JP2002065281A - Dna for targeting bikunin gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a DNA useful for targeting a bikunin gene. [An inter-α- tripsin inhibitor (hereinafter called ITI) existing in a high concentration as one of serum proteins is a complex compound consisting of 3 different kinds of protein composed of 2 heavy chains (HC1 and HC2) and a low molecular chondroitin sulfate proteoglycan called as bikunin, and the core protein of the bikunin has 2 Kunitz type tripsin inhibitor domains and the protease inhibiting activity which is the origin of name of ITI, is derived from them]. SOLUTION: This DNA for targeting the bikunin gene consisting of an artificial specific base sequence, a vector containing the DNA, and a knockout mouse, etc., having the bikunin gene produced by it are provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a DNA that can be used for targeting a bikunin gene, a vector containing the DNA, a knockout mouse for a bikunin gene produced using the DNA, and the like.

[0002]

2. Description of the Related Art One of serum proteins has a high concentration (0.4 mg).
/ ml) inter-α-trypsin inhibitor (hereinafter also referred to as ITI) contains two heavy chains (HC1 and HC1).
HC2) and bikunin (bikunin), a complex consisting of three different proteins, a small molecule chondroitin sulfate proteoglycan. The core protein of bikunin consists of two Kun.
It has an itz-type trypsin inhibitor domain, and its protease inhibitory activity, which is the name of ITI, is attributed to this. A chondroitin sulfate sugar chain, one of glycosaminoglycans, is bound to the tenth serine residue from the N-terminal of the bikunin core protein. Two heavy chains (HC1 and HC
2) is covalently linked to the side chain of the chondroitin sulfate chain.

[0003] It is known that there are pre-α-trypsin inhibitor (PαI) and inter-α-trypsin-like inhibitor (ITLI) as similar structures, forming an ITI family. These have the same core bikunin, but only one side chain HC protein.
PαI is composed of an HC3 chain having a structure different from that of ITI, and ITLI is composed of an HC2 chain.

On the other hand, hyaluronic acid (HA) is composed of glucuronic acid and N-
It is an ultra-high molecular weight glycosaminoglycan sugar chain composed of a disaccharide repeating structure of acetylglucosamine. In many cell membrane surface outer layers, there is a structure called a hyaluronic acid-rich matrix, the main component of which is hyaluronic acid, which is covalently bound to a certain protein,
(Serum-derived hyaluronic acid binding protein).
This conjugate is called a SHAP-HA complex and is contained in gel-like tissues and body fluid components rich in hyaluronic acid, such as synovial fluid in patients with rheumatism, serum from patients with chronic hepatitis and cirrhosis, and the surface of ovulated eggs. It is abundant in the uterus and endometrium. The SHAP was found to be the same substance as HC1, HC2 and HC3 of the ITI family, and was also derived from serum.

[0005] The mode of binding of these proteins to sugar chains is very unique. The α-carboxyl group of the C-terminal aspartic acid residue of the mature protein, the chondroitin sulfate N-acetylgalactosamine residue of bikunin, or the N-terminal It was found that they were linked by an ester bond to the 6-position hydroxyl group of -acetylglucosamine residue. This bond is common to HC1, HC2 and HC3. In addition, it was suggested that the SHAP-HA complex did not directly bind to the free HC chain, but rather transferred the HC chain of the ITI family molecule bound to the chondroitin sulfate chain to form a conjugate. At this time, bikunin is released from ITI, and it is excreted in urine.
It is called UTI (Urine Trypsin Inhibitor).

In order to pursue the physiological role of the SHAP-HA complex, which is considered to be important for the hyaluronic acid-rich matrix, it is conceivable to inhibit the formation of the complex. To this end, it is conceivable to delete the HC chain of the ITI family, which is SHAP, but there are a plurality of HC chains, and it is very difficult to knock out all of them. Genetic knockout of the bikunin protein, as the common component of the ITI family molecules is bikunin, to abolish all ITI family molecules, thereby inhibiting the formation of the SHAP-HA complex Was thought.

[0007]

An object of the present invention is to provide a DNA useful for targeting a bikunin gene, a vector containing the DNA, a knockout mouse of the bikunin gene produced using the DNA, and the like.

[0008]

Means for Solving the Problems As a result of intensive studies, the present inventors have provided a DNA useful for targeting a bikunin gene and a vector containing the DNA, and further used these to knock out the bikunin gene. The present invention was completed by producing a mouse or an infertile model mouse.

That is, the present invention provides a DNA comprising the nucleotide sequence of SEQ ID NO: 1 (hereinafter referred to as the DNA of the present invention). The present invention also provides a vector containing the DNA of the present invention (hereinafter, referred to as the present vector).

[0010] The present invention also provides a knockout mouse for a bikunin gene (hereinafter referred to as a mouse of the present invention) in which a portion corresponding to the bikunin gene of exon 7 of the AMBP gene in the genome is knocked out. The mouse of the present invention contains base numbers 6145 to 625 of SEQ ID NO: 1 in the genome.
Characterized by containing a base sequence represented by 0
Includes knockout mice for the bikunin gene. The mouse of the present invention preferably has the α-1-microglobulin gene normally retained, and preferably has both homologous chromosomes containing the nucleotide sequence represented by nucleotide numbers 6145 to 6250 of SEQ ID NO: 1. Particularly preferred are female mice. Further, the present invention provides an infertility model mouse comprising the female mouse of the present invention (hereinafter, the model mouse of the present invention).

[0011]

Embodiments of the present invention will be described below. <1> DNA of the Present Invention and Vector of the Present Invention The DNA of the present invention is a DNA having the nucleotide sequence of SEQ ID NO: 1 and can be used for targeting a bikunin gene, and is particularly preferably used for knocking out a bikunin gene.

The mouse bikunin gene is linked to the α-1-microglobulin (hereinafter also referred to as α-1-M) gene, which is also a serum protein, and α-1-microglobulin and bikunin bind from a common mRNA. Translated as a precursor protein. After translation, the precursor protein is cleaved by a specific protease and separated into α-1-microglobulin and bikunin. It exists on the chromosome as α-1-microglobulin / bikunin precursor gene (AMBP gene),
Consists of 10 exons, exons 1 to 7 in the first half are α
It encodes 1-microglobulin, and the latter exons 7 to 10 encode bikunin.

Exon 7 is composed of the C-terminal amino acid residue of mature α-1-microglobulin, the N-terminal portion of bikunin containing the 10th serine residue to which a chondroitin sulfate chain binds, and a cleavage site. In order to create a knockout mouse for bikunin, it is most preferable to use the position of exon 7 as a targeting site. In addition, in order to reduce the influence on the production of α-1-microglobulin and the precursor cleavage enzyme, it is preferable to preserve the cleavage site of α-1-microglobulin-bikunin precursor on the 5 ′ side of exon 7 as it is. . At the same time, it blocks the expression of bikunin molecules and binds chondroitin sulfate chains.
In order not to translate even a partial peptide near the serine residue, it is preferable to mutate the base sequence encoding the bikunin N-terminus. In particular, changing the number of the bases is particularly effective because the frame of the translation code is shifted, and even if a peptide is produced, a bikunin-like chondroitin sulfate-binding peptide is not produced.

It is preferable that the DNA used for the production of knockout mice contains a positive marker gene for positive selection. The positive marker gene is expressed when the target DNA is introduced into cells,
Thereby, sorting can be performed. It is preferable to use a neomycin resistance gene as the positive marker gene.

The DNA of the present invention is designed based on such a concept, and can be produced by the method described in Examples described later. Naturally, DNA produced by other methods is also applicable to the present invention. Included in DNA.

When the DNA of the present invention is used for producing a knockout mouse, the present invention is preferably used by incorporating it into an appropriate vector, whereby the vector of the present invention is provided. The vector to be used can be appropriately selected by those skilled in the art, but is preferably a vector containing a negative marker gene for performing negative selection.

The negative marker gene is one that can be expressed and selected when the desired homologous recombination has not occurred. As a negative marker gene, chromosome D
It is not particularly limited as long as it is expressed in a host cell when introduced into NA, and examples thereof include a thymidine kinase gene and a diphtheria toxin A gene. Preferably, it is a thymidine kinase gene.

<2> The Mouse of the Present Invention and the Model Mouse of the Present Invention The mouse of the present invention is a knockout mouse of the bikunin gene obtained by knocking out a portion corresponding to the bikunin gene of exon 7 of the AMBP gene in the genome. The mouse of the present invention contains, in the genome, base number 614 of SEQ ID NO: 1.
It is preferable to have a knockout mouse of the bikunin gene, characterized in that it contains the nucleotide sequence represented by 5 to 6250. This mouse is completely lacking bikunin gene expression (bikunin-deficient homotype (-/-);
Hereinafter, simply abbreviated as "(-/-)") or the expression level of the gene is reduced (bikunin-deficient heterotype (+ /
-); Hereinafter, simply referred to as "(+/-)") and a nucleotide sequence represented by nucleotide numbers 6145 to 6250 in SEQ ID NO: 1.

Further, the mouse of the present invention is preferably one in which the α-1-microglobulin gene adjacent to the bikunin gene is normally retained, and by using such a mouse of the present invention, a bikunin-specific functional analysis is performed. It can be performed.

It is preferable that the mouse of the present invention further comprises a nucleotide sequence represented by nucleotide numbers 6145 to 6250 of SEQ ID NO: 1 ((-/-)) in both homologous chromosomes, whereby the bikunin gene can be obtained. Can be a mouse completely lacking the expression of In addition, when such a mouse is a female mouse, particularly a (-/-) female mouse, it has been found that the pregnancy rate is remarkably low. Therefore, such a mouse of the present invention is referred to as an infertile model mouse. Thus, the model mouse of the present invention is provided.

The mouse of the present invention can be produced by the usual technique for producing a knockout mouse using the vector of the present invention and the like, and a specific example thereof is the method described in Examples below. In general, a method of introducing a vector of the present invention into ES cells (embryonic stem cells), selecting a cell into which the vector of the present invention has been incorporated, incorporating the cell into a mouse embryo, and producing a chimeric mouse is employed. .

Further, as described in the following embodiment, (+ /
When the genotype of the offspring born by crossing the-) mice is almost in accordance with Mendel's law, and there is not much difference between males and females, the mouse of the present invention has a substantially constant ratio by such crossing. Of female mice of the present invention, and (-/-) female mice of the present invention can be produced at a substantially constant rate.

[0023]

EXAMPLES The present invention will be described more specifically with reference to the following examples. However, these should not limit the technical scope of the present invention. <1> Construction of targeting vector As shown in FIG. 1, a plasmid vector containing exon 5, exon 6, and exon 7 cloned from a mouse genomic DNA library and encodes serine residue at position 7 of bikunin in exon 7 nucleotide
A mutant obtained by replacing 2 bases (AG) with 3 bases (CGC)
It is prepared using QuikChange Site-Directed MutagenesisKit (Stratagene), and at the same time, a restriction enzyme (E
co47III) A cleavage site was created. A DNA cassette containing the cleavage site treated with Eco47III, having an SV40 poly A signal containing a stop codon cut with HpaI, and further containing a neomycin resistance gene (neo) that is a positive marker and exhibits antibiotic resistance as a positive marker. Was combined. As a basic vector for constructing a targeting vector, a bluescript IISK + plasmid vector (pBluescript II SK +: manufactured by Stratagene) was used.

The mutant plasmid vector obtained by the above method was about 4.4 kb including exons 5 and 6 upstream from the targeting position (exon 7).
Since the long arm of the targeting vector was considered to be short, a DNA fragment containing exon 3 and exon 4 regions was separately prepared from mouse genomic DNA by PCR, subcloned with the Bluescript II cloning vector, and then the mutant vector and XbaI site were used. To obtain a mutant plasmid vector having a long arm of about 6 kb upstream.

The DNA fragments obtained by PCR were confirmed by sequencing to have no mutations in exon 3 and exon 4 sequences.

As a short arm, a mouse genome DN
Plasmid DNA subcloned from A clone
Exon 9 using the restriction enzymes EcoRI and PvuII.
Cut out a DNA fragment of about 1.8 kb containing
After subcloning into the Bluescript II cloning vector, it was ligated at the SalI site of the vector downstream of the neo gene of the mutant plasmid vector. The fragment (SEQ ID NO: 1) finally cut out with restriction enzymes ClaI and SalI was used as a negative marker for a vector (pBS) containing a DNA cassette containing the thymidine kinase gene (tk).
II2SX neo / tk) was inserted between the ClaI and SalI sites to construct the desired bikunin-specific targeting vector (construct) (FIG. 2; Target Constru
ct).

<2> Establishment of ES cells The above-mentioned construct was transferred to ES cells (E14) using BioRad Ge.
Electroporation was performed using ne Pulser II. That is, first, the targeting vector was cut with PvuI and the linearized DNA was treated with phenol / chloroform, precipitated with ethanol, centrifuged, washed with 70% ethanol, and dried. This dried product is mixed with TE buffer (10
The solution was dissolved in 1 mM / μl using 1 mM Tris-HCl (pH 8.0) / 1 mM EDTA to obtain a plasmid solution.

In addition, the ES whose culture solution was exchanged the day before was used.
The cells are detached from the culture dish by trypsin treatment, the cells are released as single cells by trypsin treatment and pipetting, and after counting the number of cells, the ES cells are washed with phosphate buffered saline (PBS (-)). And 5 × 10 ⁷ /
The suspension was suspended in PBS (-) so that the volume of the suspension became ml.

After 0.7 ml of the cell suspension was mixed with 30 μl of the above plasmid solution, the mixture was transferred into a disposable cuvette for electroporation and left at room temperature for 5 minutes. After that, an electric pulse (250V, 500μF) is applied to the cells.
Was added. After standing at room temperature for 5 minutes, 30 ml of ES medium was added (1.2 × 10 ⁶ cells / ml), and this was seeded with feeder cells in advance, and 10 ml of 9 ml of ES medium was added.
3 ml (3.5 × 10 ⁶ ) cells were seeded on a culture dish having a diameter of 0 mm.

After 24 hours, G418 (200 μg / m
Positive selection was started using medium containing 1). Three days later, ganciclovir was further added to a final concentration of 2 μM to start negative selection. From day 9, the ES cells in culture were observed under a microscope, and 400 colonies were collected. From these colonies, 9 clones were selected as homologous recombinants by the PCR method and Southern hybridization method described below.

<3> Preparation of knockout mouse The homologous recombinant ES cells (# 162) obtained above were incorporated into 8-cell embryos of mice (C57BL / 6) by the agglutination method to produce chimeric mice. The obtained chimeric mouse (4 males)
Was bred to female C57BL / 6 mice and the offspring (F
The DNA extracted from the tail tip of 1) was investigated by the PCR method and Southern hybridization method described below, and (+ /
-) Mouse selected. The heterozygous mice were bred with each other to prepare (-/-) mice.

<4> Identification of Bikunin-Deficient Gene by PCR Method DNA was extracted from ES cells having formed colonies or the tail tip of a pup mouse and used for analysis.

A normal bikunin gene (wild type,
Bik +) was identified using the exon 7 region in the forward direction.
Using the Gene mL PCR System 9600 (PerkinElmer) with the sequence mL-C6 (SEQ ID NO: 2) and the reverse sequence of the exon 8 region (Reverse) mL-C9 (SEQ ID NO: 3) as the primary primer under the following conditions: A PCR reaction was performed. That is, after holding at 95 ° C. for 5 minutes, denaturation at 95 ° C. for 30 seconds,
45 ° C. annealing 45 seconds and 72 ° C. extension reaction 2
Cycle for 25 minutes and finally at 72 ° C.
For 5 minutes. The obtained primary PCR reaction solution was again subjected to the forward sequence of the exon 7 region located on the inner side.
Nesting PCR was performed using mL-C7 (SEQ ID NO: 4) and the reverse sequence mL-C8 (SEQ ID NO: 5) of the exon 8 region as secondary primates. The reaction conditions were: holding at 95 ° C. for 5 minutes, denaturation at 95 ° C. for 30 seconds, annealing at 56 ° C.
A cycle consisting of 5 seconds and a 2 minute extension reaction at 72 ° C.
This was repeated 30 times, and finally an extension reaction was performed at 72 ° C. for 5 minutes.

In order to identify the (Bik-) gene in which homologous recombination has occurred, the forward sequence neo1 in the neo region was used.
(SEQ ID NO: 6) and PvuII downstream of the homologous recombination sequence
Reverse of intron region sandwiched between sites (Reverse)
Using the sequence mL-C41 (SEQ ID NO: 7) as the primary primer
A PCR reaction was performed using the e Amp PCR System 9600 (Perkin Elmer) under the following conditions. That is, 95 ° C
After 5 minutes, a cycle consisting of denaturation at 95 ° C. for 30 seconds, annealing at 54 ° C. for 45 seconds and extension reaction at 72 ° C. for 2 minutes was repeated 25 times, and finally extension reaction was performed at 72 ° C. for 5 minutes. The obtained primary PCR reaction mixture was again subjected to the forward sequence neo7 (SEQ ID NO: 8) of the neo region located further inside and the reverse sequence mL-C40 of the downstream intron region.
Nesting PC using (SEQ ID NO: 9) as a secondary primer
R performed. The reaction conditions were maintained at 95 ° C. for 5 minutes,
A cycle consisting of denaturation at 5 ° C. for 30 seconds, annealing at 56 ° C. for 45 seconds and extension reaction at 72 ° C. for 2 minutes was repeated 30 times, and finally extension reaction was performed at 72 ° C. for 5 minutes.

The second PCR reaction solution was subjected to agarose gel electrophoresis, stained with ethidium bromide, and a band was detected by ultraviolet light. A DNA fragment of about 1 kb amplified with the Bik + primer set appears as a wild type (+ / +) and a hetero type (+/-), and a DNA fragment of about 1.8 kb amplified with the Bik- primer set is homotypic ( -/-) And heterotype (+/-).

<5> Identification of Bikunin-Deficient Gene by Southern Hybridization Method DNA was extracted from ES cells that formed colonies or the tail tip of pup mice and used for analysis. Restriction enzyme
After digesting with BalI, BamHI or KpnI at 37 ℃ for 18 hours,
The DNA fragment separated by 1% agarose gel electrophoresis was transferred to a nitrocellulose membrane according to a conventional method. A DNA fragment (0.7 kbp) flanked by PvuII cleavage sites contained in the downstream region of the bikunin chromosome gene was used as a probe 1, and a DNA fragment corresponding to the neo gene region of the targeting vector was used.
(0.8 kbp) was used as probe 2. Each probe was labeled according to a conventional method, and hybridized to the transcribed DNA fragment on the nitrocellulose membrane. After sufficiently washing the nitrocellulose membrane, a band generated was detected by autoradiography.

As a result, in the case of the DNA fragment digested with BalI, a 6.6 kb band of the wild type (normal cells and normal mice) was observed when hybridized with the probe 1, whereas the recombinant cells and F1 heterozygous cells were observed. In the type, a new 3.7 kb band appears in probe 1, and in probe 2,
A band of 2.9 kb was observed together with 3.7 kb. Also, BomH
Hybridization with 1 digested fragment with probe 1 showed only a wild type 11.3 kb band.
A 10 kb band newly appeared in the recombinant. When placed on the KpnI digested fragment and hybridized with probe 1, only a 12.9 kb band was observed in the wild type, a 4.8 kb band was observed in addition to the 12.9 kb in the hetero type, and only a 4.8 kb band was observed in the homo type (-/-). A band was observed. These results indicated that the obtained recombinant had the targeting vector integrated at the position of the bikunin chromosome gene as desired.

<6> Analysis of mRNA Expression by RT-PCR In order to examine the transcription status of molecules related to bikunin knockout, RT-PCR was performed. It performed using the primer set of the following table.

Since no band was observed in (-/-) mice, it is considered that bikunin mRNA was not expressed. In contrast, in (+/-) mice and wild type (+ / +), corresponding bands were observed, indicating that the expression of bikunin mRNA is normal.

The mR of HC1 and HC2, the other components of ITI
NA maintained the same expression as normal regardless of bikunin deletion. α-1-microglobulin (α-1-M) mRNA expression was found to be equivalent to normal (wild) in heterozygous mice, but slightly decreased in homozygous mice, but mRNA was decreased.
Was surely confirmed. The primers used for mRNA expression analysis are shown below.

[0041]

<7> mRNA by Northern blotting
Confirmation of expression Northern blotting was performed using bikunin cDNA and α-1-microglobulin cDNA as probes in order to examine the expression of bikunin and α-1-microglobulin mRNA in more detail. In the wild type (+ / +), using both bikunin probe and α-1-microglobulin probe,
The 1-microglobulin / bikunin precursor mRNA was hybridized, and in the (+/-) mouse, to a lesser extent, at the same position. However, (-/-) mice did not hybridize with the bikunin probe, and α-1-
Hybridization with the microglobulin probe revealed lower molecular bands. This is the α-1 corresponding to the mutant sequence created during the construction of the targeting vector.
-May correspond to a bikunin N-terminal truncated sequence via microglobulin and cleavage site.

<8> Confirmation of Protein Expression by Western Blotting In order to examine the expression of ITI at the protein level, Western blotting was performed using an anti-UTI antibody. Wild-type (+ / +) and (+/-) mice showed a 230 kD band corresponding to ITI and a 130 kD band corresponding to PαI (pre-α-trypsin inhibitor), whereas (−) No formation of the ITI family was observed in the (-) mice.

When the expression of α-1-microglobulin protein was examined using an anti-α-1-microglobulin antibody, it was found that the wild type (+ / +) and (+/−) and (− / −) In mice, bands derived from mature α-1-microglobulin were still observed in the same pattern. This suggests that, in addition to the normal protein translation of α-1-microglobulin precursor, the cleavage protein also forms the mature protein normally.

<9> Analysis of Bikunin Gene Knockout Mice When the (+/-) mice were crossed to determine the genotype of the offspring born, they were born almost according to Mendel's law, with little difference between males and females (see Table). 1).

[0046]

[Table 1] Table 1

The (-/-) mice were born normally in both males and females, and no abnormal growth was observed. The gestational cycle and copulation behavior seen in vaginal dirt were also normal. However, when the (-/-) female mice were bred, the pregnancy rate was remarkably low regardless of the genotype of the male, and the (-/-) female mice produced an average of 8 pups, There were many mice that did not exist, and if born, about one or two mice. Male (-/-) mice did not show such a tendency and were specific to (-/-) female mice (Table 2).

[0048]

[Table 2] Table 2

Observation in the uterus on the 5th day of fertilization shows that 7 to 10 embryos line up in wild-type (+ / +) mice and (+/-) pregnant mice, whereas (-/-) The female mouse of (1) showed only one or two embryos. However, the embryo itself was growing like a normal embryo. The offspring of the offspring die in 2-3 days, but the (-/-) mice born from (+/-) mice develop normally, and grow normally when attached to surrogate parents. So (-
/-) It seems that the parenting behavior of the mother mouse is a problem. Examination of the oviducts of mated (-/-) female mice revealed that fertilized eggs were present, but the number of ovulations was low, and almost no cumulus (cumulus oophorus) formation around the egg cells was observed. Was. The egg cell itself appears normal, but the hyaluronic acid-rich matrix around the egg cell is thought to be extremely low, and the lack of bikunin prevents the formation of the ITI family and the absence of the SHAP-HA complex. Is suggested.

[0050]

The DNA of the present invention is useful for targeting bikunin gene. In this mouse, only the bikunin gene was deficient, and the α-
Since the 1-microglobulin gene is completely maintained in a normal state, it is extremely useful for bikunin and functional analysis of the bikunin gene. Further, the model mouse of the present invention
It is extremely useful as a model mouse for infertility.

[0051]

[Sequence List] SEQUENCE LISTING <110> Seikagaku Corporation <120> Bikunin gene targeting DNA <130> J200002400 <140> <141> <160> 19 <170> PatentIn Ver. 2.1 <210 > 1 <211> 9274 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: modified mouse genomic DNA <220> <221> UNSURE <222> 4104 <223> n = a or g or c or t <400> 1 atcgatggaa agttcctcta ccacaaatcc agtaagtgag aggacacacc actgtctctt 60 ccttcccctt tctctccccg ctccaccctt gcagccttcc ttcccattta tctaagtagt 120 catccatcct tcccccaatt gcatcactgc catcttggca cccacagccc cccagtcacc 180 atatgaccag aactgggaga ggtgcagaat gccaaggatg acatcaacgg ctccccaccc 240 cacagcccca tgcttgtgtt ttgtggcaga gggatatggg cccaaattta ctcagagatt 300 tagcaactgt ctgttaactg tgctgtgtgt gccaaatcat tgtgtgtggg atgcacgccg 360 tggacggtga gcacgggaga gattgagtgc atgcgcaata gtaagcaaat gaacgggcag 420 atgagttcat gggtcatctg aaagacaggt ggagggagtg atggagggag aacagaagga 480 aggaaagaca gacagatggg aaagaag aag gaagaaagga aagctggcca tggtggttta 540 gcttaatatt ctcaagcaat gagttggagg gagaagggtc agaagttcaa agtcgtccct 600 ggctacatag caagctaaaa ggccagactg agcttaaatt agactctgct tttaaataaa 660 agagggagaa aagaacagga actggggagg gggaggagag ttagtgggca agagaggagg 720 agaacagaag ggtgggagga aggaagagag gaatgagaag aggaaggtag gaaaaaagag 780 agggcaggca ccagggaggg agagagacag agaaaaggaa acaagagcgg acttagagtg 840 ggtggatgga gtggaggtgg gtgagttgat gcggtctagc agctggagag agggatgctg 900 ggtgagcaga tgttgatccc ttagaagggc catgtggctg tggggatgga tgtggacacg 960 ctgagtggag gatgagtgat gggcctccag ggtgaagagc ctaaccactg cctccttcgg 1020 tctacctgca cctcccacac agaatggaac ataaccttgg aatcctatgt ggtccacacc 1080 aactatgacg aatatgccat tttccttacc aagaagtcca gccaccacca cgggctcacc 1140 atcactgcca agctctatgg taaaggcctt aagcatgagt ctgttagagc tggggtgggg 1200 tgggggcagt gattattcat ctatgaaccc tgggtgggaa ggagcaggaa gcttgtctct 1260 tctttatgag agaactcccg gttagggtgg gacaatgggc cctggccttg aatagctcag 1320 catcctgggt gaagtagaat tctcacacca tctctgaaag ccactcctag agccagggag 1380 acagttcaat gattaaagtg cttgccacca agaatgaggc caaattggga ttcccagaac 1440 ccacacaaac accaggtggg tgtggccatc tgcctgtaat tctagccagc cagcaggggg 1500 tctgtcagga tctgtcaggg gatcctaccc aagctggcaa gggacacagg tgtatctgtg 1560 ggttctgagt ttgactgaga gaccctaccc caatggtagg agagcaatgt aagccgattc 1620 cttcttcaac ccctggcttc cacctgcata ggtgcccaca tagacaaaca catgtgcgtg 1680 catgcatgca tgcacacctg tgcacacccc tccctaccat gaaaatggaa aggagccagg 1740 agaaagtgac tctagagtca tcgaggtcca gacaaagcca atgggaatag ccctgaataa 1800 ggacccagga cctatggtgg gcccagtctc tctcttggag acccatcttt caatggtatg 1860 ccaaggggag ttaatgggcc agtcctttcc acacttttca ctggacacag tccagctggc 1920 caggaagcct gggactctca gcttctcctc tgacttctgt ttgcaggtcg ggagccacag 1980 ctgagggaca gccttctgca ggagttcaag gatgtggccc tgaatgtggg catctctgag 2040 aactccatca tttttatgcc tgacagaggt aagtggtatt ttgcctgact ctgaattttg 2100 gttctcctct ccttcctgtc ccatagagag catccccagt ccctgcactc agagacaagc 2160 aaggccatgg tcctctccct ctctacagag actgagagag ctcc tgggtt tgcccaagtc 2220 tcagaacatg cactagcaga ggcagcactt aacgaattcc attcttatcc cagggtcaca 2280 agtctccatg agggtatcca cagacactgc cgaagaggca ggaaagcctt ggggttgagg 2340 gcacagactc cacagtcacc gtacccacat tgaagcttag tggctcaagc acagagtttt 2400 gacttctggc tagccatcct agacccttac ttacctcacc tgtcaaatgg gataataatg 2460 cgctccctat caaagcccac tgtgaggggt ctgtgagaga aaaaaagcca ttagaggatg 2520 cagcatgatg ctttggaagt gcccctgtca atgatctcct gaggatactg acagagtgat 2580 gctggtctgt taactgtccc atcaaaggct ttgagtgcag tgaggagggc cctccatgaa 2640 gtgctctaaa tcacaggaca cgatggccac agaaatctgg gtccaccctc cagagcctgg 2700 gtctcttgct cactcttctt ttgtgtcctc tgcattaggg gaatgtgtcc ctggggatcg 2760 ggaggtggag cccacatcaa ttgccgtgag tatctcttcc acccttcttc aacagcccca 2820 ctacagacat tctgtagaga ctggcacctt actgatgttt aagaactggc cagcaacacc 2880 agtttcttgg gcacaggagc agagacaatg tcagtgtctc tggggagagg gatgaggctc 2940 acaagcacag aaccacagtg gcctgtcaca ttgcatggga cagtgttgag cttgcaaatg 3000 ttccccagaa gcctatccac atagggagag tggaaggctt gaccaaggtg ttggctttta 3060 ggatgaccgt ggctgcctct ttggagcact taaaattcag accaacatga ctattgcagt 3120 ccccagtggt gacaaaactc tgggtccagt ggccagccaa agttgcttct gcattgatgt 3180 gatctttaaa acctcatcct taagggatgc cccaagaaaa attcagtaat ctctccctgg 3240 agagagacca ggtagatctc ctccagggac ttgatcttca cgtccttgtg caggtggctc 3300 agcttggtga caagagtcac tcctcatctt cagctttgtc tccatgtgtt aaaggaagca 3360 tgccagagat gaccactcag acatatgaat agccaattga aagtctttat tccaatcagc 3420 gggaaagtgc tcaggacttg agtgtagccc cgagtgtcca gaacacaggg tttttttttt 3480 taaaaaggca gaaaccatga cctcacatct cactcagcag ttgcatgggg gttgggggtt 3540 ggggccttct cttctcaaaa gcacatagtt tgaagcaagc acttttaaca gaagctaaga 3600 aaagttagct aggaggcccc aacctttgat tctgagaacg gagatgggta attttccatg 3660 ggattctcca tcaaggaggc catatcctag ttaaagatga aatagcctct gaaagaacac 3720 aaagtggagg agcgtctgca ctgtcctaac ccactatcca taagccctgt gccccaggcc 3780 atgggccaag cctcagccat aaccacggcc ttgctgactc ctctgtggaa gacgccacgc 3840 ctctcaagct ggagttcctg ggtcttctgg gcctttctgc actgaatcac ttacc atttg 3900 tattttccca aggaagaagc tacatttttt tcattttgtt tttatttaat gttcactggt 3960 gttttacctg taggtatgtt catgtgctat gtgtgcacta tctacagtgg ccagtagagg 4020 gcaatagata ccctagaact gggtttcaga cagttatgag ccatcacgtt agtgttggaa 4080 accaaacctg ggtcgtctgc aagnatcaac ttctgagtga cctctcaagt accacaccgc 4140 cttgtcactt ttaaaaagtt cttttaattt ggcatttatt tatttattta tttatttatt 4200 tatttatttg agacaaccgc cttgtcactt ttaaaaagtt cttttaattt ggcatttatt 4260 tatttatttg agacaatgcc tcgctgtgta gctccggctg tcctggaact cacaatatag 4320 agcaggctgg ccttgaactc agagagatct gcctgcctct gcctcccaag tgctgggatt 4380 aaaggcatgc gccaacacaa ccacctggta actatttatt ttgaaatgta tgcttcgctg 4440 gctttatatg ctagtctccc tcaattttct tctttattta gaaaatttag tttaaaaaaa 4500 aaaagtcaaa atccaatgac gtaccccagc tgagatagca gaacatggct agagctttca 4560 cagagccatt tgggtccatt accaggtgtt tgctaaggca tctacatata acccatgtct 4620 ctttattgcc taactggtat tgataccagt agaaagcagg gtgggaggta atcatattta 4680 tctgctttat cataatttcc ttacaattac attttacttc tggcctttaa gtttcctttt 4740 ctctggtgga aatgtttgcc ataacaaaca tttttcctcc caagaaaaac acatgcggca 4800 tatttctggg tccttgctgt cttagtgttt tgttcactgt gatgaaacac catgagcata 4860 agcaatccgg aaaagggctt atttggttta ctcttccacg tccctgttca tcattgaagg 4920 aagtcaggaa gggactctca cggggcagga ccctggaggc aggagctgat gcggagccca 4980 cggaggggtg atgcttcctg gcttgctcct tgtagctcag ccaccctgat tacttacaga 5040 agccaggatc ctgccaccta caatgggatg ggtctaccca catcaatccc tagttacgag 5100 aatgctctac tggcttgctt gcagcctgat tttatggagg cattttctca gttgaggtcc 5160 ttgcccctca gagagcttgt gtcaagttga cgtaagaacc ggtcagcatt cttacccagc 5220 ttagactgta tttctgtgag tccgtcatac cgtcagcagc ttgggtgaaa ggagaatggc 5280 tgcttctcac gatctccatc atttactctt gagcgtcttc ctgcgtttca atcctcccgt 5340 ttaccctgcg ggtctccagg ctcccggccc ttcttcctta cagtgtctat tcggaggtgt 5400 ttgtctgtct taattttgac gattttatgt ttttcctcac ggatggacaa cccaactcct 5460 tcttcaactc actaaagttt tcttctattg tttctagctt atttgtcttc ctcccacaac 5520 gcccaggatg tggatttatt tcctgcactg tctaaatcac aattcattat ctcagctatg 5580 aggtgaaacc aaacacctct tccaactcta atggtttcca tcactgatgg tggctcttac 5640 accaatagac actactgatc ttttactggt ggcttaattt ttttttcata tttcttgctc 5700 tcctacttct ttatccttgt gagttgtgaa tagtcctttc actgtaatcc aatttcttgg 5760 agcatctgta ttatctgact tgtttccctt ttctccatct ctcctttctc attaaagaaa 5820 gcctgtatgt caccaggtta agatgaatct ggaagagacc acgggccctg acggttttag 5880 ttacccctta aaagaagaaa caatctcaga ccaccataga actacctctt taagccttag 5940 accactcctg tgagcagaca ggctacctgg tgaccaatgt ctccatcccc atcccttctt 6000 taactggtct tgagcccttt ctgtgaattc agattgacgc tggggctagc atctcactag 6060 gcagtctggt ccgtttccac agctacaagg attcaggtga gcaacagcaa tgaagtctct 6120 gcaaagctgt tttcttctct ccccagagag cccggcgggc agtgctgccc caagagcaac 6180 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat 6240 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat 6300 catgtctgga tcccccgggc tgcaggaatt cgatatcaag cttatcgatc cgaacaaacg 6360 acccaacacc cgtgcgtttt attctgtctt tttattgccg atcccctcag aagaactcgt 6420 caagaa ggcg atagaaggcg atgcgctgcg aatcgggagc ggcgataccg taaagcacga 6480 ggaagcggtc agcccattcg ccgccaagct cttcagcaat atcacgggta gccaacgcta 6540 tgtcctgata gcggtccgcc acacccagcc ggccgcagtc gatgaatcca gaaaagcggc 6600 cattttccac catgatattc ggcaagcagg catcgccatg ggtcacgacg agatcctcgc 6660 cgtcgggcat gcgcgccttg agcctggcga acagttcggc tggcgcgagc ccctgatgct 6720 cttcgtccag atcatcctga tcgacaagac cggcttccat ccgagtacgt gctcgctcga 6780 tgcgatgttt cgcttggtgg tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc 6840 gcattgcatc agccatgatg gatactttct cggcaggagc aaggtgagat gacaggagat 6900 cctgccccgg cacttcgccc aatagcagcc agtcccttcc cgcttcagtg acaacgtcga 6960 gcacagctgc gcaaggaacg cccgtcgtgg ccagccacga tagccgcgct gcctcgtcct 7020 gcagttcatt cagggcaccg gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg 7080 ctgacagccg gaacacggcg gcatcagagc agccgattgt ctgttgtgcc cagtcatagc 7140 cgaatagcct ctccacccaa gcggccggag aacctgcgtg caatccatct tgttcaatgg 7200 ccgatcccat attggctgca gggtcgctcg gtgttcgagg ccacacgcgt caccttaata 7260 tgcgaagtgg a cctcggacc gcgccgcccc gactgcatct gcgtgttcga attcgccaat 7320 gacaagacgc tgggcggggt ttgctcgaca ttgggtggaa acattccagg cctgggtgga 7380 gaggcttttt gcttcctctt gcaaaaccac actgctcgac attgggtgga aacattccag 7440 gcctgggtgg agaggctttt tgcttcctct tgaaaaccac actgctcgaa gcttggctgg 7500 acgtaaactc ctcttcagac ctaataactt cgtatagcat acattatacg aagttatatt 7560 aagggttatt gaatatgatc ggaattccgg tcgacggtat cgataagctt gatatcgaat 7620 tccacatgta gatgcaggtg gcctgggtca gtcatattct gtttgtttgt ttgtttctgg 7680 ggacccatcc agtttcccag tctgtagaag aaggacctgc aatctagatg tcaggctccc 7740 ctggccctgt ctattgtcct gtgcatgtcc caacagcagg tgcaagacta actctccatg 7800 tccctctttc ctccacagcg gcctgcaatc tccccatagt ccaaggcccc tgccgagcct 7860 tcataaagct ctgggcattt gatgcagcac aagggaagtg catccaattc cactacgggg 7920 gctgcaaagg caacggcaac aaattctact ctgagaagga atgcaaagag tactgtggag 7980 tccctggtga tggtaggagg ccctcgggag tccagggagg caggcgaggg ggtcctggag 8040 agtaaacgct agggactgga gagtcaaatg atggactctt tcccaggtga gcagcctggt 8100 ccactctctg ggcctc agct tcccacctgg gaaataggcc aagcccagga atatgccctc 8160 gctggtccta gctggaccag agaagatgca aaggtcattt agcaagtgac atttcattct 8220 tacccaagga cccattctaa gtcattctct atctgcaggg tacgaggaac taatacgcag 8280 ttgaaggtgc cagtctgcaa gccagagggt agccactgtt tgtcacagcg cagtccagct 8340 tagatgatct ggacccaaat aaaacaagtt gtcacttcct agcattctgt gtgttggcgt 8400 ctcattgtca caataagggt actggagggg aagggcatga gtaaaccagc cttcccagga 8460 gagaagatgc atagtttgca atgtgacagc atcctgcagt ccaagatcct ggctgtacat 8520 catttcaaac acaacagtcc ccaactcttc agtagtggag tgtggttatt ttgctttata 8580 aacataacat aagaaagaaa acccagtaga tgatggtggt acatgccttt aatcccaaca 8640 cttgggaggc aggcagatct ctgtgagttc aaggccagag tggtctacag agtgaactcc 8700 aggacagcca gggctacaca gagaagcctt gtctcaaaca acaacaacaa caacaacaac 8760 aaccaacaaa accaaaaaaa agaaagcaaa aaccaaaaaa ctccaaccaa ccaaccaacc 8820 aaccaaccaa ccaaccaacc aaccaaccaa acaaacaaca acaacaaaaa gaaactgagt 8880 ctttaaaaag ggcaacagca tttactagac actattctca attctaagat cagcccccac 8940 cccaaaccca gaaactctga c acttgtttc tagaagaggc tgcacaaggg tcacaaagat 9000 gcaaggacaa ataagtagtg gtgtcctgca gtgcccaagt acccgaggct acccccaatc 9060 cttcccacat gcagctcttt ctagtgtttt cttctgagca gtcagtttgt ccctttaccc 9120 attcaaaatc acccagagct gcagcccctg ctctttctcc cccttgacct gtctcctggg 9180 gtcctcagca gccccccata tgccctggtg gactatcccc tggtcttcct ggtacatctg 9240 caacaaccag atcaagctta tcgataccgt cgac 9274 <210> 2 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for genomic PCR (forward) <400> 2 ccaagagagt gaggggtcag ggact 25 <210> 3 <211> 25 <212> DNA <213> Artificial Sequence <220 > <223> Description of Artificial Sequence: primer for genomic PCR (reverse) <400> 3 gtaatacctc tcttgcatcc ctagg 25 <210> 4 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for genomic PCR (forward, nested) <400> 4 gagccactaa taactgggac cctca 25 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequen ce: primer for genomic PCR (reverse, nested) <400> 5 caggggcctt ctgagtaatt gagct 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for genomic PCR (forward) <400> 6 cgacattggg tggaaacatt ccagg 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for genomic PCR (reverse) <400> 7 tggggtactg tgtggatgca gttag 25 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for genomic PCR (forward, nested) <400> 8 cgaagcttgg ctggacgtaa actcc 25 <210> 9 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for genomic PCR (reverse, nested) <400> 9 attctccagg gcatgggcat aggcc 25 <210> 10 < 211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (forward) <400> 10 caagaattca gggcaacctg 20 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (reverse) <400> 11 ttctcgagca cagcctggtc cctcc 25 <210> 12 <211> 24 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: primer for RT-PCR (forward) <400> 12 acggatcctc tcagctcaag aaat 24 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (reverse) <400> 13 ccctcgagtt tccaagatga 20 <210> 14 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (forward ) <400> 14 ccaagagagt gaggggtcag ggact 25 <210> 15 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (reverse) <400> 15 gggtccagat catctaagct ggact 25 <210> 16 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (forward) <400> 16 gctgatcatg cgtcaacact gc 22 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: primer for RT-PCR (reverse) <400> 17 cgtgatcatc tggcaattga cgtgggc 27 <210> 18 <211> 25 <212> DNA <213> Artificial Sequence <220> < 223> Description of Artificial Sequence: primer for RT-PCR (forward) <400> 18 cctggaatgt ttccacccaa tgtcg 25 <210> 19 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : primer for RT-PCR (reverse) <400> 19 ccatgatatt cggcaagcag gcatc 25

[Brief description of the drawings]

FIG. 1 is a diagram showing a site where a mutation is introduced in a DNA of the present invention.

FIG. 2 is a diagram showing a schematic configuration of the DNA of the present invention.

Claims (8)

[Reference number] J200002400 [Claims] 1. A D comprising the nucleotide sequence of SEQ ID NO: 1.
NA. 2. A vector comprising the DNA according to claim 1. 3. Exon 7 of the AMBP gene in the genome
Knocked out the portion corresponding to the bikunin gene of
Knockout mouse of bikunin gene. 4. The nucleotide sequence of SEQ ID NO: 1
The knockout mouse for a bikunin gene according to claim 3, which comprises a nucleotide sequence represented by 45 to 6250. 5. The method according to claim 3, wherein the α-1-microglobulin gene is maintained normally.
2. The knockout mouse according to 1. 6. The knockout mouse according to any one of claims 3 to 5, wherein both homologous chromosomes contain the nucleotide sequence represented by nucleotide numbers 6145 to 6250 of SEQ ID NO: 1. . 7. The knockout mouse according to any one of claims 3 to 6, which is a female mouse. 8. An infertile model mouse comprising the knockout mouse according to claim 7.