JP2000116382A - Dna for targeting hyaluronic acid synthase gene - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gene which can be used for targeting hyaluronic acid synthase 1 (HAS1) gene. SOLUTION: This is a DNA containing an introduction DNA which is to be introduced into a chromosomal DNA of a host cell, the first homologous region and the second homologous region which were linked to the 5'-side and 3'-side of the introduction DNA, respectively, and negative marker genes which were linked to the 5'-side of the first homologous region and the 3'-side of the second homologous region and can be expressed in the host cell, wherein the introduction DNA is constructed in such a way that it contains a positive marker gene which can be expressed in the host cell, and the introduction DNA and the 5'-side of the first homologous region and the 3'-side of the second homologous region which were linked to the introduction DNA and a region coding for an intracellular loop of hyaluronic acid synthase 1 of the chromosomal DNA can cause the recombination.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a DN used for gene targeting of a hyaluronic acid synthase gene.
About A.

[0002]

2. Description of the Related Art Hyaluronic acid (hereinafter, also referred to as "HA") is a high molecular weight glycosaminoglycan, and is a main constituent molecule of extracellular matrix such as articular cartilage. H
In A, glucuronic acid is converted to N-acetylglucosamine by β-acetylglucosamine.
It is composed of 1,3-linked disaccharide units (GlcUAβ1-3GlcNAc: GlcUA represents glucuronic acid, and GlcNAc represents N-acetylglucosamine) by β-1,4 linkage repetition. HAS ”) in vivo.

[0003] HA, together with the HA binding protein, interacts with cell surface receptors to regulate many cell behaviors such as cell adhesion, migration, differentiation and the like. In addition, HA is involved in many phenomena such as morphogenesis, regeneration, wound healing, tumor invasion, and cancer metastasis. These functions depend on many factors, such as HA concentration and chain length. For example, high molecular weight HA, at high concentrations, has been shown to inhibit endothelial cell growth, while low molecular weight HA stimulates cell growth and induces angiogenesis. Similarly, high molecular weight HA, at high concentrations, inhibits the growth of synovial cells and certain fibroblasts, whereas low molecular weight HA stimulates the growth of these cells at low concentrations.

It is considered that an effective means for elucidating the cell regulation mechanism of HA is to elucidate the mechanism of HA synthesis and to develop a method for controlling the synthesis of HA.

With respect to HAS, HAS 1, 2 and 3 have been identified, and it has been clarified that they differ in the enzymatic activity and the molecular size of the synthesized hyaluronic acid (Japanese Patent Application Laid-Open No. 9-224677, WO97 / 38113). Issue International Brochure, Spicer, AP, Augustine, ML, and
Mcdonald, JA (1996) J. Biol. Chem. 271, 23400-23.
406; Spicer, AP, Olson, JS and Mcdonald, JA
(1997) J. Biol. Chem. 272, 8957-8961; WO 98/00551, International Publication Pamphlet).

[0006]

The object of the present invention is to provide an HA
An object of the present invention is to provide a DNA that can be used for gene targeting of the HAS1 gene for controlling the activity of HAS1 among S and enabling the functional analysis of hyaluronic acid at an individual level.

[0007]

As a result of the research, the present inventors have found that the activity of HAS1 can be efficiently reduced by homologous recombination using DNA having a homologous region set so that a mutation can be introduced at a specific position in the HAS1 gene. They found that they could control well and completed the present invention.

[0008] That is, the present invention relates to the chromosome D of the host cell.
The introduced DNA to be introduced into the NA, the first homologous region DNA and the second homologous region DNA linked to the 5 ′ side and the 3 ′ side of the introduced DNA, and the 5 ′ side and the second homologous region DNA of the first homologous region DNA. A DNA linked to the 3 ′ side of the homologous region DNA and containing a negative marker gene that can be expressed in a host cell, wherein the introduced DNA contains a positive marker gene that can be expressed in a host cell; DNA and the first and second homologous region DNAs linked to the introduced DNA and the region encoding the intracellular loop of hyaluronan synthase 1 of the chromosomal DNA may cause homologous recombination. Provided is a DNA (hereinafter, also referred to as the DNA of the present invention) constructed so as to be capable of being used.

In the DNA of the present invention, the region encoding the intracellular loop of HAS1 is preferably at least a part of exon 5 of the HAS1 gene.

[0010] The host cell is preferably a mouse-derived cell. When the host cell is a mouse-derived cell, the first homologous region DNA can be preferably excised from the chromosomal DNA of the hyaluronic acid synthase 1 gene by digestion with restriction enzymes BamHI and HindIII, and DNA containing at least a part of exon 4 of the gene, and the second homologous region DNA is preferably a chromosomal DNA of the hyaluronic acid synthase 1 gene.
Can be cut out by digestion with the restriction enzymes HpaI and HindIII and the hyaluronan synthase gene 1
Is a DNA containing a stop codon.

[0011] The introduced DNA is preferably a positive marker gene that can be expressed in a host cell. The positive marker gene is preferably a neomycin resistance gene. The negative marker gene is preferably a diphtheria toxin A gene.

The present invention also provides a targeting vector containing the DNA of the present invention.

[0013]

Embodiments of the present invention will be described below. The DNA of the present invention can be used as a targeting vector for gene targeting of the HAS1 gene. For gene targeting,
This includes gene knockout, conditional gene targeting, point mutation introduction, knockin, and the like.

The DNA of the present invention is a chromosomal DNA of a host cell.
In order to introduce the introduced DNA into the region encoding the intracellular loop of HAS1 of the chromosomal DNA by homologous recombination, the homologous region DNA capable of causing homologous recombination should be 5 ′ and 3 ′ of the introduced DNA. It is connected to the 'side. That is, the introduced DNA, the first homologous region DNA and the second homologous region DNA linked to the 5 ′ and 3 ′ sides of the introduced DNA, and the region encoding the intracellular loop of hyaluronic acid synthase 1 of the chromosomal DNA. Are characterized in that the first homologous region DNA and the second homologous region DNA are constructed so that homologous recombination can occur.

In this specification, HAS1 is HAS
Among them, those identified as HAS1 (mouse: JP-A-9-224677, human: WO97 / 38113, International Publication Pamphlet, and other vertebrates: Spicer, AP et al.
(1998), J. Biol. Chem., 273, 1923-1932) and high homology (usually 85%) in the amino acid sequence.
Above).

The HAS1 intracellular loop means a portion where HAS1 protrudes into the cell from the cell membrane (see FIG. 1). Although this portion does not have a strict boundary, for example, in mouse HAS1, in the amino acid sequence shown in SEQ ID NO: 2, from the 227th to 228th position from the N-terminal to the 409th to 420th position from the N-terminal. Area. In mouse HAS1, the amino acid sequence encoded by exon 5 corresponds to the 360th to 583th positions from the N-terminal in the amino acid sequence shown in SEQ ID NO: 2.

As used herein, the term “introduction” means that a part of chromosomal DNA is replaced or inserted into chromosomal DNA by homologous recombination.

Since the active site of HAS1 is located on the intracellular loop (particularly the C-terminal region thereof), introduction of the introduced DNA into the region encoding the intracellular loop of HAS1 in the chromosomal DNA will result in the loop portion. The activity of HAS1 can be efficiently controlled by modifying the portion that encodes In addition, since transcription of the HAS1 chromosomal gene in the promoter region is normally started, the effect on other expression systems, particularly the expression of HAS2 and HAS3, is considered to be reduced. It is preferable to introduce the introduced DNA so as to modify the intracellular loop in at least a part of the 5 exon region (exon 5). For example, in the case of knockout, the introduced D
NA can be introduced.

The homologous region DN enabling such an introduction
Selection of A can be performed based on known information on chromosomal DNA of HAS1. That is, a D having a nucleotide sequence homologous to the nucleotide sequence before and after the nucleotide sequence to be modified
NA may be selected as the first homologous region DNA and the second homologous region DNA. For example, in the case of mouse HAS1, the first homologous region DNA is obtained from the chromosomal DNA of the hyaluronic acid synthase 1 gene by using the restriction enzymes BamHI and BamHI. About 2.1 kb DNA which can be excised by digestion with HindIII and contains at least a part of exon 4 of hyaluronic acid synthase 1 gene;
The second homologous region DNA was obtained from the chromosomal DNA of the hyaluronic acid synthase 1 gene by using the restriction enzymes HpaI and HindIII.
About 4.2 kb of DN that can be excised by digestion with E. coli and contains the stop codon of hyaluronic acid synthase gene 1
A. In the amino acid sequence of HAS1,
It is expected that there is a difference between the species and a difference within the same species (the presence of the corresponding gene).
It is easy for those skilled in the art to select NA.

Elements constituting the DNA of the present invention other than the above-mentioned homologous region DNA may be the same as those of the conventional targeting vector (for example, "Gene Targeting File ~ '97", Experimental Medicine Special Edition, Vol. 14). ,
No. 20 (1996), published by Yodosha, "Bio-Manual Series Gene Targeting-Generation of Mutant Mice Using ES Cells-", Experimental Medicine Supplement, Shinichi Aizawa, published by Yodosha (1995), U.S. Patent No. 5,464,764).

The introduced DNA is appropriately selected according to the purpose of gene targeting. When the purpose is knockout, it may consist of only the positive marker gene.

The positive marker gene is used for positive selection, and is contained in the introduced DNA. The positive marker gene is not limited as long as it is a gene that can be expressed in a host cell and can be positively selected.
Examples include a neomycin resistance gene, a hygromycin B resistance gene, a blasticidin S resistance gene, and the like. Preferably, the positive marker gene is a neomycin resistance gene. Usually, expression is enabled in a host cell by linking with an appropriate promoter. As such a promoter, those having a strong expression activity in a host cell are preferable. For example, when a cell derived from a mammal is used as the host cell, a phosphoglycerate kinase 1 promoter and the like can be mentioned.

The negative marker gene is used for negative selection, and can be expressed and selected when the desired homologous recombination has not occurred. In the DNA of the present invention, it is located 5 ′ to the first homologous region DNA and 3 ′ to the second homologous region DNA. The negative marker gene is not particularly limited as long as it is expressed in a host cell when introduced into chromosomal DNA.
For example, a thymidine kinase gene, a diphtheria toxin A gene and the like can be mentioned. Preferably, diphtheria toxin A
Is a gene. The negative marker gene can be expressed in a host cell by linking it to an appropriate promoter. As such a promoter, one having an appropriate expression activity is preferable, and when a cell derived from a mammal is used as a host cell, thymidine kinase (TK) is used.
Promoters and the like.

Further, in combination with the above-mentioned positive marker and negative marker, elements used for polyA selection can be incorporated and used for selection of recombinants.

The host cell may be one used for conventional gene targeting, and can be used as a large number of cells in culture, and is usually a cell capable of regenerating into an individual animal after being selected. Embryonic stem cells (ES cells) are used. The cells are usually derived from a mammal, preferably a mouse.

The introduction of the DNA into the host cell and the identification of the homologous recombinant can be performed in the same manner as in the conventional method. For example, the method of introducing the DNA into the host cell includes a method by electroporation. Examples of the method for identifying a homologous recombinant include PCR and Southern hybridization.

A knockout animal can be prepared from a homologous recombinant obtained using the DNA of the present invention in the same manner as in a conventional method.

[0028]

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. Identification of active center of mouse HAS1 <1> Construction of wild-type mouse HAS1 expression vector Mouse HAS1 obtained by the method described in JP-A-9-224677 and having the nucleotide sequence of SEQ ID NO: 1
cDNA was used.

The mouse HAS1 cDNA was ligated with a restriction enzyme BglII adapter according to a conventional method, and
AG-CMV-2 vector (Eastman Kodak)
Subcloned into the BglII site of pFLAG-HA
S1 was constructed. The pFLAG-CMV-2 vector is
It contains a CMV promoter and a sequence encoding a FLAG peptide consisting of 8 amino acids, and the peptide encoded by the cloned fragment is produced as a fusion protein with FLAG in cells into which the vector has been introduced.

<2> Construction of Site-Specific Mutant HAS Expression Vector Using a Quick-Change ^™ site-specific mutation kit, HAS1 has a mutation (Asp242Glu) in which the Asp residue at position 242 is replaced with a Glu residue, position 344. As
Mutation that replaces p residue with Glu residue (Asp344Gl
u) A mutation in which the Gln residue at position 380 is replaced with an Asn residue (Gln380Asn), and the Arg residue at position 383 is L
Mutation that replaces ys residue (Arg383Lys), 38
Mutation that replaces the Trp residue at position 4 with a Tyr residue (Trp residue)
384 Tyr) or a Gly residue at position 312 by Pro
A mutation was introduced into pFLAG-HAS1 so as to introduce a mutation (Gly312Pro) that substitutes for a residue.

In the HAS protein, the Asp residue at position 242, the Asp residue at position 344, and the Gln at position 380
The positions of the residues, Arg residue at position 383 and Trp residue at position 384 are shown in FIG.

Furthermore, in HAS1, a mutation (Thr337Se) in which the Thr residue at position 337 is replaced with a Ser residue is used.
r), and in addition to the mutation, a His residue at position 338 was replaced with Ly.
Mutation that substitutes s residue (Thr337Ser, His3
A mutation that introduced 38 Lys (= T337S, H338K) was introduced into pFLAG-HAS1.

The introduction of the desired mutation was confirmed by nucleotide sequencing.

<3> HA synthesis assay (1) COS of FLAG-tag labeled HAS expression vector
-1 Introduction into cells The FLAG-tag-labeled vector expressing wild-type HAS1 and mutant HAS1, constructed as described above,
COS-1 cells were transfected by electroporation. As a control, pFLAG-C
The MV-2 vector was transfected into COS-1 cells. Electroporation is performed using a commercially available device (Gen
e Pulser, Bio-Rad) according to the device manual. The transfected cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and 2 mM L-glutamine at 37 ° C. for 3 days.

(2) Measurement of HA synthesis amount A crude membrane fraction was prepared from the cultured cells, and 0.2 ml of 25
mM HEPES-NaOH (pH 7.1), 5 mM dithiothreitol, 15 mM MgCl ₂ , 1 mM UDP-
GlcNAc (Sigma), 0.1 mM UDP-Glc
A (Nakarai Tesque), and 2.5μ
CiUDP- [ ¹⁴ C] GlcA (28.5 mCi / mm
ol, DuPont NEN). The amount of protein was quantified using a BIO-RAD protein assay kit (Bio-Rad). HAS activity is Ng, KF
and Schwartzm, NB (1989) J. Biol. Chem., 264, 1
The measurement was performed by modifying the method described in 1776-11783. That is, after keeping the above suspension at 37 ° C. for 1 hour, 1 TRU of Streptomyces hyaluronidase (Seikagaku Corporation) was added or not at 37 ° C.
Incubated further for hours. Then, 1% (W /
V) SDS was added and boiled.

The HA having radioactivity was used as HiLoad1.
Separation was performed by chromatography using 30 / pg 6/60 Superdex (Pharmacia Biotech). Elution was performed using 0.2 M ammonium acetate,
Each 1 ml was fractionated, and the radioactivity was measured. Radioactivity incorporated into HA was measured by calculating a count of Streptomyces hyaluronidase sensitivity. HAS activity (picomoles / min / μg protein) was calculated from the radioactivity incorporated into the HA (Table 1).

[0038]

[Table 1] Table 1 ─────────────────── Mutant relative activity (%) ────────────────── ─ Wild-type HAS1 100 Asp242Glu <1 Gly312Pro 100-110 Thr337Ser 150 T337S, H338K 580 Asp344Glu <1 Gln380Asn 10-20 Arg383Lys 10-20 Trp1Tyr < ─

The use of a mutant HAS protein in which a specific amino acid on the intracellular loop has been substituted with another amino acid results in an extreme change in the relative activity of the mutant HAS protein with respect to the wild-type HAS protein.
1 was found to be on the intracellular loop (particularly its C-terminal region), and by mutating the portion of the gene on the chromosome that encodes the loop, HA
It was suggested that the activity of S1 could be controlled.

2. Isolation and identification of mouse HAS1 chromosomal gene Mouse HAS1 chromosomal gene is mouse HAS1 cDNA
Hybridization using mouse as a probe, the method of Yamada et al. (Yamad
a, Y., Itano, N., Zako, M., Yoshida, M., Lenas,
P., Niimi, A., Ueda, M., and Kimata, K., Biochem.
J., (1998) 330, pp1223-1227), and its structural analysis was performed using restriction enzymes. The result of the structural analysis is
It conformed to the structure of the chromosomal DNA described in the literature (FIG. 1).

3. Design of Targeting Vector for Knockout <1> Construction of Targeting Vector Particularly, the active site of HAS1 is located on the intracellular loop (the region from the vicinity of the 5 'end of exon 2 to the vicinity of 5' of exon 5 of chromosomal DNA). What was considered to be in the terminal region (5 ′ region of exon 5);
It is believed that the normal initiation of transcription in the promoter region of the chromosomal gene of AS1 reduces the effect on the expression of other expression systems, particularly HAS2 and HAS3.
A targeting vector was designed in which the fifth exon region, which is the final region of the AS1 translation region, and the fourth intron region upstream thereof were replaced with a positive marker gene (FIG. 2).

As the positive marker, antibiotic resistance was adopted, and phosphoglycerate kinase 1 (PGK-1) was used.
And a DNA cassette (PGKneo) containing the neomycin resistance gene (neo) downstream thereof (Gene (Amst), (1987) 60, 65-74 and Nucleic Aci).
ds Res., (1991) 19, 5755-5761). Since the positive marker gene contains a restriction enzyme BamH1 cleavage site, the HA of the chromosome is homologously recombined as described later.
This is advantageous for reconfirming that the targeting vector has been replaced by the S1 gene.

Furthermore, a toxin-producing ability was adopted as a negative marker, and a DNA cassette containing the TK promoter and the diphtheria toxin A gene (DTA) downstream thereof was used.
With respect to the positive marker gene, it was set at two locations, upstream and downstream, across the homologous recombination region.

The basic vector for construction is Bluescript II SK + plasmid vector (pBluescr
ipt II SK +: manufactured by Stratagene).

The PGKneopUC plasmid (pKJ-1: Dr. Mike) was inserted into the EcoRI site and the HindIII site of the Bluescript II SK + plasmid vector.
Bluescript II into which a positive marker gene was inserted by subcloning the PGKneopA cassette cloned from McBurney (granted from University of Ottawa)
An SK + plasmid vector was obtained. Ec of the vector
BamH1-H of chromosomal HAS1 gene at oRI site
An indIII fragment (including part of the third exon to part of the fourth intron; region III) was blunt-inserted,
Then, the chromosome H was added to the HindIII site in the vector.
The HpaI-HindIII fragment of the AS1 gene (including a part of the fifth exon and the part after it; region V) was blunt-inserted. Furthermore, a cassette having a TK promoter and a diphtheria toxin A gene (DTA) downstream thereof (M
Thanks to r. Piccotto (Pasteur Institute), Nature, 1
995,374, 65-67) was cut out with a restriction enzyme NotI to obtain a negative marker gene fragment, and the negative marker gene fragment was inserted into the NotI site of the above vector, and then blunted and inserted into the blunted XhoI site of the above vector. . An outline of the construction is shown in FIG.

<2> Electroporation The above construct was prepared according to the method of Andrew et al.
Spicer, Gerald J. Rowse, Thomas K. Lindner, and Sa
ndra J. Gendler, J. Biol. Chem., (1995) 270, 50, 3
[0093-30], ES cells (GK129) were electroporated using a BTX electroporator.
That is, the targeting vector of the above <1> was cut with SacII and linearized DNA (25 mg), treated with phenol / chloroform, precipitated with ethanol, centrifuged,
The precipitate was washed with 70% ethanol and dried. The dried product is washed with TE buffer (10 mM Tris-HCl (pH 8.0) / 1 mM EDTA) 3
Dissolved in 00 μl. ES whose culture solution was changed the day before
The cells are detached from the culture dish by trypsin treatment, the cells are released as single cells by trypsin treatment and pipetting, the number of cells is counted, and the ES cells are washed with phosphate-buffered saline (PBS). The cells were suspended in PBS so as to be × 10 ⁷ / ml. After 500 μl of the cell suspension was mixed with the plasmid solution, it was transferred into a disposable cuvette for electroporation and left at room temperature for 5 minutes. Thereafter, the cells were pulsed (400 V, 25 m
F) was added. After 5 minutes at room temperature, 12.5 ml of E
S medium was added (1.5 × 10 ⁵ / ml) and 2 ml (3 × 1
They were seeded the cells of 0 ^6). Then, G41 from 36 hours later
Positive selection was started using a medium containing 8 (150 mg / ml).

<3> Identification of homologous recombinant The ES cells in the culture were observed under a microscope, and 432 colonies were collected. From these colonies, two clones (# 166 and # 185) were selected as homologous recombinants by PCR and Southern hybridization.

(1) Identification by PCR method DNA was extracted from ES cells forming each colony according to a conventional method. Based on the sequence contained in the third exon and the sequence contained in the positive marker PGKneo region, 20 to 40 mer oligonucleotides (FIG. 2)
Medium, primers # 1 and # 2) were synthesized, and using them as primers, GeneAmpPCRSystem960
0 (PerkinElmer) under the following conditions:
A CR reaction was performed. That is, after holding at 80 ° C. for 2 minutes, a cycle consisting of denaturation at 94 ° C. for 10 seconds, annealing at 65 ° C. for 30 seconds and extension reaction at 68 ° C. for 5 minutes is repeated 10 times, and the same cycle is further repeated for 5 minutes. 25 minutes, extending 15 minutes per cycle from minutes to 25 times,
Finally, an extension reaction was performed at 72 ° C. for 10 minutes. DN amplified by PCR from ES cells derived from each colony
A fragment was subjected to agarose gel electrophoresis, stained with ethidium bromide, and a band was detected by ultraviolet light. A DNA fragment of about 2.1 kb generated by the amplification was cut out, and the nucleotide sequence was analyzed. As a result, DNA fragments derived from two ES cell colonies showed the third intron of HAS1 and the fourth intron of HAS1.
It is clear that the exon and a part of the sequence of the fourth intron inserted adjacent to the positive marker are included,
ES cells forming these two colonies (# 166 and
# 185) was found to be the desired homologous recombinant.

(2) Identification by Southern Hybridization Method Total DNA was prepared from the selected colonies (# 166 and # 185), and the DNA was converted to the restriction enzyme HindIII (3 U / mg) or B
After digestion with amHI (3 U / mg) at 37 ° C. for 18 hours, the DN
The A digest was subjected to 1% agarose gel electrophoresis according to a conventional method. And DNA separated by agarose gel electrophoresis
The fragment was transferred to a nitrocellulose membrane.

The second intron in the HAS1 chromosome gene (the region between the second and third exons)
, A DNA fragment of about 1 kb cut out with EcoRI was designated as "probe 3"("3" in FIG. 2), and the fourth exon of the targeting vector and the positive marker PGK
A DNA fragment of about 1.7 kb that spanned the neo region and was cut out with PstI and BamHI was used as an “internal probe” (“int” in FIG. 2). Each probe was radioactively labeled with ³² P according to a conventional method, and hybridized to the nitrocellulose membrane onto which the DNA fragment was transferred. After sufficiently washing the nitrocellulose membrane, a band generated was detected by autoradiography.

As a result, in the case of the HindIII digested fragment, only a band of about 3.5 kb was observed in normal cells (wild type) when hybridized with probe 3 or hybridized with the internal probe. In the recombinant cells, a band of about 9 kb was newly observed in each of the two colonies (# 166 and # 185). Also, BamHI
By hybridizing with the internal probe in the digested fragment, only a band of about 14 kb was observed in normal cells, while a band of about 2.5 kb was observed in both colonies of the recombinant cells.

From these results, the two recombinants were:
It was supported by the targeting vector that the desired homologous recombination occurred at the position of the HAS1 chromosome gene.

HAS1 expressed by this homologous recombination
Is believed to be deleted, thus knocking out the HAS1 gene.

[0054]

According to the present invention, there is provided a DNA which can be used for gene targeting of the HAS1 gene for controlling the activity of HAS1 and enabling the functional analysis of hyaluronic acid at an individual level.

[0055]

[Sequence List] <110> Seikagaku Corporation <120> DNA for gene targeting of the hyaluronan synthase gene <130> P-6032 <160> 2 <210> 1 <211> 2102 <212> DNA <213> Mus musculus <220> <221> CDS <222> 49..1800 <400> 1 CTAAAGAGAA CAAGACGGAG AAGAGAGAAT CCAGGAGGAC CCACAGCC ATG AGA CAG 57 Met Arg Gln 1 GAC ATG CCA AAG CCC TCA GAG GCA GCG CGT TGC TGC TCT GGC CTG GCC 105 Asp Met Pro Lys Pro Ser Glu Ala Ala Arg Cys Cys Ser Gly Leu Ala 5 10 15 AGG CGA GCA CTC ACG ATC ATC TTT GCC CTG CTC ATC CTG GGC CTC ATG 153 Arg Arg Ala Leu Thr Ile Ile Phe Ala Leu Leu Ile Leu Gly Leu Met 20 25 30 35 ACC TGG GCC TAC GCC GCA GGC GTT CCT CTG GCT TCA GAT CGC TAT GGA 201 Thr Trp Ala Tyr Ala Ala Gly Val Pro Leu Ala Ser Asp Arg Tyr Gly 40 45 50 CTC CTG GCC TTT GGC CTC TAT GGG GCA TTC CTC AGC GCA CAC CTA GTG 249 Leu Leu Ala Phe Gly Leu Tyr Gly Ala Phe Leu Ser Ala His Leu Val 55 60 65 GCA CAG AGC CTC TTC GCT TAC CTG GAG CAC CGA AGG GTG GCA GCG GCT 297 Ala Gln Ser Leu Phe Ala Tyr Leu Glu His Arg Arg Val Ala Ala Ala 70 75 80 GCG CGG CGC TCC TTG GCG AAG GGG CCC CTG GAT GCG GCC ACT GCA CGC 345 Ala Arg Arg Ser Leu Ala Lys Gly Pro Leu Asp Ala Ala Thr Ala Arg 85 90 95 AGC GTG GCA CTC ACC ATC TCA GCC TAC CAA GAG GAT CCC GCT TAC CTG 393 Ser Val Ala Leu Thr Ile Ser Ala Tyr Gln Glu Asp Pro Ala Tyr Leu 100 105 110 115 CGC CAG TGC TTG ACC TCC GCG CGC GCC TTG CTG TAC CCG CAC ACG AGG 441 Arg Gln Cys Leu Thr Ser Ala Arg Ala Leu Leu Tyr Pro His Thr Arg 120 125 130 TTA CGC GTG CTC ATG GTG GTG GAC GGC AAC CGC GCT GAG GAT CTG TAC 489 Leu Arg Val Leu Met Val Val Asp Gly Asn Arg Ala Glu Asp Leu Tyr 135 140 145 ATG GTG GAC ATG TTC CGA GAA GTC TTC GCC GAT GAG GAC CCC GCC ACT 537 Met Val Asp Met Phe Arg Glu Val Phe Ala Asp Glu Asp Pro Ala Thr 150 155 160 TAT GTG TGG GAT GGC AAC TAC CAT CAT CAG CCC TGG GAA CCA GCG GAG GCT 585 Tyr Val Trp Asp Gly Asn Tyr His Gln Pro Trp Glu Pro Ala Glu Ala 165 170 175 ACG GGC GCT GTC GGT GAA GGT GCC TAC CGG GAG GTG GAG GCG GAG GAC 633 Thr Gly Ala Val Gly Glu Gl y Ala Tyr Arg Glu Val Glu Ala Glu Asp 180 185 190 195 CCC GGG CGG TTG GCG GTG GAG GCG CTG GTG AGA ACA CGC AGG TGC GTG 681 Pro Gly Arg Leu Ala Val Glu Ala Leu Val Arg Thr Arg Arg Cys Val 200 205 210 TGC GTG GCT CAG CGT TGG GGC GGC AAA CGT GAG GTC ATG TAC ACA GCT 729 Cys Val Ala Gln Arg Trp Gly Gly Lys Arg Glu Val Met Tyr Thr Ala 215 220 225 TTC AAG GCA CTG GGC GAC TCC GTG GAC TAC GTG CAG GTC TGT GAC TCA 777 Phe Lys Ala Leu Gly Asp Ser Val Asp Tyr Val Gln Val Cys Asp Ser 230 235 240 GAC ACA AGA CTA GAC CCC ATG GCA CTG CTG GAG CTT GTG CGA GTG TTG 825 Asp Thr Arg Leu Asp Pro Met Ala Leu Leu Glu Leu Val Arg Val Leu 245 250 255 GAT GAA GAC CCC CGG GTA GGG GCT GTT GGA GGG GAT GTG AGG ATC CTT 873 Asp Glu Asp Pro Arg Val Gly Ala Val Gly Gly Asp Val Arg Ile Leu 260 265 270 275 275 AAC CCT CTG GAC TCC TGG GTC AGC TTC TTG AGC AGT CTT CGA TAC TGG 921 Asn Pro Leu Asp Ser Trp Val Ser Phe Leu Ser Ser Leu Arg Tyr Trp 280 285 290 GTA GCC TTC AAT GTG GAA CGA GCT TGT CAG AGC TAC TTC CAC TGT GTG 969 Val Ala Ph e Asn Val Glu Arg Ala Cys Gln Ser Tyr Phe His Cys Val 295 300 305 TCC TGC ATC AGT GGT CCT CTG GGT CTA TAC AGA AAC AAT CTC CTG CAG 1017 Ser Cys Ile Ser Gly Pro Leu Gly Leu Tyr Arg Asn Asn Leu Leu Gln 310 315 320 CAG TTC TTG GAG GCC TGG TAC AAC CAA AAG TTC CTG GGC ACC CAC TGC 1065 Gln Phe Leu Glu Ala Trp Tyr Asn Gln Lys Phe Leu Gly Thr His Cys 325 330 335 ACA TTT GGG GAT GAC AGG CAC CTC ACC AAC CGA ATG CTT AGC ATG GGC 1113 Thr Phe Gly Asp Asp Arg His Leu Thr Asn Arg Met Leu Ser Met Gly 340 345 350 350 355 TAT GCT ACC AAG TAT ACC TCG CGC TCC AGA TGC TAC TCG GAG ACG CCC 1161 Tyr Ala Thr Lys Tyr Thr Ser Arg Ser Arg Cys Tyr Ser Glu Thr Pro 360 365 370 TCC TCC TTC CTT CGT TGG TTG AGC CAA CAG ACC CGC TGG TCC AAA TCT 1209 Ser Ser Phe Leu Arg Trp Leu Ser Gln Gln Thr Arg Trp Ser Lys Ser 375 380 385 TAC TTC CGA GAG TGG CTA TAC AAT GCT CTG TGG TGG CAT CGC CAC CAC 1257 Tyr Phe Arg Glu Trp Leu Tyr Asn Ala Leu Trp Trp His Arg His His 390 395 400 GCA TGG ATG ACC TAT GAA GCG GTG GTC TCG GGC CTC TTC CCT TTC TTC 1305 Ala Trp Met Thr Tyr Glu Ala Val Val Ser Gly Leu Phe Pro Phe Phe 405 410 415 GTG GCT GCC ACG GTG TTG AGG CTC TTC TAT GCA GGG CGC CCG TGG GCT 1353 Val Ala Ala Thr Val Leu Arg Leu Phe Tyr Ala Gly Arg Pro Trp Ala 420 425 430 435 435 CTG CTC TGG GTG CTG CTC TGT GTG CAG GGC GTA GCA CTG GCA AAG GCA 1401 Leu Leu Trp Val Leu Leu Cys Val Gln Gly Val Ala Leu Ala Lys Ala 440 445 450 GCC TTT GCA GCC TGG CTG CGT GGC TGC GTG CGC ATG GTG CTG CTG TCA 1449 Ala Phe Ala Ala Trp Leu Arg Gly Cys Val Arg Met Val Leu Leu Ser 455 460 465 CTC TAT GCA CCA CTC TAC ATG TGC GGC CTC CTG CCT GCC AAA TTC CTA 1497 Leu Tyr Ala Pro Leu Tyr Met Cys Gly Leu Leu Pro Ala Lys Phe Leu 470 475 480 GCG TTG GTT ACC ATG AAT CAA AGT GGT TGG GGT ACC TCG GGC CGG AAG 1545 Ala Leu Val Thr Met Asn Gln Ser Gly Trp Gly Thr Ser Gly Arg Lys 485 490 495 AAA CTG GCT GCT AAC TAT GTC CCC GTG TTG CCC CTG GCA CTC TGG GCT 1593 Lys Leu Ala Ala Asn Tyr Val Pro Val Leu Pro Leu Ala Leu Trp Ala 500 505 510 515 CTA CTG CTG CTT GGA GGC CTG GCC CGC A GT GTG GCC CAG GAG GCC AGA 1641 Leu Leu Leu Leu Gly Gly Leu Ala Arg Ser Val Ala Gln Glu Ala Arg 520 525 530 GCT GAC TGG AGT GGC CCA TCC CGA GCA GCT GAA GCC TAC CAC CTT GCT 1689 Ala Asp Trp Ser Gly Pro Ser Arg Ala Ala Glu Ala Tyr His Leu Ala 535 540 545 GCT GGG GCT GGT GCC TAT GTG GCC TAC TGG GTG GTA ATG TTA ACT ATC 1737 Ala Gly Ala Gly Ala Tyr Val Ala Tyr Trp Val Val Met Leu Thr Ile 550 555 560 TAC TGG GTA GGT GTG AGG AGG CTG TGC AGA CGT CGG AGC GGT GGT TAC 1785 Tyr Trp Val Gly Val Arg Arg Leu Cys Arg Arg Arg Ser Gly Gly Tyr 565 570 575 CGT GTC CAA GTA TGAGTCCGGG CATGAAGATG CAGCTGAGGG CTCTAGAAGG18 CTCTAGAAGG GTGAGGCACC CCCTGGAGTC ATGGCTTTCT GGGGACTCAG TTTACCTTCT 1897 CTTTGAAATG AGGGAGTTAT TTAAGATTCT TCAATATGGA CTGTATTGGA ACTCAGATCT 1957 AGGTCTCTGG GGAGGGGTAA TTTATTGATC AGGGAGTGGG ATTGTAGGAC CAGGGCCAGA 2017 ACGTCTCCAT TTTCTCCCTG TTGCTATTTA ATGTTTGTAC TGTAAGATTA TATAATAAAT 2077 TTTTTATTTA TTTTCTTCTT TAGAG 2102 <210> 2 <211> 583 <212> PRT <213> Mus musculus <400 > 2 Met Arg Gln Asp Met Pro Lys Pro Ser Glu Ala Ala Arg Cys Cys Ser 1 5 10 15 Gly Leu Ala Arg Arg Ala Leu Thr Ile Ile Phe Ala Leu Leu Ile Leu 20 25 30 Gly Leu Met Thr Trp Ala Tyr Ala Ala Gly Val Pro Leu Ala Ser Asp 35 40 45 Arg Tyr Gly Leu Leu Ala Phe Gly Leu Tyr Gly Ala Phe Leu Ser Ala 50 55 60 His Leu Val Ala Gln Ser Leu Phe Ala Tyr Leu Glu His Arg Arg Val 65 70 75 80 Ala Ala Ala Ala Arg Arg Ser Leu Ala Lys Gly Pro Leu Asp Ala Ala 85 90 95 Thr Ala Arg Ser Val Ala Leu Thr Ile Ser Ala Tyr Gln Glu Asp Pro 100 105 110 Ala Tyr Leu Arg Gln Cys Leu Thr Ser Ala Arg Ala Leu Leu Tyr Pro 115 120 125 His Thr Arg Leu Arg Val Leu Met Val Val Asp Gly Asn Arg Ala Glu 130 135 140 Asp Leu Tyr Met Val Asp Met Phe Arg Glu Val Phe Ala Asp Glu Asp 145 150 155 160 Pro Ala Thr Tyr Val Trp Asp Gly Asn Tyr His Gln Pro Trp Glu Pro 165 170 175 Ala Glu Ala Thr Gly Ala Val Gly Glu Gly Ala Tyr Arg Glu Val Glu 180 185 190 Ala Glu Asp Pro Gly Arg Leu Ala Val Glu Ala Leu Val Arg Thr Arg 195 200 205 Arg Cys Val Cys Val Al a Gln Arg Trp Gly Gly Lys Arg Glu Val Met 210 215 220 Tyr Thr Ala Phe Lys Ala Leu Gly Asp Ser Val Asp Tyr Val Gln Val 225 230 235 240 Cys Asp Ser Asp Thr Arg Leu Asp Pro Met Ala Leu Leu Glu Leu Val 245 250 255 Arg Val Leu Asp Glu Asp Pro Arg Val Gly Ala Val Gly Gly Asp Val 260 265 270 Arg Ile Leu Asn Pro Leu Asp Ser Trp Val Ser Phe Leu Ser Ser Leu 275 280 285 Arg Tyr Trp Val Ala Phe Asn Val Glu Arg Ala Cys Gln Ser Tyr Phe 290 295 300 His Cys Val Ser Cys Ile Ser Gly Pro Leu Gly Leu Tyr Arg Asn Asn 305 310 315 320 Leu Leu Gln Gln Phe Leu Glu Ala Trp Tyr Asn Gln Lys Phe Leu Gly 325 330 335 Thr His Cys Thr Phe Gly Asp Asp Arg His Leu Thr Asn Arg Met Leu 340 345 350 350 Ser Met Gly Tyr Ala Thr Lys Tyr Thr Ser Arg Ser Arg Cys Tyr Ser 355 360 365 Glu Thr Pro Ser Ser Phe Leu Arg Trp Leu Ser Gln Gln Thr Arg Trp 370 375 380 Ser Lys Ser Tyr Phe Arg Glu Trp Leu Tyr Asn Ala Leu Trp Trp His 385 390 395 400 Arg His His Ala Trp Met Thr Tyr Glu Ala Val Val Ser Gly Leu Phe 405 410 415 Pro Phe Phe Val Ala Al a Thr Val Leu Arg Leu Phe Tyr Ala Gly Arg 420 425 430 Pro Trp Ala Leu Leu Trp Val Leu Leu Cys Val Gln Gly Val Ala Leu 435 440 445 Ala Lys Ala Ala Phe Ala Ala Trp Leu Arg Gly Cys Val Arg Met Val 450 455 460 Leu Leu Ser Leu Tyr Ala Pro Leu Tyr Met Cys Gly Leu Leu Pro Ala 465 470 475 480 Lys Phe Leu Ala Leu Val Thr Met Asn Gln Ser Gly Trp Gly Thr Ser 485 490 495 Gly Arg Lys Lys Leu Ala Ala Asn Tyr Val Pro Val Leu Pro Leu Ala 500 505 510 Leu Trp Ala Leu Leu Leu Leu Gly Gly Leu Ala Arg Ser Val Ala Gln 515 520 525 Glu Ala Arg Ala Asp Trp Ser Gly Pro Ser Arg Ala Ala Glu Ala Tyr 530 535 535 His Leu Ala Ala Gly Ala Gly Ala Tyr Val Ala Tyr Trp Val Val Met 545 550 555 560 Leu Thr Ile Tyr Trp Val Gly Val Arg Arg Leu Cys Arg Arg Arg Ser 565 570 575 Gly Gly Tyr Arg Val Gln Val 580

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing the structure of a mouse HAS1 gene (top) and the structure of mouse HAS1 in the cell membrane (bottom). In the gene structure, E1 to E5 indicate exons, and the upper line indicates a restriction enzyme cleavage site. In the HAS1 structure, the lower side is inside the cell.

FIG. 2: Chromosomal DNA of mouse HAS1 gene, targeting vector, and chromosome DN of mutant gene
It is a schematic diagram which shows the structure of A.

FIG. 3 is an explanatory diagram of construction of a targeting vector.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) (C12N 15/09 ZNA C12R 1:91) (C12N 5/10 C12R 1:91) F-term (Reference) 2G045 AA35 CB01 DA13 DA20 FB01 FB02 FB07 4B024 BA07 BA10 CA01 CA03 CA04 CA07 CA09 CA20 DA02 FA02 FA10 GA14 GA18 GA25 HA13 HA14 4B050 CC04 CC05 DD11 LL01 LL03 LL10 4B063 QQ08 QQ42 QR08 QR32 QR40 QR42 QR56 QR62QS01

Claims (8)

[Claims] 1. An introduced DNA to be introduced into a chromosomal DNA of a host cell, and a first homologous region DNA and a second homologous region DNA linked to the 5 ′ and 3 ′ sides of the introduced DNA, respectively.
And the 5'-side of the first homologous region DNA and the second homologous region DNA
DNA containing a negative marker gene that can be expressed in a host cell, linked to the 3 ′ side of
NA contains a positive marker gene that can be expressed in a host cell, and contains the introduced DNA and the first homologous region DNA and the second homologous region D linked to the introduced DNA.
DNA constructed so that homologous recombination between NA and a region encoding the intracellular loop of hyaluronic acid synthase 1 of the chromosomal DNA can occur. 2. The DNA according to claim 1, wherein the region encoding the intracellular loop of hyaluronan synthase 1 is at least a part of exon 5 of the hyaluronan synthase 1 gene. 3. The DNA according to claim 1, wherein the host cell is a mouse-derived cell. 4. The method according to claim 1, wherein the first homologous region DNA is obtained from the chromosomal DNA of the hyaluronic acid synthase 1 gene by using the restriction enzyme
DNA that can be cut out by digestion with HI and HindIII and contains at least a part of exon 4 of the hyaluronic acid synthase 1 gene,
NA is the chromosomal DNA of the hyaluronic acid synthase 1 gene
Can be cut out by digestion with the restriction enzymes HpaI and HindIII and the hyaluronan synthase gene 1
4. The DNA according to claim 3, which is a DNA containing a stop codon of
A. 5. The DNA according to claim 1, wherein the introduced DNA comprises a positive marker gene that can be expressed in a host cell. 6. The DNA according to any one of claims 1 to 5, wherein the positive marker gene is a neomycin resistance gene. 7. The DNA according to claim 1, wherein the negative marker gene is a diphtheria toxin A gene. 8. D according to any one of claims 1 to 7,
A targeting vector containing NA.