'-CC CGA CCA-5 is Universal Genome Walker Kit
Was used.

(3) Polymerase chain extension reaction (PCR) The PCR method uses mouse Dlx5-specific primer (DSP) 1 and adapter primer (AP) 1 at a final concentration of 0.5 μM using about 1 ng of each DL library as a template. Expand High Fidelity PCR system (Boehring
er Mannheim, Amsterdam, Netherlands). The reaction was incubated at 94 ° C for 2 minutes, then at 94 ° C for 30 seconds, 57 ° C for 30 seconds, 68 ° C for 3 seconds.
10 minutes at 94 ° C for 30 seconds, 60 ° C for 30 seconds.
Twenty cycles of 4 minutes at 68 ° C. for 20 seconds, and the final extension step was performed at 68 ° C. for 7 minutes. Further, the 1 obtained as described above
The above-mentioned reaction was performed again using DSP2 and AP2 at a final concentration of 0.5 μM using 1/1000 of the first PCR product as a template. The amplified PCR product is 1
% Electrophoresis on an agarose gel to confirm its formation (FIG. 1).

(4) Plasmid pCR-DP2.3, p
Construction of CR-DP1.0 and pCR-DP1.5

In step (3), 4 μl of the reaction solution in which generation of a PCR product was observed was added to a pCRII vector (Invitrogen).
Inc., San Diego, CA: 1 μl (12.5 ng)
Was ligated with T4 DNA ligase. The plasmid obtained by ligating the PCR product generated from DL-1
CR-DP2.3 (FIG. 2), DL-3 was converted to pCR-DP
1.0 (FIG. 3) and DL-5 were named pCR-DP1.5 (FIG. 4), respectively.

(5) Sequence analysis

The plasmid of (4) was transformed into E. coli JM109 which was made competent by treatment with calcium chloride (hereinafter referred to as competent), and was transformed (Molecular cloning 249-251, Cold Spring Ho
rbor Laboratory, 1982), 5-bromo-4-chloro-
3-Indolyl-β-galactopyranoside (hereinafter referred to as X-ga
l) 50 μg / ml and 50 μg / ml ampicillin
Containing LB agar medium (NaCl 15 per liter (l))
g, yeast extract 5 g, Bacto tryptone 10 g, glucose 1 g, Bacto agar 18 g), and white colonies not expressing β-galactosidase were selected. DNA was prepared from this colony, and the Sanger method using fluorescently labeled dideoxynucleotide (F. Sanger et.al, Proc. Natl. Acad. Sci. USA, 74, 5463).
-5467, 1987), the entire nucleotide sequence of these PCR products was determined. Sequence analysis revealed that the PC containing the initiation codon of the mouse Dlx5 gene and the 5 'untranslated region
It was revealed that the R product amplified the same region (SEQ ID NOs: 1 to 3).

Example 2 5 'of mouse Dlx5 gene
Examination of promoter activity in the side untranslated region (1) Construction of a hybrid gene with firefly luciferase Firefly luciferase expression vector (pGL3 promoter ve
ctor) 5 μg with NheI and HindIII at 37 ° C.
After digestion with 1% agarose gel electrophoresis.
An equivalent amount of phenol: chloroform (1: 1), which was blunt-ended with DNA polymerase and saturated with TNE buffer, was added, and the mixture was gently suspended, centrifuged at 8,000 rpm for 5 minutes, and the upper aqueous layer separated into two layers was removed. Ethanol precipitation was performed by adding 1/10 volume of 3M sodium acetate solution (pH 4.5) and 2 volumes of ethanol, and the precipitated DNA
After washing with 70% ethanol and drying,
lTE buffer was added and dissolved. Further, an equivalent amount of phenol: chloroform (1: 1) which had been dephosphorylated with alkaline phosphatase and saturated with TNE buffer was added, gently suspended, and centrifuged at 8,000 rpm for 5 minutes.
Take the upper aqueous layer, which is divided into layers, and add 1/10 volume of 3M
A sodium acetate solution (pH 4.5) and a 2-fold amount of ethanol were added to perform ethanol precipitation, and
After washing with ethanol and drying, 10 μl of T
E buffer was added and dissolved to obtain an expression vector (pLuc) having no promoter region. On the other hand, pCR-D
P2.3, pCR-DP1.0 and pCR-DP1.5
Was digested with SmaI at 30 ° C. overnight and approximately 2.3 kb, 1.0 kb and 1.5 kb on a 1% agarose gel, respectively.
Fragments containing the untranslated region at the 5 'end of the mouse Dlx5 gene to be migrated in the vicinity (DP2.3, DP1.0 and DP1.5)
Was collected and ligated with the above-described pLuc and T4 DNA ligase to integrate the incorporated expression vector pDP2.3-Lu.
c. (FIG. 5), pDP1.0-Luc. (FIG. 6), and pDP1.5-Luc. (FIG. 7) was obtained.

Similarly, pCR-DP2.3 was converted to Spe
Fragment DP0.3 obtained by digestion with I and SmaI
Was introduced into the pLuc vector to express the expression vector pDP0.
3-Luc (FIG. 8) was obtained. These expression vectors were transformed into competent Escherichia coli JM109, and cultured on LB agar medium containing 50 μg / ml of ampicillin to obtain single cell colonies.

(2) Large-scale preparation of plasmid pool: One colony obtained in the above (1) was treated with 50 μl of ampicillin.
g / ml of sterilized LB liquid medium (NaCl per 1.0 litter)
(15 g, 5 g of yeast extract, 10 g of bacto-tryptone, 1 g of glucose) were inoculated into a 500 ml baffled Erlenmeyer flask and cultured overnight at 37 ° C. with shaking. Plasmids were prepared from the E. coli by the method of Holmes and Quigley et al. (Molecular cloning, 1.34-1.35, 1989) and used as a plasmid pool.

(3) Confirmation of promoter activity: Confirmation of promoter activity of each expression vector was performed using MC3T3-E1.
Various expression vectors were transfected into a cell line, and the luciferase activity of the cell lysate was measured. Mouse osteoblast-like cell line (MC3T3-E1) has 1
0% fetal bovine serum (FBS, GIBCO BRL) and 1% penicillin (10,000 U / ml) / streptomycin (10,000 μl)
g / ml) solution (GIBCO BRL)
5 in imum essential medium (α-MEM, GIBCO BRL)
The cells were cultured at 37 ° C. in the presence of% carbon dioxide. Cells are about 2 × 1
0 ⁴ cells / well in 24 Uerupure - DOO After overnight incubation at (Corning Co.), an expression vector of 250ng per well, Sea 250ng pansy luciferase expression vector (Promega) and 1.5μl of FuGENE6 Transfecti
Gene was introduced by adding a mixture of on reagent (Boehringer Mannheim). After culturing for 48 hours, the cells in each well were collected, washed with PBS (Ca ⁺⁺ , Mg ⁺⁺ free), suspended in a cell lysis solution (Promega), and left at room temperature for 10 minutes. The supernatant after centrifugation at 15000 rpm for 5 minutes was measured for the luminescence intensity of the cell lysate using a dual luciferase reporter system (Promega) according to the manufacturer's instructions using a scintillation counter (Aloka). Nomarized relati was calculated by correcting the emission intensity of firefly luciferase with the emission intensity of seapan luciferase.
ve luminescence unit (RLU).

As a result, luciferase activity was observed in each of the various expression vectors, and it was confirmed that the promoter activity was present in this region (Table 1).

[0043]

[Table 1]

(4) Preparation of transformed cells:
Seed in × 10 ⁵ cells / 60mm dish, cultured overnight,
1.5 μg of expression vector (pDP2.3-Luc), 1 μg of seapangil luciferase expression vector, 200 ng of neomycin resistance gene expression vector (pSV2neo) and 9
μl of FuGENE 6 transfection reagent (Boehringer M
annheim) was introduced. After culturing for 48 hours, trypsin digest and collect cells,
The seeds were seeded on several 100 mm dishes. 48 hours later, G418
(0.5 to 1.0 mg / ml), and the medium was further replaced. Thereafter, the medium was replaced with a medium supplemented with G418 every three days, and the cells were cultured until colonies were formed. Cells forming colonies by trypsin digestion were collected, and their expression traits were evaluated. Cells expressing luciferase activity and responding to the drug were selected.

[0045]

According to the present invention, there is provided a gene that controls the expression of a transcription control factor. Use of this gene enables production of useful proteins, evaluation of promoter activating substances, and the like.

[0046]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 2-57 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: Genomic DNA Origin Organism name: Myomopha Mus Sequence AAATTTATAG TAAATAAAAT TGGGGCCTGT GATGGGATCA CCCATCTCTC TGCACTTTTT 60 TTTTTTATA CCTCTGAACA CTTTGACCAG CTCAAGGCAT TGATTTGTAA GGCCCCATGG 120 ATTAGGTGAG CATCTTTAGA GCAGACCGCA ATCATAATCC AGTAAGGTCA GCAGTACGTT 180 TGCCTCCTTG AAGACACTCA GTGCAAATCT AGAGCCTGGA CAGCCACATG AAAATCAAAG 240 GCAAGGGATC AGTGTCAAGG GACAGCAAAC ATTTGTTAAA CCCACACATC TGTGTCAGAA 300 GGGAAATCTA GAGACACCAA GGCTGCAAGT AGGGACTCCA GATGAAGAAA GGGGTGGCCC 360 TGCTGCATGC TAGCTCCCTC TCGTTGTTGA CAAAAGCTGA AGTTGTACAG CCAAGGAAAA 420 GCAACAGTAG CCTGGGTTTA TGGGGCCCAT GTGCACCTGA GGCACGTGTT GCTGCTTCCA 480 GTTGCAGCTC CAGATTCCAG AGAGGGACTG ACCTCCTGCT TCCAGCTCTC TGACAGAGGC 540 TTGGAGTCCT TGGTTGCCAA AACAAGAACA AAGTAACCTC AGACAAAACT CAGGCAGATT 600 TTCACTTCTT CCCCTGGCAA AAGGGGAACC AGAAGAGGAA CCAGAAGATT CCACCCTGAA 660 CATTCAATGA CACTCCCAGG TTCCTGGCCA AAAGGAGGCC CAAAATAGGA TACAACTCAT 720 TTTTGAGGGG AGTAGACACA GCATTGAGTG AGGATCAGAA CACATTCCTA AGGCCTCACT 780 CAATCATAAC TAGAGGAAAA GCCCATGTTG ACAAAATAAA CTTCTTCTGG TGGTCTGTGG 840 CCAGTGTATG AGTCTTGAAC CCTGCTCCCT TTGTACATAA ACGAAGATGC TCAGTAGGCA 900 GGAAATAATG GGGGATGCTA CAGAACCCAA ACTCAAGAAT TGAGAGGGGG ATTGGAGTGG 960 ATGTAGGTAA TGGAACCAGG AGAGGGTCGT CCATGATCGA AACCCCATCT CCCAAGCGGA 1020 CTTGGTTCCT TGCCTACTTT TCGGTCTTCA TACTCCATTT GGTCTGGAGG CACACTATAG 1080 GTCACACACC CAAATCAATT CACGTTTGCA GGAATCTGGC TCCTGAACTG AGACAAGAGC 1140 CATTCTAGAA GCTTCCAAGT AGTCGAGGAT ATTGAGCAAA AAACCAAAAG GCTGCTCAGC 1200 TCTCTGGGAA AGCCAACTAA GAGAGTTCAG GCAGTGGCAA GGTATTTCAG GAATGAAAGA 1260 CATTGATTTT CAGCAGTCCC TGTAAAGACT TCAGTTGCCA CTGGGGACGT CTAGATTCTT 1320 GGACATCTCT GTGGAGTCCT GAGAAACAGG GGCTTCTGCA AAAGCGCTTG CTGCGTGTCC 1380 AACTGTTTTC CAGTTTACAA ATTTTCAACA TAAATTAGTT GCAACTTGCT TCCCATCATC 1440 CCACTAAGAC AATGCTGTTC TCCATTTGTT CTCACTGCCA TCTAACACTG CTCCTCCTCT 1500 GCACAC AGTC CCACAGCGGT GCTCTCTCCT ACAGTCTCCG TCGGAGGAGG GGGGAGAGGA 1560 CTGGTAGGTG TGGAGCAAGA AAAGAGCATC AAGAACCGCA TCCTCTAAAC TGTACTTTTG 1620 AGATTGCTGA TTGGAACACG TTGTCTTCAA ACAGCACGGA GCTAGGCCCT GGGACTGCTT 1680 GGTTTGTCGA AGCTATTCAC TAAAAAGAGA TCGACTGGGA CCCTCCCTCC CCCAACAGGG 1740 AACAACTCAT ATTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC 1800 TCTCTCTCTC TCACACACAC ACACACACAC TCTCTCTCTC TCTCTCTCCT TGTACGCCTC 1860 TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTATCTA TCTATCTTTA 1920 AGCAATGCTT TGTTGTGCTA AAACTAGTTG GACGAGTTAG GGTGTTGCTC TGCTCCCGAA 1980 GCCCTGGCCA ATGGAACCCG ATCCACCCCC GCTGTGCGTA AGCGCGCAAT TAAGGATTTA 2040 ACCAAGGCTG TGCTGCGCCT CAGCCAGCAG TCAGCCTGCG GGAGACAGAG CCTCCACGAC 2100 TCCCAGCCTC CTCCTCCTCC TCCTCCCAGC CGCCGCCGCC GCCTCCTCCT CTTCCTCCTC 2160 CTCCCTCGCT CCCACAGCCA TGTCTGCTTA GACCAGAGCA GCTCCACAGC CAATTCAGGC 2220 AGCAGCGGCC GCCGCAGTAG AAGAACAGCC ACCGCCC 2257

[0047]

[Sequence list]

 SEQ ID NO: 2 Sequence length: 963 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Myomopha Mus Sequence TTGCCACTGG GGACGTCTAG ATTCTTGGAC ATCTCTGTGG AGTCCTGAGA AACAGGGGCT GCTCTGCTAGCTGCTCAGCT GTTTTCCAGT TTACAAATTT TCAACATAAA 120 TTAGTTGCAA CTTGCTTCCC ATCATCCCAC TAAGACAATG CTGTTCTCCA TTTGTTCTCA 180 CTGCCATCTA ACACTGCTCC TCCTCTGCAC ACAGTCCCAC AGCGGTGCTC TCTCCTACAG 240 TCTCCGTCGG AGGAGGGGGG AGAGGACTGG TAGGTGTGGA GCAAGAAAAG AGCATCAAGA 300 ACCGCATCCT CTAAACTGTA CTTTTGAGAT TGCTGATTGG AACACGTTGT CTTCAAACAG 360 CACGGAGCTA GGCCCTGGGA CTGCTTGGTT TGTCGAAGCT ATTCACTAAA AAGAGATCGA 420 CTGGGACCCT CCCTCCCCCA ACAGGGAACA ACTCATATTC TCTCTCTCTC TCTCTCTCTC 480 TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCAC ACACACACAC ACACACTCTC 540 TCTCTCTCTC TCTCCTTGTA CGCCTCTCTC TCTCTCTCTC TCTCTCTCTC TCTCTCTCTC 600 TCTCTCTCTC TATCTATCTA TCTTTAAGCA ATGCTTTGTT GTGCTAAAAC TAGTTGGACG 66 0 AGTTAGGGTG TTGCTCTGCT CCCGAAGCCC TGGCCAATGG AACCCGATCC ACCCCCGCTG 720 TGCGTAAGCG CGCAATTAAG GATTTAACCA AGGCTGTGCT GCGCCTCAGC CAGCAGTCAG 780 CCTGCGGGAG ACAGAGCCTC CACGACTCCC AGCCTCCTCC TCCTCCTCCT CCCAGCCGCC 840 GCCGCCGCCT CCTCCTCTTC CTCCTCCTCC CTCGCTCCCA CAGCCATGTC TGCTTAGACC 900 AGAGCAGCTC CACAGCCAAT TCAGGCAGCA GCGGCCGCCG CAGTAGAAGA ACAGCCACCG 960 CCC 963

[0048]

[Sequence list]

 SEQ ID NO: 3 Sequence length: 1484 Sequence type: Nucleic acid Number of strands: Double-stranded Topology: Linear Sequence type: Genomic DNA Origin Organism name: Myomorpha Mus Sequence CCTCACTCAA TCATAACTAG AGGAAAAGCC CATGTTGACA AAATAAACTT CTTCTGGTGC 60 TCTGTGGCGTGT 60 GCTCCCTTTG TACATAAACG AAGATGCTCA 120 GTAGGCAGGA AATAATGGGG GATGCTACAG AACCCAAACT CAAGAATTGA GAGGGGGATT 180 GGAGTGGATG TAGGTAATGG AACCAGGAGA GGGTCGTCCA TGATCGAAAC CCCATCTCCC 240 AAGCGGACTT GGTTCCTTGC CTACTTTTCG GTCTTCATAC TCCATTTGGT CTGGAGGCAC 300 ACTATAGGTC ACACACCCAA ATCAATTCAC GTTTGCAGGA ATCTGGCTCC TGAACTGAGA 360 CAAGAGCCAT TCTAGAAGCT TCCAAGTAGT CGAGGATATT GAGCAAAAAA CCAAAAGGCT 420 GCTCAGCTCT CTGGGAAAGC CAACTAAGAG AGTTCAGGCA GTGGCAAGGT ATTTCAGGAA 480 TGAAAGACAT TGATTTTCAG CAGTCCCTGT AAAGACTTCA GTTGCCACTG GGGACGTCTA 540 GATTCTTGGA CATCTCTGTG GAGTCCTGAG AAACAGGGGC TTCTGCAAAA GCGCTTGCTG 600 CGTGTCCAAC TGTTTTCCAG TTTACAAATT TTCAACATAA ATTAGTTGCA ACTTGCTTCC 660 CATCATCCCA CTAAGACAAT GCTGTTCTCC ATTTGTTCTC ACTGCCATCT AACACTGCTC 720 CTCCTCTGCA CACAGTCCCA CAGCGGTGCT CTCTCCTACA GTCTCCGTCG GAGGAGGGGG 780 GAGAGGACTG GTAGGTGTGG AGCAAGAAAA GAGCATCAAG AACCGCATCC TCTAAACTGT 840 ACTTTTGAGA TTGCTGATTG GAACACGTTG TCTTCAAACA GCACGGAGCT AGGCCCTGGG 900 ACTGCTTGGT TTGTCGAAGC TATTCACTAA AAAGAGATCG ACTGGGACCC TCCCTCCCCC 960 AACAGGGAAC AACTCATATT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT 1020 CTCTCTCTCT CTCTCTCTCA CACACACACA CACACACTCT CTCTCTCTCT CTCTCCTTGT 1080 ACGCCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTCTCTCTCT CTATCTATCT 1140 ATCTTTAAGC AATGCTTTGT TGTGCTAAAA CTAGTTGGAC GAGTTAGGGT GTTGCTCTGC 1200 TCCCGAAGCC CTGGCCAATG GAACCCGATC CACCCCCGCT GTGCGTAAGC GCGCAATTAA 1260 GGATTTAACC AAGGCTGTGC TGCGCCTCAG CCAGCAGTCA GCCTGCGGGA GACAGAGCCT 1320 CCACGACTCC CAGCCTCCTC CTCCTCCTCC TCCCAGCCGC CGCCGCCGCC TCCTCCTCTT 1380 CCTCCTCCTC CCTCGCTCCC ACAGCCATGT CTGCTTAGAC CAGAGCAGCT CCACAGCCAA 1440 TTCAGGCAGC AGCGGCCGCC GCAGTAGAAG AACAGCCACC GCCC 1484

[0049]

[Sequence List] SEQ ID NO: 4 Sequence length: 314 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: Genomic DNA Origin Organism name: Myomorpha Mus Sequence CTAGTTGGAC GAGTTAGGGT GTTGCTCTGC TCCCGAAGCC CTGGCCAATG GAACCCGATC 60 CACCCCCGCT GTGCGTAAGC GCGCAATTAA GGATTTAACC AAGGCTGTGC TGCGCCTCAG 120 CCAGCAGTCA GCCTGCGGGA GACAGAGCCT CCACGACTCC CAGCCTCCTC CTCCTCCTCC 180 TCCCAGCCGC CGCCGCCGCC TCCTCCTCCC CCTCCTCCTC CCTCAGCCAGCCAGCCAGCCAGCCAGCCAGCACGACCCAGCAGCAG

[Brief description of the drawings]

FIG. 1 shows a PCR product generated by subjecting mouse genomic DNA to restriction enzyme digestion, adding a DL adapter, performing two rounds of PCR using a prepared DL library as a template, and performing 1% agarose gel electrophoresis. Lane 1 is λ
The size marker obtained by digesting the DNA with EcoT14I, lane 2 contains a 2.3 kbp DNA fragment containing the P
The CR product, a DNA fragment of 0.5 kbp, is a gene product unrelated to the application. Lane 3 is a gene product unrelated to this application. Lane 4 is the PCR product shown in SEQ ID NO: 2. Lane 5 was not amplified in this study. Lane 6 is the PCR product shown in SEQ ID NO: 3. Lane 7 is a size marker obtained by digesting φ × 174 with HincII.

FIG. 2: DL-1 PCR product and p
The following describes the method for preparing pCR-Dp2.3 by performing a ligation reaction of CRII vector with T4 DNA ligase.

FIG. 3: DL-3 PCR product and pC shown in SEQ ID NO: 2
A method for preparing pCR-Dp1.0 obtained by ligating an RII vector with T4 DNA ligase will be described.

FIG. 4. DL-5 PCR product and pC shown in SEQ ID NO: 3
A method for preparing pCR-Dp1.5 by ligating an RII vector with T4 DNA ligase will be described.

FIG. 5. 1 from SmaI digest of pCR-DP2.3
2.3 kbp recovered by% agarose gel electrophoresis
DNA fragment and Nh of pGL3 promoter vector
pL obtained by blunt-ending digested products of eI and HindIII
The method for preparing pDp2.3-Luc obtained by ligating a uc vector with T4 DNA ligase will be described.

FIG. 6. 1% from SmaI digest of pCR-DP1.0
The ligation reaction was carried out by using T4 DNA ligase between the 1.0 kbp DNA fragment and the pLuc vector recovered by agarose gel electrophoresis, and the resulting pDp1.0-Luc was obtained.
The production method of is shown.

FIG. 7: 1% from SmaI digest of pCR-DP1.5
A method for preparing a pD1.5-Luc obtained by performing a ligation reaction using a T4 DNA ligase with a 1.5 kbp DNA fragment and a pLuc vector recovered by agarose gel electrophoresis.

FIG. 8 shows that a 0.3 kbp DNA fragment and a pLuc vector recovered from a SpeI and SmaI digest of pCR-DP2.3 by 1% agarose gel electrophoresis were used for T4D.
The ligation reaction is carried out with NA ligase and the resulting pDp
A method for producing 0.3-Luc will be described.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁶ Identification code FI C12N 5/10 C12P 21/02 C C12P 21/02 C12Q 1/68 A C12Q 1/68 C12N 5/00 B // (C12N 5 / 10 C12R 1:91) (C12P 21/02 C12R 1:91)

Claims (28)

[Claims] 1. A DNA fragment having at least the nucleotide sequence of SEQ ID NO: 4. 2. A DNA fragment having at least the nucleotide sequence of SEQ ID NO: 3. 3. A DNA fragment having at least the nucleotide sequence of SEQ ID NO: 2. 4. A DNA fragment having at least the nucleotide sequence of SEQ ID NO: 1. 5. A DNA which hybridizes with a DNA fragment having the nucleotide sequence of SEQ ID NO: 1, 2, 3 or 4.
DNA that is a fragment and has promoter activity
fragment. 6. A hybridization buffer (50 ° C.)
６０60 ° C.), and the hybridized nylon membrane was washed with 0.1% to 4.0 × S containing 0.05% SDS.
The DNA fragment having promoter activity according to claim 5, which is obtained by washing with an SC solution and hybridizing with a probe. 7. A nylon membrane immersed in a hybridization buffer and hybridized at 55.degree.
The probe is washed with a 2.0 × SSC solution containing 05% SDS at room temperature, and further washed with a 0.1 × SSC solution containing 0.1% SDS at 55 ° C. to hybridize with the probe. The DNA fragment having promoter activity according to claim 5, which is obtained under the following conditions: 8. D according to any one of claims 1 to 5,
Promoter with NA fragment. 9. The promoter according to claim 8, which is derived from a mouse cell. 10. A plasmid or vector having the DNA fragment according to any one of claims 1 to 7. 11. A DNA comprising the DNA fragment according to claim 1 ligated upstream of a reporter gene.
A vector or plasmid containing the fragment. 12. A DNA comprising the DNA fragment according to any one of claims 1 to 4 linked upstream of a reporter gene.
A transformed cell into which the fragment has been introduced. 13. The vector or plasmid according to claim 10, wherein the reporter gene is a DNA encoding a luciferase protein. 14. The transformed cell according to claim 12, wherein the reporter gene is a DNA encoding a luciferase protein. 15. The transformed cell according to claim 12, wherein the transformed cell is an osteoblast. 16. The transformed cell according to claim 15, wherein the transformed cell is an MC3T3-E1 cell. 17. The method according to claim 12, wherein
A gene-mutated animal having the transformed cell according to Item. 18. The genetically modified animal according to claim 17, which has the DNA fragment according to claim 1 on a chromosome. 19. The method according to claim 12, wherein
A method for evaluating a substance that promotes the differentiation of osteoblasts, comprising using the transformed cell described in the above section. 20. A method for evaluating a substance that promotes bone formation, comprising using the genetically modified animal according to claim 17. 21. A method for evaluating a substance that promotes bone differentiation by adding a test substance to a cell culture medium of the transformed cell according to any one of claims 12 and 14 to 16. 22. A substance selected or evaluated by performing the method according to any one of claims 19 to 21. 23. An osteogenesis promoter comprising a substance selected by the method according to claim 22 or a substance evaluated by the method as an active ingredient. 24. A method for gene therapy using the vector or plasmid according to any one of claims 10, 11 and 13. A gene therapy method using the transformed cell according to any one of claims 12 to 16. 26. A protein expressed using the promoter according to claim 5. 27. A eukaryotic cell obtained by linking a gene encoding a useful substance protein to the vector or plasmid according to claim 10, 11 or 13 so that the gene can be expressed, and transforming the vector or plasmid with the recombinant DNA sequence. A method for producing a protein, comprising using: 28. The method according to claim 27, wherein the protein to be expressed is a transcription factor of a bone morphogenetic protein.