JP3646161B2 - Method for producing non-human animal enhanced foreign phase metabolism system by Keap1 gene modification - Google Patents

Description

【図面の簡単な説明】
【図１】ターゲティングベクター、野生型Ｋｅａｐ１遺伝子座、及びターゲティングベクターを用いた相同組換えにより得られると予想される、破壊されたＫｅａｐ１遺伝子座（組換え体）の構造を示す。
【図２】ＥＳ細胞（図２Ａ）及びＦ２マウス（図２Ｂ）におけるサザンブロット解析の結果を示す。
【図３】ＭＥＦの全ＲＮＡのＲＮＡブロット解析によるＫｅａｐ１破壊の確認を示す。
【図４】免疫ブロット分析による抗β−ガラクトシダーゼ抗体を用いたＬａｃＺｃＤＮＡノックインの確認を示す。
【図５】ＲＮＡブロット分析による代表的な異物代謝系第二相酵素群の発現を示す。
【図６】ＣＢＢ染色及び免疫ブロット解析による異物代謝系第二相酵素群の発現を示す。
【図７】免疫ブロット法による生後１０日目の肝臓核抽出物におけるＮｒｆ２発現を示す。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for producing a non-human animal in which a foreign substance metabolic system phase II enzyme group is constantly activated.
[0002]
[Prior art]
With the development of industry, environmental chemical substances with carcinogenicity and teratogenicity are increasing year by year, and improving the ability of these chemical substances to be released from the body is a basic technology that leads to the development of safer livestock animals. It is. The production of non-human animals such as domestic animals, which have enhanced metabolic ability for these chemical substances and environmental chemical substances are difficult to remain in the body, is a very interesting issue.
[0003]
When a living body is exposed to a xenobiotic, a group of detoxification enzymes is induced in the living body, and these enzymes convert the xenobiotic into a more water-soluble derivative. Such a biological reaction to environmental chemicals consists of a first phase of the foreign body metabolic system and a subsequent second phase reaction. The first phase reaction is a functional group addition reaction mainly by a cytochrome P-450 monooxidase system, which converts a chemically inactive compound into a reactive metabolic intermediate. The subsequent second phase reaction is a reaction with an enzyme such as glutathione S transferase (GST) or NAD (P) H: quinone oxidoreductase (NQO1), and the intermediate obtained in the first phase is generally more Converts to a less toxic derivative.
[0004]
It has been clarified that induction of the first phase of the foreign body metabolic system is controlled by an allyl hydrocarbon receptor (AhR: also known as dioxin receptor) which is a receptor transcription factor.
[0005]
On the other hand, several compounds have been known as drugs that activate the second phase of the foreign body metabolism system, but the mechanism of action has not been known. In addition, the induction was transient, and it was necessary to continuously administer the drug in order to induce constant activation.
[0006]
Moreover, although the transcription factor that controls the second phase of the xenobiotic metabolism system has not been clarified, the inventors of the present case have already confirmed that Nrf2 is a transcriptional activator of the xenobiotic metabolism type 2 phase enzyme group Reported ahead of. Furthermore, the inventors of the present case control the second phase of the xenobiotic metabolic system by controlling the nuclear translocation of Nrf2 having the strongest transcriptional activity in the CNC group (Cap'n 'color family). (Itoh, K. et al., Genes & Development 13: 76-86, 1999). That is, the following molecular mechanism involved in the Keap1-Nrf2 functional complex was proposed. Under non-stress conditions, Nrf2 binds to the β-propeller domain of Keap1 located in the cytoplasm, but it is protective against oxidative stress or stimuli such as phase II substrates metabolized by phase I detoxification enzymes. Occurs, Nrf2 is released from Keap1 and quickly translocates to the nucleus. As a result, Nrf2 forms a heterodimer with another bZip partner such as small Maf (hereinafter also referred to as sMaf) (Marini, MG et al., J Biol Chem 272, 16490-16497, 1997; Motohashi, H. et al., Nucleic Acids Res 25, 2953-2959, 1997). As a result, the heterodimer is an antioxidant response element (hereinafter also referred to as ARE) present in the gene regulatory region of the target gene (hereinafter also referred to as GRR) (Xie, T. et al., J Biol Chem 270, 6894-6900, 1995; Venugopal, R. and Jaiswal, AK, Oncogene 17, 3145-3156, 1998) through target gene expression (eg, phase 2 detoxification enzymes and oxidative stress-reducing proteins) Transactivate.
[0007]
In the individual level analysis of established Nrf2-deficient mice and the analysis of primary cultured macrophages isolated from the mice, inducible expression of their target genes was not observed (Itoh, K. et al., Biochem Biophys Res Commun 236). , 313-322, 1997; Itoh, K. et al., Free Radic Res 31, 319-324, 1999).
[0008]
[Problems to be solved by the invention]
Therefore, the purpose of the present invention is to elucidate the role of Keap1 and the function and control mechanism of Keap1-Nrf2 functional complex in vivo by paying attention to the transcription factor Nrf2 and cytoplasmic protein Keap1 that control the second phase of the foreign body metabolism system. It is to create a viable non-human animal in which the second phase of the foreign body metabolism system is constantly activated.
[0009]
[Means for Solving the Problems]
In order to solve the above problems, the present invention provides a method for producing a Keap1 hetero-deficient non-human animal using an embryonic stem cell clone in which the Keap1 gene is disrupted, and a Keap1 hetero-deficient non-human animal produced using the method. A method of producing a Keap1 homo-deficient non-human animal in which the second phase of the foreign body metabolic system is constitutively activated by mating, and Keap1 hetero-deficient non-human animal and Nrf2 homo-deficient mouse are mated and viable Keap1 Method for producing hetero deficient Nrf2 hetero deficient non-human animal, and Keap 1 hetero deficient Nrf 2 hetero deficient non-human animal produced by mating Keap 1 hetero deficient Nrf 2 hetero deficient non-human animal produced using said method Or a viable Keap1 homo-deficient Nrf2 heterozygous with constitutively activated foreign substance metabolism system second phase Provides methods for making losses nonhuman animal.
[0010]
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides a method for producing a non-human animal in which the second phase of the xenobiotic metabolism system is constantly activated. Specifically, a non-human animal in which a foreign substance metabolic system phase II enzyme group is constantly expressed is produced by deleting Keap1.
[0011]
In the present specification, the “foreign substance metabolic system” means a system that detoxifies the substance taken into the living body by exposure to water.
The “foreign substance metabolizing system second phase enzyme group” means a metabolic intermediate having a protocarcinogenicity produced by the action of a foreign substance metabolizing system first phase enzyme group such as cytochrome P-450 monooxidase, which is more toxic. A group of metabolic enzymes that metabolize to low intermediates. Examples of the second phase enzyme include glutathione S transferase such as GSTπ and GSTμ (hereinafter also referred to as GST), NAD (P) H: quinone oxidoreductase (hereinafter also referred to as NQO1), and epoxide hydratase. Can be mentioned.
[0012]
Examples of environmental chemical substances that cause a foreign body metabolic system response include carcinogenic substances, teratogenic substances, chronic toxic substances, subacute toxic substances, and acute toxic substances.
[0013]
As used herein, “constantly activated” refers to the second phase, even under non-stimulating conditions such as when administering a second phase substrate metabolized by oxidative stress or first phase detoxification enzyme. It means that the enzyme group is not inducible but expressed constantly.
[0014]
In the present specification, “non-human animals” means animals other than humans, for example, mammals other than humans (mouse, rat, rabbit, dog, cat, monkey, sheep, pig, cow, horse, etc.), Examples include birds (for example, chickens), amphibians (for example, frogs), reptiles, and insects.
[0015]
According to one embodiment of the present invention, by producing a mouse deficient in Keap1, the function of Keap1, which is an intracellular regulatory protein of Nrf2, can be clarified in vivo.
Here, “Nrf2” is a transcription factor of the CNC group as described above, and is a protein capable of forming a heterodimer with a small Maf group protein and binding to an antioxidant reaction sequence. Means a group control factor.
[0016]
“Keap1” ( K elch-like E CH A sociating P rotein1) is a protein that was cloned using a yeast two-hybrid system using a Neh2 domain conserved between species in Nrf2 as a molecule that directly binds to Nrf2 (Itoh, K. et al., Genes Dev 13, 76-86, 1999). Keap1 is a cytoplasmic regulatory protein of Nrf2, and suppresses Nrf2 activity by localizing Nrf2 in the cytoplasm.
[0017]
A Keap1 gene knockout non-human animal can be obtained by the following method. First, for example, embryonic stem cells in which at least one allele has been disrupted by homologous recombination (hereinafter also referred to as ES cells) are injected into blastocysts of fertilized eggs to obtain chimeric non-human animals. The resulting chimeric animal can be crossed with a corresponding wild-type non-human animal to produce a heterozygous non-human animal in which only one of the alleles is disrupted (hereinafter also referred to as Keap1 +/−). Furthermore, these obtained heterozygous non-human animals can be bred to produce homozygous non-human animals in which both alleles are disrupted (hereinafter also referred to as Keap1 − / −). Wild type is expressed as Keap1 + / +.
[0018]
In the case of a mouse, E14, CCE, J1, or T2 can be used as the ES cell.
Homologous recombination of ES cells can be performed, for example, by introducing recombinant DNA into which an arbitrary DNA sequence capable of interfering with Keap1 expression is inserted into ES cells. As the arbitrary DNA sequence capable of interfering with the expression of Keap1, it is preferable to use a drug resistance gene (for example, neomycin resistance gene (neo)) that can confirm introduction of recombinant DNA into ES cells, and a reporter gene. Although it is preferable to use (β-galactosidase (lacZ)) or the like, it is not particularly limited.
[0019]
Examples of the DNA introduction method include an electroporation method and a microinjection method. The Keap1 gene disruption in ES cells can be confirmed by, for example, PCR and Southern blot hybridization according to a conventional method.
[0020]
Examples of a method for producing a chimeric non-human animal having ES cells in which the Keap1 gene is disrupted by homologous recombination include a blastocyst microinjection method. In the blastocyst injection method, a desired chimeric non-human animal is produced by microinjecting ES cells in which Keap1 is disrupted into a non-human animal blastocyst and transplanting the treated blastocyst into a pseudopregnant non-human animal. can do.
[0021]
A heterozygous non-human animal obtained by mating a chimeric non-human animal obtained as described above with a corresponding wild-type non-human animal, or obtained by further mating the heterozygous non-human animal The homozygous non-human animal can be confirmed for its genotype by, for example, Southern blotting using DNA collected from the tail. In addition, the presence or absence of expression of the Keap1 gene can be confirmed by RNA blot analysis.
[0022]
The present invention also provides a method for producing a viable Keap1 heterodeficient Nrf2 heterodeficient non-human animal by crossing a Keap1 heterodeficient nonhuman animal and an Nrf2 homodeficient nonhuman animal.
Furthermore, the present invention creates a viable non-human animal in which the Keap1 hetero-deficient Nrf2 hetero-deficient non-human animals produced by the above method are bred with each other to constantly express the foreign phase II enzyme group. Provide a way to do it.
[0023]
A method for producing a Keap1-Nrf2 compound mutant non-human animal is obtained by mating a Keap1 +/− nonhuman animal and an Nrf2 − / − nonhuman animal obtained by the method according to the present invention as described above to obtain a wild type nonhuman animal. No difference Keap1 +/− Nrf2 +/− (which means that a heterodeficient for Keap1, ie, only one allele is destroyed, a heterodeficient for Nrf2, ie, only one allele is destroyed, Hereinafter, a non-human animal having a genotype of K1N1) is produced. By mating the non-human animals, non-human animals having all genotypes can be produced, and therefore non-human animals lacking both Keap1-Nrf2 can be easily obtained.
[0024]
According to a method for producing Nrf2 − / − non-human animals, embryo stem cells in which at least one allele has been disrupted by homologous recombination are first injected into a blastocyst of a fertilized egg according to the same method as Keap1 − / −. Alternatively, a chimera non-human animal in which ES cell-derived cells and embryo-derived cells are mixed is obtained by associating with mulberry embryos and then transplanting into pseudopregnant non-human animals. The resulting chimeric animal can be crossed with a corresponding wild-type non-human animal to produce a heterozygous non-human animal (hereinafter also referred to as Nrf2 +/−) in which only one of the alleles has been disrupted. Furthermore, these obtained heterozygous non-human animals can be bred to produce a homozygous non-human animal (hereinafter also referred to as Nrf2 − / −) in which both alleles are disrupted (Japanese Patent Application). Hei 11-064772). The wild type is expressed as Nrf2 + / +.
[0025]
Regarding K1N1 non-human animals, K0N0 non-human animals (Keap1-/-Nrf2-/-non-human animals), K0N2 non-human animals (Keap1-/-Nrf2 + / +), etc. obtained by the above method, for example, from the tail The genotype can be confirmed by Southern blotting using the collected DNA.
[0026]
Hereinafter, the present invention will be described in more detail with reference to examples. However, the scope of the present invention should not be construed as being limited thereby in any way.
[0027]
【Example】
Example 1: Production of Keap1-deficient mice
(1) Preparation of targeting vector
Two phage clones containing the full-length Keap1 gene and 5 ′ and 3 ′ flanking sequences long enough to create a targeting vector by screening the 129 · SvJ mouse genomic library (Stratagene) (Hereinafter referred to as clone 1 and clone 2) were isolated.
[0028]
Among the two phage clones obtained, clone 1 was cloned into pBluescript plasmid (Stratagene) using clone 1 suitable for targeting vector preparation, that is, having flanking sequence sufficient to perform homologous recombination. Was made. This targeting vector contains a nuclear localization signal (hereinafter also referred to as NLS) -β-galactosidase (lacZ) gene in an open reading frame (hereinafter also referred to as ORF) region of Keap1 cDNA between HindIII and SmaI sites. It is inserted and designed to replace the gene with the Keap1 gene. In order to select a transformant, a neomycin resistance (neo) gene is arranged downstream of the NLS-lacZ gene, and a diphtheria toxin A (DT-A) gene (for negative selection of heterologous recombinants) ( (Distributed by Dr. M. Taketoh, University of Tokyo) was inserted downstream of the Keap1 gene.
[0029]
The vector contains 5 ′ and 3 ′ genomic sequences, 5.5 kb and 2.7 kb, respectively, and is designed to place a 5.7 kb NLS-lacZ cDNA adjacent to neomycin. Yes. This targeting vector is designed to delete a sequence encoding the 197th amino acid residue from the 8th to 204th of Keap1, and the 7 amino acid residues on the N-terminal side of Keap1 are linked to NLS-LacZ. doing.
[0030]
FIG. 1 shows the structure of a targeting vector, a wild-type Keap1 locus, and a disrupted Keap1 locus (recombinant) expected to be obtained by homologous recombination using the targeting vector.
[0031]
(2) Introduction and confirmation of targeting vector into ES cells
25 μg of the targeting vector obtained in Example 1 (1) was linearized. ES cell (E14) approx. 1 × 10 ⁷ 20 μg of the linearized targeting vector was added to 0.6 mL of the medium in which the cells were suspended, and the ES cells were electroporated using Gene Pulser (Bio-Rad) under the condition of 0.21 kV / 0.5 F. The gene was introduced. To the cell suspension, 30 mL of a medium containing 0.3 mg / mL-G418 (Gibco) was added and cultured at 37 ° C. for 9 days.
[0032]
About 360 colonies of G418-resistant ES cells were obtained, high molecular weight DNA was prepared from the ES clones according to a conventional method, and recombinants were screened by polymerase chain reaction (hereinafter also referred to as PCR). PCR is a screening brimer that can anneal to the neo gene 1: 5′-TCAGAGCAGCCCGATTGTCTGTTTGCCCCAGTCAT-3 ′ (sequence of SEQ ID NO: 1 in the sequence listing), and a primer that can be annealed with the Keap1 gene outside the targeting vector Using 2: 5′-ACCCACGTCGATGCACGCGTGGAACACCTCGGG-3 ′ (sequence of SEQ ID NO: 2 in the Sequence Listing), 30 cycles of 98 ° C. for 10 seconds and 68 ° C. for 5 minutes were performed.
[0033]
As described above, when PCR was performed, among the 360 clones, 17 positive clones (clone that produced a DNA fragment of about 3.5 kb) in which the desired homologous recombination occurred were found. These positive clones were analyzed in more detail by Southern blotting to determine the genotype. As a result, Keap1 disruption was observed at any of the 17 ES clone loci, and it was confirmed that all of them were Keap1 heterozygous. It was. FIG. 2A shows the results of Southern blotting performed on wild type (+ / +), ES clone 1 (ES1) and ES clone 2 (ES2). In the analysis by Southern blotting, a DNA fragment (wild type: ˜7.7 kb, recombinant: ˜8.7 kb) generated when genomic DNA is digested with the restriction enzyme EcoRI is placed outside the targeting 5 ′. Hybridization with a probe (EcoRI-Xbal fragment: 140 bp) and targeting of a DNA fragment (wild type: ~ 4.5 kb, recombinant type: ~ 6.6 kb) generated when digesting genomic DNA with the restriction enzyme SacI This was carried out by hybridization with a 3 ′ probe (EcoRI-SacI fragment 200 bp) placed outside the vector (see FIG. 1).
[0034]
(3) Creation of Keap1 gene disrupted mice
Among the 17 types of clones obtained in Example 1 (2), 3 types of clones for ES clone germline chimera production (hereinafter referred to as ES clone 1, ES clone 2 and ES clone 3 respectively) were used. Chimeric mice were prepared by the blastocyst microinjection method. That is, one of the three types of ES cells was microinjected into C57BL / 6J mouse blastocysts, and the treated blastocysts were transplanted into pseudopregnant mice to produce chimeric mice.
[0035]
Using a chimeric mouse male produced using two of the three types of ES clones (ES clone 1 and ES clone 2), this was crossed with a C57BL / 6J female, and Agouti's hair color was changed. Obtained F1 offspring. Transmission of the mutant gene to the germline in F1 progeny was confirmed by Southern blot analysis of the tail genome. Furthermore, among the F1 progeny, F2 progeny were produced by crossing Keap1 heterozygous mutants, and the genotype was determined by Southern blotting in the same manner.
[0036]
FIG. 2B shows the result of Southern blot analysis performed on F2 progeny produced by crossing F1 produced using the ES clone. In FIG. 2B, + / + represents a wild type mouse, +/− represents a Keap1 hetero-deficient mouse, and − / − represents a Keap1 homo-deficient mouse. As a result of Southern blot analysis, a band of a DNA fragment derived from a disrupted Keap1 locus, which is expected to be obtained by homologous recombination, is observed. Therefore, F2 progeny have heterozygous and homozygous mice with respect to the Keap1 locus. It was confirmed that the disruption of the Keap1 gene occurred in these mice.
[0037]
(4) RNA blot analysis of Keap1 knockout mice
Embryonic fibroblasts (hereinafter also referred to as MEF) are taken out from the Keap1 hetero-deficient mouse and Keap1 homo-deficient mouse obtained in Example 1 (3), and wild-type mice, and RNA is extracted using ISOGEN (Nippon Gene). separated. 25 μg of the RNA was electrophoresed on a formamide / 1.2% agarose gel, transferred to a nylon membrane (HybondN +, Amarsham), and then BamHI- containing the complete ORF of mouse Keap1 cDNA derived from the pCMV-ECFP-mKeap1 expression vector. The XbaI fragment (1924 bp) was radiolabeled using a random primer label kit (Takara Shuzo) and used as a probe. 50% formamide, 4 × SSC (20 × SSC means an aqueous solution containing 3M NaCl and 0.3M sodium citrate: pH 7.0), 5 × Denhardt's reagent, 0.2% sodium dodecyl sulfate (hereinafter, The above-mentioned transfer membrane and the probe were hybridized at 42 ° C. overnight. The treated transfer membrane was first washed at 58 ° C. for 10 minutes in 2 × SSC containing 0.5% SDS, then washed at 58 ° C. for 1 hour, and finally 0.4% containing 0.1% SDS. X Washed in SSC at 60 ° C for 0.5 hour.
[0038]
A GAPDH probe was used as a control. Similar to the above Keap1 cDNA probe, the transfer membrane was hybridized with the GAPDH probe, washed in 0.5 × SSC containing 0.5% SDS at 60 ° C. for 20 minutes, and then 0.2% SDS. Washing was performed in 0.2 × SSC containing 0.1% SDS at 60 ° C. for 45 minutes and finally in 0.2 × SSC containing 0.1% SDS at room temperature for 20 minutes.
The transfer film was exposed to X-ray film (BioMax or XOR, Kodak) at −80 ° C. overnight using an intensifying screen.
[0039]
The results of RNA blot analysis performed as described above are shown in FIG. In FIG. 3, + / + represents a wild type mouse, +/− represents a Keap1 hetero-deficient mouse, and − / − represents a Keap1 homo-deficient mouse. The lower panel in FIG. 3 means a control. As a result, the level of Keap1 RNA present in the MEF of Keap1 hetero-deficient mice was about half of the level present in MEF of wild-type mice, and no Keap1 RNA was detected in the MEF of Keap1 homo-deficient mice. This shows that Keap1 destruction occurs in Keap1 hetero-deficient mice and Keap1 homo-deficient mice.
[0040]
(5) Confirmation of lacZ cDNA knock-in by immunoblot analysis
Total nuclear protein of liver extracted from wild-type mice, Keap1 hetero-deficient mice and Keap1 homo-deficient mice obtained in Example 1 (3) was analyzed using the dignum method (Dignam, JD, Methods Enzymol 182, 194-203, 1990). It was prepared by. The protein concentration of the nuclear extract obtained by the above method was determined using a protein assay reagent (BioRad), and bovine serum albumin was used as a standard substance.
[0041]
An equal volume of 2 × SDS sample buffer (100 mM Tris-HCl: pH 6.8, 200 mM dithiothreitol (hereinafter also referred to as DTT), 4% SDS, 0.1 to liver nuclear extract (20 μg protein) % Bromophenol blue, 20% glycerol) and immediately boiled for 5 minutes. This was separated with an appropriate SDS-polyacrylamide gel (hereinafter also referred to as PAGE), and further transferred to a PVDF membrane (Millipore). Subsequently, immunostaining was performed using an anti-β-galactosidase antibody (ICN).
[0042]
The immunostaining method is shown below. Using 2% normal serum / TBS-T (10 mM Tris-HCl: pH 8.0, 150 mM sodium chloride, 0.05% Tween 20) from the same animal as 3% skim milk and secondary antibody source, Block for 1 hour, then react the treated transfer membrane with anti-β-galactosidase antibody in 5% skim milk / TBS-T for 1 hour with an appropriate secondary antibody conjugated to horseradish peroxidase Detection was performed using an ECL color development kit (Amarsham). A band control of nuclear lamin B detected by an anti-lamin B polyclonal antibody (Santa Cruz) was used.
[0043]
The results of immunoblot analysis performed as described above are shown in FIG. In FIG. 4, + / + represents a wild type mouse, +/− represents a Keap1 hetero-deficient mouse, and − / − represents a Keap1 homo-deficient mouse. As a result, the NLS-LacZ level in the liver extract of the Keap1 homo-deficient mouse is about twice the NLS-LacZ level in the liver extract of the Keap1 hetero-deficient mouse. NLS-LacZ was not detected. From this, it was confirmed that the lacZ gene was knocked into Keap1 hetero-deficient mice and Keap1 homo-deficient mice.
[0044]
Furthermore, using β-galactosidase staining method (see Maniplating the Mouse Embryo, A Laboratory Method 2nd Edition), NLS-LacZ expression in Keap1 +/− mice or Keap1 − / − mice overlaps with cells of tissues expressing Keap1 mRNA. In addition, it was confirmed that LacZ is located in the nucleus (data not shown).
[0045]
(6) Observation of phenotype of Keap1-deficient mice
No difference was found in the phenotypes of hetero-deficient mice and wild-type mice born from the same mother obtained by F1 mating. However, several different phenotypes were found in homo-deficient mice.
[0046]
Homo-deficient Keap1 − / − mice were born normally with no difference in body size or behavior from wild-type mice or hetero-deficient mice born from the same maternal body. However, growth delay was observed from the fourth day of life. On the 5th and 6th days after birth, dandruff was found all over the body surface. In addition, compared to wild-type mice and hetero-deficient mice, homo-deficient mice clearly had a thick keratin layer on the ventral skin of the mice. On the other hand, no abnormality was found in the way the hair and teeth grew. The homo-deficient mice were debilitated and all died within 3 weeks.
[0047]
(7) Anatomical analysis of Keap1-deficient mice
Anatomical analysis revealed that the size of each organ of the Keap1 homo-deficient mouse was proportional to the size of the body. Although no histological abnormalities were observed in all organs except the esophagus and cardia, an abnormal keratin layer was observed on the inner wall of the esophagus and cardia as well as the skin. Furthermore, a huge tumor was observed in the cardia of the stomach of Keap1 homo-deficient mice. Analysis of stomach sections revealed that the tumor was composed of multiple layers of keratin. From the experiment of bromodeoxyuridine (BrdU) uptake using individuals, the proportion of immature squamous epithelial cells in the esophagus and cardia sinus of Keap1 homo-deficient mice is not significantly different from that of wild-type mice and heterozygous mice Okay (data not shown).
[0048]
(8) Immunohistochemical analysis in Keap1-deficient mice
For each genotype mouse, the expression of differentiation markers and proliferation markers related to epidermal cells was analyzed by immunohistochemical analysis.
Esophageal sections of wild-type mice, Keap1 heterodeficient mice, and Keap1 homodeficient mice were treated with antibodies against several types of cytokeratins. The primary antibodies against each cytokeratin used are anti-cK1, anti-cK13, anti-Loricrin, and anti-cK14 (all of which were kindly provided by Dr. Dennis Loop). The prepared sections were observed with a microscope (Leica DMRD, DMRE).
[0049]
As a result, intense changes were observed in some cytokeratins in wild-type mice, Keap1 hetero-deficient mice and Keap1 homo-deficient mice born from the same maternal body. In Keap1 homo-deficient mice, an increase was observed in cytokeratin cK1, and in particular, the differentiation marker Loricrin was significantly increased. On the other hand, the expression of cytokeratin cK13 decreased, and cK14 showed almost the same expression level as that of wild type mice.
[0050]
(9) Confirmation of cytokeratin changes by immunoblot analysis
RIPA buffer containing protease inhibitor cocktail (Biolab) (50 mM Tris-HCl: pH 8.0, 150 mM sodium chloride, 0.02% sodium azide, 0.1% SDS, 1% Nonidet P-40, 0.5% The esophagus 5 mm excised from each genotype mouse was homogenized in sodium deoxycholate.
[0051]
Except for the SDS concentration of the sample buffer being 10% SDS, the protein concentration was measured and the homogenized esophagus (5 μg of protein amount) was used as described in Example 1 (5). Immunoblot analysis was performed as described in The antibodies used were anti-cK6 (kindly from Dr. Dennis) and anti-Loricrin, and the band obtained with anti-cK14 (COVANCE) was used as a control.
As a result, an increase was observed in cytokeratin cK6 in Keap1 homo-deficient mice, and similarly to the result of Example 1 (8), Loricrin was remarkably increased, and cK14 showed almost the same expression level as that of wild-type mice. .
[0052]
As can be seen in the results of Example 1 (8) and Example 1 (9), it is not clear why some changes in cytokeratin occurred in Keap1 homo-deficient mice, but these changes are not related to Keap1 homo-deficiency. It is expected to contribute to the growth delay and short life of mice.
[0053]
(10) Histochemical analysis
The livers of mice 10 days after each genotype were stained with hematoxylin-eosin (hereinafter also referred to as HE staining).
As a result, from the histochemical analysis of the HE-stained sections, no difference was observed in all of the wild type, the Keap1 hetero-deficient mouse and the Keap1 homo-deficient mouse.
[0054]
(11) Confirmation of the expression of phase II detoxification enzymes by RNA blot analysis
As mentioned above, Nrf2 plays an important role in the inducible expression of phase 2 detoxification enzymes in the nucleus. Keap1 also traps Nrf2 in the cytoplasm and prevents Nrf2 from entering the nucleus in a normal cell state. From these viewpoints, it was considered that the relationship between Keap1 and the expression of the second phase enzyme group was very interesting.
[0055]
Therefore, the expression of the second phase detoxification enzyme group in the liver of Keap1-deficient mice was examined.
First, the expression (NQO1 and GSTπ) of several types of second-phase enzymes was confirmed by RNA blot analysis.
[0056]
Similar to Example 1 (4), Keap1 hetero-deficient mouse, Keap1 homo-deficient mouse, and wild type mouse, embryonic fibroblasts (MEF), total amount of embryo on day 12 (E12.5), newborn and postnatal day 10 Total RNA was isolated from day-old liver (P10 liver). Here, the entire ORF region of NQO1 and a part of the ORF region of GSTπ were used as probes. GAPDH was used as a control.
[0057]
The results of RNA blot analysis are shown in FIG. In FIG. 5, Wt represents a wild type mouse, Ht represents a Keap1 hetero-deficient mouse, and Hm represents a Keap1 homo-deficient mouse. As a result, the expression of NQO1 and GSTπ genes in Keap1 heterodeficient mice was remarkably increased in all RNAs such as MEF and liver as compared to wild type mice and Keap1 heterodeficient mice. From this, it was found that the expression of NQO1 and GSTπ, which are the second detoxification enzymes, increased in Keap1-deficient mice.
[0058]
According to the findings so far, the expression of these second phase enzyme group genes is usually induced via the first phase, but surprisingly, in the Kap1 homozygous mice, the RNAs of these second phase enzymes are constitutive. It turned out to be a high level.
[0059]
Therefore, in Keap1 homo-deficient mice, Nrf2 appears to be constitutively activated by translocating to the nucleus without being trapped by Keap1 even under normal cell conditions. Since the expression of Nrf2 and Keap1 is usually detected everywhere in the body, this increase phenomenon of Nrf2 due to Keap1 deficiency can occur not only in detoxified metabolic organs but also in various tissues. Therefore, deletion of Keap1 may also change the expression level of an unknown Nrf2 target gene, which may cause death of Keap1 homo-deficient mice.
[0060]
(12) Confirmation of the expression of phase II detoxification enzymes by CBB staining and immunoblot analysis
RIPA buffer containing protease inhibitor cocktail (Biolab) (50 mM Tris-HCl: pH 8.0, 150 mM sodium chloride, 0.02% sodium azide, 0.1% SDS, 1% Nonidet P-40, 0.5% 0.2 g of liver extracted from 10 days old mice of each genotype was homogenized in sodium deoxycholate).
The protein concentration of the liver extract obtained by the above-described method was determined using a protein assay reagent (BioRad), and bovine serum albumin was used as a standard substance.
[0061]
An equal volume of 2 × SDS sample buffer (100 mM Tris-HCl: pH 6.8, 200 mM DTT, 4% SDS, 0.1% bromophenol blue, 20% glycerol) was added to the liver extract (10 μg protein). Later, it was immediately boiled for 5 minutes. This was separated with an appropriate SDS-polyacrylamide gel (hereinafter also referred to as PAGE) and stained with Coomassie Brilliant Blue (hereinafter also referred to as CBB). A broad prestained marker (Bio-Rad) was used as a size marker.
The results of CBB staining are shown in the upper panel of FIG. As a result, clear induction of GST protein was confirmed. The induction was strong enough to be confirmed by protein staining with CBB dye.
[0062]
Furthermore, according to the description of Example 1 (5), immunoblot analysis was performed according to the description of Example 1 (5) using the liver extract (protein amount of 10 μg) obtained as described above. The expression level of GST protein was analyzed. The mouse GST subunits (α, μ, and π classes) were immunostained using a rabbit antibody that specifically reacts with each previously reported subunit (Itoh, K. et al., Biochem Biophys Res). Commun 236, 313-322, 1997). The Nuclear Lamin B band was used as a control band.
[0063]
The result is shown in FIG. In FIG. 6, Wt represents a wild type mouse, Ht represents a Keap1 hetero-deficient mouse, and Hm represents a Keap1 homo-deficient mouse. Regarding constitutively expressed GSTα, mice of all genotypes showed equal expression levels, but normally inducibly expressed GSTμ and GSTπ were significantly increased in the livers of Keap1 homo-deficient mice in both males and females.
Therefore, it has been clarified that deletion of Keap1 steadily increases the foreign substance metabolism system phase II detoxification enzyme group.
[0064]
(13) Confirmation of Nrf2 expression by immunoblotting
It can be expected that Nrf2 is automatically transferred to the nucleus without being trapped in the cytoplasm due to the destruction of Keap1. Therefore, Nrf2 is considered to accumulate in the nucleus. To verify this hypothesis, Nrf2 expression was confirmed by immunoblotting.
[0065]
According to Example 1 (5), immunoblotting analysis was performed on the nuclear extract of the liver of a 10-day-old mouse having each genotype. The anti-Nrf2 antibody used was produced by immunizing Japanese white rabbits with the N-terminal region recombinant polypeptide of Nrf2 using the adjuvant system (RIBI Immunochem Research, Inc). In addition, 293T-derived cell total protein transfected with pEFmock or pEFNrf2 expression vector using an anti-Nrf2 polyclonal antibody was used as a reference substance for Nrf2 detection, and total protein of D3T-treated mouse liver was used as a control for liver Nrf2 detection. As a loading control, the Nuclear Lamin B band was used.
[0066]
The result is shown in FIG. In FIG. 7, Wt represents a wild type mouse, Ht represents a Keap1 hetero-deficient mouse, and Hm represents a Keap1 homo-deficient mouse. Nrf2 expression increased in the liver of Keap1 homo-deficient mice.
Therefore, it was found that the high level expression of the foreign substance metabolic system phase II detoxification enzyme in Keap1 disrupted mice was due to constitutive activation of Nrf2.
[0067]
From the results of Example 1 above, it is possible to increase the expression of Nrf2 in non-human animals by disrupting the Keap1 gene, and to constitutively activate the foreign phase II enzyme group accordingly. It became clear that
However, the non-human animal produced in Example 1 and constantly active in the second-phase enzyme group has a definite disadvantage that growth is delayed and it is short-lived.
[0068]
Example 2: Production of Keap1-Nrf2-deficient mice
As described in Example 1, Keap1 homozygous mice have growth delay and various different phenotypes compared to wild type, and eventually die. As described above, lack of Keap1 results in a state of constant activation without Nrf2 control, suggesting that this contributes to these phenotypes and death. Therefore, the present inventors considered that various phenotypes and death can be avoided by further reducing the expression of Nrf2 from the Keap1 homo-deficient mouse, and in the Keap1 homo-deficient mouse, the mouse in which the Nrf2 gene is also half-deleted Decided to create.
[0069]
(1) Creation of Nrf2-deficient mice
1) Creation of targeting vector
Screening the 129 · SvJ mouse genomic library (Stratagene) using the full length Nrf2 cDNA as a probe, and including 5 ′ and 3 ′ flanking sequences long enough to generate a targeting vector, Two genomic phage clones with overlapping sequences were obtained.
Of the two genomic clones, a targeting vector was generated using a phage clone containing a 2.5 kb 5 'flanking sequence and a 9.5 kb 3' flanking sequence. This targeting vector is designed to replace the SV40NLS-lacZ recombinant gene with the b-Zip region of the Nrf2 gene. In order to select a transformant, a neomycin resistance (neo) gene is arranged downstream of the NLS-lacZ gene, and a diphtheria toxin (DT-A) gene is used for negative selection against a heterologous recombinant. It is designed to be placed upstream of the Nrf2 gene.
[0070]
2) Introduction and confirmation of targeting vector into ES cells
The targeting vector obtained in 1) of Example 2 (1) was linearized with a restriction enzyme and 4 × 10 4 ⁷ Genes were introduced into individual ES cells using an electroporation apparatus. Thereafter, the cells were cultured for 9 days at 37 ° C. in a medium containing 0.3 mg / mL-G418 (Gibco). As a result, about 1,000 G418-resistant ES cell colonies were obtained. From among these 96 colonies, a high molecular weight DNA was prepared by a conventional method, and a primary screening of a recombinant was performed by a PCR method. As a result, there were 21 clones in which the desired homologous recombination occurred. When these were analyzed in detail by Southern blotting, it was confirmed that the 21 clones were all heterozygous for the Nrf2 locus.
[0071]
3) Two types of ES clones (hereinafter referred to as ES cell clone No. 68 and cell ES clone No. 20) among the 21 types of clones obtained in 2) of Example 2 (1) above are germ cells. Chimeric mice were produced by two different methods shown below, which were used for the production of the chimeras. In the first method, that is, the blastocyst microinjection method, ES cell clone No. 5 was added to the blastocyst on day 3.5 of C57BL / 6J mice. 68 was microinjected and the treated blastocysts were transplanted into ICR pseudopregnant mice. In the second method, that is, the association method, C57BL / 6J mouse day 2.5 morula and ES clone no. 20 were associated and transplanted into ICR pseudopregnant mice. ES cell clone no. 68 or ES cell clone no. F1 offspring exhibiting a chinchilla color was obtained by crossing a male chimeric mouse produced using 20 with a female ICR mouse or female BALB / cA mouse. Transmission of mutant gene units in the F1 progeny to the germline was determined by Southern blot analysis of tail DNA. Furthermore, among the F1 progeny, F2 progeny were generated by crossing heterozygous mice with respect to the Nrf2 locus, and the genotype was confirmed by Southern blot analysis. In addition, it has already been confirmed that the Nrf2 homo-deficient mouse obtained by this can survive.
[0072]
(2) Production of Keap1-Nrf2 gene-deficient mice
The Kap1 heterodeficient mouse obtained in Example 1 (3) and the Nrf2 homodeficient mouse obtained in Example 2 (1) were crossed to produce a mouse having the K1N1 genotype. Furthermore, the K1N1 mice were crossed to produce mice of various genotypes.
[0073]
The genotype of each mouse was determined by PCR using the following primers. Nrf2 genotyping was initiated by a reaction at 96 ° C. for 2 minutes, and then PCR was performed by performing 35 cycles of 96 ° C. for 20 seconds, 59 ° C. for 30 seconds, and 72 ° C. for 45 seconds. The primers used were primer 3: 5′-TGGACGGGACTATTGAAGGCTG-3 ′ (sequence of SEQ ID NO: 3 in the sequence listing) and primer 4: 5′-GCCGCCCTTTTCAGTAGATGAGG-3 ′ (SEQ ID NO: of the sequence listing) that can anneal to the Nrf2 gene. 4), primer 5: 5′-GCGGATTGACCGTAATGGGATAGG-3 ′ (sequence of SEQ ID NO: 5 in the sequence listing) that can anneal to the lacZ gene located in the knock-in allele. The 734 bp and 511 bp band PCR products show the wild type allele and the mutant allele, respectively.
[0074]
The Keap1 genotype was started by a reaction at 94 ° C. for 1 minute, and then PCR was performed by performing 30 cycles of 94 ° C. for 30 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 1 minute. The primers used were primer 6: 5′-CGGGATCCCCCATGAGAAGGCTTATTGAGTTC-3 ′ (sequence of SEQ ID NO: 6 in the sequence listing) and primer 7: 5′-GAAGTGCATGTATATACACTCCCC-3 ′ (SEQ ID NO: of the sequence listing) that can be annealed with the Keap1 gene 7), primer 8: 5′-TCAGAGCAGCCCGATTGTCTGTTGGCCCAGTCA-3 ′ (sequence of SEQ ID NO: 8 in the sequence listing) that can anneal to the neo gene located in the knock-in allele. PCR products of 236 bp and 429 bp bands show the wild type allele and the mutant allele, respectively.
[0075]
(3) Observation of phenotype of Keap1-Nrf2-deficient mice
The Keap1 − / − Nrf2 + / + (K0N2) mice born from the same mother died several weeks after the Keap1 homo-deficient mice obtained in Example 1 (3). However, not only K0N0 mice but also K0N1 mice that are heterodeficient for Nrf2 were able to survive without dying. These K0N0 and K0N1 mice do not recognize any difference compared to the wild type, and the growth delay and dandruff seen in the Keap1 homo-deficient mice are not observed, and an abnormal keratin layer in the esophagus is not observed. It was.
Therefore, it was found that Nrf2 is deeply involved in the phenotype of Keap1 homo-deficient mice and contributes to death.
[0076]
(4) Histochemical analysis
The esophagus of K0N0 and K2N2 mice was observed with HE staining. From the histochemical analysis of HE-stained sections, the phenotype seen in Keap1 homo-deficient mice was not seen in K0N0.
[0077]
(5) Phase 2 enzyme gene expression
According to the description in Example 1 (11), the expression of the second phase enzyme gene in the K0N0 mouse and the K0N1 mouse was examined. As a result, the constitutive second phase enzyme group found in the liver extract of the Keap1 homozygous mouse Gene expression was comparable in K0N0 mice to normal expression levels in wild type mice. This expression level was dependent on the proportion of the Nrf2 gene (data not shown).
Therefore, it is suggested that GST expression is controlled by Nrf2.
[0078]
From the results of Example 2 above, Nrf2 causes a phenotype in Keap1 homo-deficient mice, causes an increase in abnormal keratin layer that is a cause of death, and is involved in the expression of foreign phase 2 metabolizing enzymes. Became clear.
In addition, it was revealed that K0N0 mice and K0N1 mice with reduced Nrf2 never die and can survive, but K0N0 mice are completely deficient in the Nrf2 gene and thus exposed to foreign substances. If you do, you no longer have the ability to metabolize it. On the other hand, K0N1 mice in which only half of the Nrf2 gene is deleted have excellent ability to excrete foreign substances because Nrf2 is constantly activated, and death is caused by overexpression of Nrf2. There is no.
[0079]
Furthermore, a mouse that is half-deficient in the Keap1 gene and a mouse that is also half-deficient in the Nrf2 gene (K1N1 mouse) grows and reproduces normally and is useful for the proliferation of K0N1 mice.
[0080]
Therefore, for the first time, it has become possible to produce a non-human animal that is constantly activated and capable of activating the foreign substance metabolism phase II enzyme group.
Moreover, it can be seen from the results of Example 1 and Example 2 that the complex of Keap1 and Nrf2 affects the death of the mouse individual. By destroying Keap1, the Keap1-Nrf2 complex is not formed, and Nrf2 moves to the nucleus. This causes overexpression of Nrf2 to cause various phenotypes and death to the individual. Therefore, it can be seen that Keap1 is an essential cytoplasmic molecule for individual survival and for regulating the function of Nrf2.
[0081]
Furthermore, since Nrf2-deficient mice can survive without expressing an inducible gene of a phase II enzyme, Keap1 plays a role in cellular response to xenobiotic stimulation and oxidative stimulation in the Keap1-Nrf2 complex, and the Keap1-Nrf2 complex Can play a very important role in maintaining the detoxification system.
[0082]
【The invention's effect】
By disrupting the Keap1 gene or preventing the interaction between Keap1 and Nrf2, the expression of Nrf2 is increased in non-human animals, and a foreign phase II enzyme group such as GSTπ and GSTμ is made constant. Can be activated.
[0083]
Further, in Keap1-disrupted mice, further deletion of one of the alleles of the Nrf2 gene allows survival in which the second phase of the foreign body metabolism system is constantly activated without overexpression of Nrf2 and without the need for drug administration. Possible non-human animals can be created.
[0084]
Furthermore, the present invention triggers the development of a powerful inducer of the foreign substance metabolism system phase II enzymes by blocking the interaction between Keap1 and Nrf2, thereby enabling cancer prevention and prevention of drug side effects.
[0085]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 shows the structure of a disrupted Keap1 locus (recombinant) expected to be obtained by homologous recombination using a targeting vector, wild-type Keap1 locus, and targeting vector.
FIG. 2 shows the results of Southern blot analysis in ES cells (FIG. 2A) and F2 mice (FIG. 2B).
FIG. 3 shows confirmation of Keap1 disruption by RNA blot analysis of total MEF RNA.
FIG. 4 shows confirmation of LacZ cDNA knock-in using anti-β-galactosidase antibody by immunoblot analysis.
FIG. 5 shows the expression of representative foreign body metabolic system phase II enzymes by RNA blot analysis.
FIG. 6 shows the expression of foreign substance metabolic system phase II enzymes by CBB staining and immunoblot analysis.
FIG. 7 shows Nrf2 expression in 10-day-old liver nuclear extracts by immunoblotting.

Claims (5)

  A viable non-human animal in which the second phase of the xenobiotic metabolism system is constitutively activated, and has a genotype of Keap1 homo-deficiency and Nrf2 hetero-deficiency.   A nonhuman animal that is a nonhuman animal for use as a parent animal for producing the nonhuman animal according to claim 1 and has a genotype of Keap1 heterodeficiency and Nrf2 heterodeficiency. A method for producing a viable non-human animal according to claim 2 comprising:
Creating a Keap1 heterodeficient non-human animal from an embryonic stem cell clone having a disrupted Keap1 gene;
A non-human animal obtained in the first step is crossed with a Nrf2 homo-deficient non-human animal to produce a non-human animal having a viable Keap1 hetero-deficient and Nrf2 hetero-deficient genotype Process and;
A method comprising: A method for breeding a viable non-human animal according to claim 2 comprising:
A method comprising the step of mating the non-human animals according to claim 2 to produce a viable non-human animal offspring having the same genotype as the parent. A method for producing a viable non-human animal in which the second phase of the xenobiotic metabolism system according to claim 1 is constantly activated:
A method comprising the step of mating the non-human animals according to claim 2 to produce a non-human animal having a Kap1 homo-deficient and Nrf2 hetero-deficient genotype.

Images