WO2015068411A1 - Genetically-modified non-human animal - Google Patents

Genetically-modified non-human animal Download PDF

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WO2015068411A1
WO2015068411A1 PCT/JP2014/058342 JP2014058342W WO2015068411A1 WO 2015068411 A1 WO2015068411 A1 WO 2015068411A1 JP 2014058342 W JP2014058342 W JP 2014058342W WO 2015068411 A1 WO2015068411 A1 WO 2015068411A1
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oxytocin receptor
base sequence
gene
human animal
site
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French (fr)
Japanese (ja)
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克彦 西森
志寿 日出間
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国立大学法人東北大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New breeds of animals
    • A01K67/027—New breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/20—Animal model comprising regulated expression system
    • A01K2217/206—Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0356—Animal model for processes and diseases of the central nervous system, e.g. stress, learning, schizophrenia, pain, epilepsy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention mainly relates to non-human animals having a specific base sequence.
  • Oxytocin is a 9-amino acid neuropeptide hormone that is thought to contribute to various physiological actions and reproductive behavior of animals through the oxytocin receptor (OTXR). So far, oxytocin receptor knockout mice have been created and have been shown to exhibit a phenotype that causes abnormal milk ejection (Patent Document 1, Non-Patent Document 1). In addition, knockout mice have been shown to exhibit a social behavioral disorder phenotype. However, further research is needed on the molecular mechanism of the oxytocin-oxytocin receptor.
  • Non-Patent Documents 2 to 4 In recent years, attention has been focused on that oxytocin exhibits an antidepressant action, an autism action, and an anti-schizophrenia action via an oxytocin receptor.
  • OTXR knockout mice are known to exhibit a haploinsufficient phenotype.
  • Haploinsufficiency is a phenomenon in which when one of the sister chromosomes is mutated, the amount of protein produced is insufficient, so that one chromosomal chromosome cannot be used alone and the phenotype is inherited dominantly.
  • Oxytocin receptor knockout mice exhibit behavioral abnormalities such as reduced social cognitive ability, increased aggression, and decreased maternal behavior.
  • Oxytocin receptor haplotype knockout mice one of the chromosomes is wild type and the other is knockout type
  • the main object of the present invention is to provide a non-human animal that can be used for elucidation of mechanisms such as physiological actions involving the oxytocin-oxytocin receptor system, development of new drugs based on the pharmacological action of oxytocin, and pharmacological tests. Let it be an issue.
  • the present inventor has made (i) a base sequence encoding an oxytocin receptor protein under the control of the promoter of the oxytocin receptor gene at at least one of the oxytocin receptor loci, (ii) an IRES sequence, And (iii) it has been found that a non-human animal having a base sequence encoding a site-specific recombinase can solve the above problem.
  • the present invention includes the following aspects.
  • Item 1 at least one of the oxytocin receptor loci, (i) a base sequence encoding an oxytocin receptor protein under the control of a promoter of the oxytocin receptor gene, A non-human animal having (ii) an IRES sequence and (iii) a base sequence encoding a site-specific recombinase.
  • Item 2 The non-human animal according to Item 1, wherein the IRES sequence is an IRES sequence derived from a human eIF4G gene.
  • the coding region of exon 3 of the oxytocin receptor locus is (I) a base sequence encoding an oxytocin receptor protein, Item 3.
  • the non-human animal according to Item 1 or 2 which is substituted with a base sequence comprising an IRES sequence and (iii) a base sequence encoding a site-specific recombinase.
  • Item 4 The non-human animal according to any one of Items 1 to 3, wherein the site-specific recombinase is Cre.
  • Item 5 The non-human animal according to any one of Items 1 to 4, wherein the oxytocin receptor protein has a tag sequence on the C-terminal side.
  • Item 6 The non-human animal according to any one of Items 1 to 5, further comprising a base sequence whose gene function can be modified by the site-specific recombinant enzyme.
  • Item 7 (I) a base sequence encoding an oxytocin receptor protein under the control of a promoter of the oxytocin receptor gene in at least one of the oxytocin receptor gene loci, (Ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombinase, and (II) A method for analyzing the function of an oxytocin receptor and / or a gene whose function can be altered, comprising using a non-human animal having a base sequence whose gene function can be altered by the site-specific recombinant enzyme.
  • Item 8 A vector comprising the following base sequence: (I) a base sequence encoding an oxytocin receptor protein, (Ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombination enzyme (iv) a base sequence that can be recombined with the base sequence of the oxytocin receptor locus.
  • the present invention provides a non-human animal that can be used as a new tool that can be used for elucidation of mechanisms such as physiological actions involved in the oxytocin-oxytocin receptor system, development of new drugs based on the pharmacological action of oxytocin, and pharmacological tests. Is done.
  • the structure of the knock-in vector used in the examples and the structure of the gene obtained by homologous recombination are shown.
  • the result analyzed by Southern blot hybridization is shown. 1: Oxtr-Cre +/- mouse, 2: Oxtr-Cre-Neo +/- mouse, 3: wild type (Wt) mouse.
  • the analysis result of milk injection ability is shown. Specifically, the effect of genotype on parturition and maternal behavior is shown.
  • (A) Hippocampus CA3 region and (B) DEn) (Dorsal endopiriform nucleus) staining are shown.
  • the right figure shows the enlarged view (Enlargedlargeview) of the left figure.
  • the result of X-gal staining in the brain of Oxtr-Cre +/ ⁇ : ROSA26 +/ ⁇ mouse (male) is shown.
  • LS Lateral septum
  • C Hippocampal CA3 region and DEn (Dorsal endopiriform nucleus),
  • DEn Dorsal endopiriform nucleus
  • Each staining is shown.
  • the sequence of the knock-in construct is shown.
  • oxytocin receptor Opytocin receptor
  • HA tag HA tag
  • human eIF4G IRES sequence Human eIF4G IRES sequence
  • Cre recombinase The sequence of the knock-in construct is shown (continued). Includes the FRT sequence (FRT site) and the base sequence of PGK-neo. The structure of AAV-loxP-WGA virus is shown. An arrow (Coding) indicates the direction of the code area.
  • Non-human animal The non-human animal of the present invention comprises (i) a base sequence encoding an oxytocin receptor protein under the control of a promoter of the oxytocin receptor gene at at least one oxytocin receptor gene locus,
  • the main features are (ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombinase.
  • Non-human animal may be any kind of non-human animal as long as it has an oxytocin receptor locus.
  • mammals other than humans that are closely related to humans are preferred. Examples of such mammals include apes such as monkeys, rodents (murines), rabbits, cats, dogs, horses, cows and the like.
  • Rodents such as mice, rats, guinea pigs and hamsters are preferable from the viewpoint of easy handling as experimental animals, and mice are more preferable from the viewpoint of easy genetic recombination operations.
  • a mouse a mouse belonging to an inbred strain such as C56BL / 6, C57BL / 6, BALB / c is particularly preferable.
  • the non-human animal of the present invention has the above base sequences (i) to (iii) under the control of the promoter of the oxytocin receptor gene at at least one of the oxytocin receptor loci. That is, the non-human animal of the present invention is a genetically modified non-human animal (transgenic non-human animal). It can be said that the above (i) to (iii) are knocked-in non-human animals. In this specification, it may be described as “the knock-in animal of the present invention”.
  • the non-human animal of the present invention may have an endogenous oxytocin receptor even if it has the base sequences of (i) to (iii) above (hetero) at one of the endogenous oxytocin receptor loci. Both of the loci may be those having the base sequences (i) to (iii) (homo).
  • the oxytocin gene and the oxytocin locus oxytocin receptor gene are known genes.
  • the oxytocin receptor protein encoded by the oxytocin receptor gene is a seven-transmembrane GTP-binding protein (G protein) -coupled receptor having oxytocin as a ligand.
  • G protein GTP-binding protein
  • the oxytocin receptor gene is highly conserved in fish (eg, zebrafish), birds (eg, chickens) and mammals (eg, humans, cows, mice).
  • the nucleotide sequence of the oxytocin receptor gene mRNA and the amino acid sequence of the oxytocin receptor protein are registered in GenBank provided by the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • the mouse is registered with the following accession number (if multiple revisions are registered, it is understood to refer to the latest revision):
  • Mouse oxytocin receptor protein NP_001074616
  • the oxytocin receptor locus encoding the oxytocin receptor is present on chromosome 6 in mice, for example.
  • the mouse oxytocin receptor locus is composed of four exons and three introns, and sequences encoding oxytocin receptor proteins are present in the third and fourth exons.
  • a base sequence encoding oxytocin receptor protein a cDNA sequence of oxytocin receptor can be mentioned as a suitable example.
  • the nucleotide sequence may be interrupted by a natural or artificial intron sequence (for example, the third intron of the oxytocin receptor).
  • the base sequence encoding the mouse oxytocin receptor is exemplified in SEQ ID NO: 1.
  • the oxytocin receptor encoded by the base sequence (i) is preferably derived from the non-human animal into which it is introduced, but is not limited thereto. .
  • the oxytocin receptor encoded by the base sequence is (x) one having an amino acid sequence of a natural oxytocin receptor, (y) one or more (for example, about 20 or less in the amino acid sequence of a natural oxytocin receptor; About 10 or less; about 9, 8, 7, 6, 5, 4, 3, 2) including amino acids substituted, added or deleted and having a function as an oxytocin receptor.
  • the oxytocin receptor protein may have a tag sequence at its terminal (amino terminal (N terminal) or carboxy terminal (C terminal), preferably C terminal).
  • tag sequence is not particularly limited, but HA tag (YPYDVPDYA) (SEQ ID NO: 2), FLAG tag (DYKDDDDK) (SEQ ID NO: 3), Myc tag (EQKLISEEDL) (SEQ ID NO: 4), V5 tag (GKPIPNPLLGLDST) (SEQ ID NO: 5) and the like are exemplified.
  • IRES sequence refers to a sequence that allows translation initiation independent of the CAP structure of mRNA. Since the non-human animal of the present invention has an IRES sequence, a base sequence encoding a protein downstream of IRES is also efficiently translated.
  • IRES sequence a known one can be used.
  • IRES sequence derived from picornavirus eIF4G (eukaryotic initiation factor-4G) gene, PDGF2 (platelet-derived growth factor 2) gene, VEGF (vascular endothelial growth factor) gene, IGF-II (insulin-like growth factor) II
  • examples include IRES sequences derived from genes such as genes.
  • the IRES sequence derived from the human eIF4G gene has a viewpoint that the expression level of the oxytocin receptor protein encoded by the base sequence (i) is comparable to the expression level of the wild-type oxytocin receptor protein.
  • SEQ ID NO: 6 shows the base sequence of the IRES sequence derived from the human eIF4G gene.
  • site-specific recombination enzyme refers to an enzyme that causes recombination of specific DNA at a specific recognition sequence (base sequence) or between specific recognition sequences.
  • specific examples of the site-specific recombinase include Cre recombinase and FLP recombinase.
  • the recognition sequence for Cre recombinase includes the loxP sequence, and the recognition sequence for FLP recombinase includes the FRT sequence.
  • the amino acid sequence of the site-specific recombination enzyme and the base sequence encoding it are known.
  • the base sequence encoding Cre recombinase is exemplified in SEQ ID NO: 7.
  • the base sequences (i) to (iii) are arranged under the control of the promoter of the oxytocin receptor gene. That is, the transcription product containing the nucleotide sequences (i) to (iii) described above is expressed with the same expression pattern (expression time, expression cell, expression intensity, etc.) as the wild-type oxytocin receptor gene. It only has to be done.
  • the base sequences (i) to (iii) are integrally transferred to the same transcription product, that is, transferred in a polycistronic (for example, bicistronic) manner.
  • the oxytocin receptor protein encoded by (i) and the site-specific recombinant enzyme encoded by (iii) are preferably expressed as separate proteins. In this case, the above (i) and (iii) each have a termination codon separately.
  • the non-human animal of the present invention preferably has the base sequences (i) to (iii) in this order, but is not limited thereto. For example, you may arrange
  • the coding region of the third exon of the oxytocin receptor locus is substituted with the above (i) to (iii).
  • the non-human animal of the present invention may further have a base sequence whose gene function can be modified by the site-specific recombinant enzyme.
  • a base sequence whose gene function can be modified all or part of the base sequence encoding a gene whose function on the genome is modified is sandwiched between the recognition sequences of the two site-specific recombinant enzymes. What is present is exemplified.
  • the site-specific recombination enzyme is Cre recombinase
  • all or part of the base sequence encoding the gene whose function is modified is sandwiched between loxP sequences.
  • the non-human animal of the present invention can be produced using a known genetic recombination technique.
  • a chimeric embryo obtained by introducing an embryonic stem cell (ES cell) in which the above (i) to (iii) are knocked in under the control of the promoter of the oxytocin receptor gene into a blastocyst is obtained from a female of the subject animal.
  • An example is a method of obtaining a chimeric animal by implantation in the uterus.
  • the knocked-in ES cell can be prepared using, for example, a vector having the following sequence: (I) a base sequence encoding an oxytocin receptor protein, (Ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombination enzyme (iv) a base sequence that can be recombined with the base sequence of the oxytocin receptor locus.
  • the base sequence capable of homologous recombination with the base sequence of the oxytocin receptor gene locus preferably refers to the 5'-side base sequence and the 3'-side base sequence of the insertion position.
  • the above-mentioned vector includes marker positive selection (eg, drug resistance gene such as neomycin resistance gene) for negative selection, and marker gene (eg, HSV (herpes simplex virus) thymidine kinase) for positive selection.
  • marker gene eg, HSV (herpes simplex virus) thymidine kinase
  • Tk diphtheria toxin
  • DT diphtheria toxin
  • the marker gene is preferably sandwiched between recognition sequences of the enzyme so that it can be removed by site-specific recombinant enzymes such as FLP recombinase and Cre recombinase.
  • the non-human animal of the present invention has a base sequence whose gene function can be modified by the site-specific recombination enzyme, for example, it has the obtained base sequences (i) to (iii). It can be obtained by mating a non-human animal with a non-human animal having a base sequence whose gene function can be modified.
  • the present invention also provides a function analysis method for an oxytocin receptor and / or a gene whose function can be altered, characterized by using a non-human animal having the following (I) and (II): provide: (I) at least one of the oxytocin receptor gene loci, under the control of the promoter of the oxytocin receptor gene (i) a base sequence encoding an oxytocin receptor protein, (Ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombinase, and (II) A base sequence whose gene function can be modified by the site-specific recombinant enzyme.
  • the function of a predetermined gene is specifically altered in cells in which the oxytocin receptor is expressed.
  • the function of the gene is lost in cells in which the oxytocin receptor is expressed.
  • the knock-in animal of the present invention described in the column “1.” is infected with the AAV-loxP-WGA virus shown in FIG. 9 (particularly, an oxytocin receptor expression site such as in the brain is preferable).
  • a non-human animal having the above (I) and (II) is produced.
  • the target protein Since the AAV-loxP-WGA virus shown in FIG. 9 has a base sequence encoding the target protein in the opposite direction to the direction of the promoter, the target protein is usually not expressed.
  • WGA wheat germ agglutinin
  • the target protein may be a single protein or a fusion protein of two or more proteins. In the case of a fusion protein, a fluorescent protein is an example of the fusion protein.
  • the base sequence encoding the target protein is a recognition sequence of a pair of site-specific recombinant enzymes (here, the loxP sequence is taken as an example, but is not limited thereto).
  • the loxP sequence is preferably sandwiched between irreversible inversion types such as a combination of loxPJTZ17 and loxP71.
  • the site-specific recombinase When the site-specific recombinase is expressed at the site of infection (here, Cre recombinase is taken as an example, but is not limited thereto), the site-specific recombinase is used as a recognition sequence pair. By causing a recombination reaction between them, the direction of the base sequence encoding the target protein is reversed, and the target protein is expressed.
  • AAV-loxP-WGA shown in FIG. 9 When AAV-loxP-WGA shown in FIG. 9 is used, recombination occurs only in cells expressing Cre recombinase (ie, cells expressing oxytocin receptor), and WGA-TdTomato is expressed.
  • TdTomato is a fluorescent protein. Since the WGA protein moves anterogradely between neurons, it moves to the target of neurons expressing the oxytocin receptor. For example, by visualizing the WGA protein by antibody staining, the target neuron can be identified and information on the network formed by the neuron expressing the oxytocin receptor can be obtained. Alternatively, visualization can also be performed with a fluorescent protein.
  • the target protein can be appropriately selected by those skilled in the art according to the purpose of analysis. For example, by using a rabies virus instead of the WGA protein, it is possible to visualize the projection source of a neuron from its retrograde nature.
  • nerves expressing the oxytocin receptor can be specifically removed by using a cytotoxic protein such as cholera toxin or thymidine kinase as the target protein.
  • a photoreceptor molecule such as channelrhodopsin (active) or halorhodopsin (inhibitory) is used as a target protein to express the photoreceptor molecule in a neuron that expresses the oxytocin receptor.
  • a photoreceptor molecule such as channelrhodopsin (active) or halorhodopsin (inhibitory) is used as a target protein to express the photoreceptor molecule in a neuron that expresses the oxytocin receptor.
  • Oxtr-Cre knock-in vector (Preparation of Oxtr-Cre knock-in vector) Oxtr cDNA and Cre recinbinase cDNA are inserted into the exon 3 region where the ORF (Open Reading Frame; protein coding region) start point of the oxytocin receptor (Oxtr) gene is present. Oxtr and Cre recinbinase are expressed by controlling the expression of the Oxtr gene. An Oxtr-Cre knock-in vector was constructed.
  • coli Ampicillin resistance gene required for conversion, drug resistance gene and its promoter (pgk-neo) necessary for positive selection, FRT sequence to remove pgk-neo when knock-in mice are made, for negative selection A 20 kb knock-in vector consisting of parts such as the thymidine kinase (tk) gene (mc1tk) of HSV (herpes simplex virus) having a property of killing cells by changing a nucleic acid analog into a cytotoxic substance as a gene of .
  • tk thymidine kinase
  • HSV herpes simplex virus
  • knock-in vector into ES cells
  • the knock-in vector was purified and linearized by restriction enzyme SalI digestion. Thereafter, the above knock-in vector was introduced into the ES cell line E14TG2a using the electroporation technique. Next, ES cell line colonies in which homologous recombination occurred were isolated and cultured by drug selection, and colonies in which homologous recombination occurred were screened using the Southern blot hybridization technique.
  • FIG. 1 shows the structure of the used knock-in vector and the structure of the gene obtained by homologous recombination.
  • the structure of the wild-type gene is shown at the top, the structure of the knock-in vector at the second stage, and the structure obtained as a result of homologous recombination at the bottom.
  • E1 represents exon 1
  • E2 represents exon 2
  • E3 represents exon 3.
  • XbaI represents an XbaI digestion site
  • XhoI represents an XhoI digestion site
  • SphI represents an SphI digestion site.
  • the sequence of the knock-in vector is shown in SEQ ID NO: 8 and FIG.
  • ES cells that had undergone homologous recombination were introduced into mouse blastocysts.
  • a blastocyst derived from a BL6 strain mouse into which ES cells were introduced was transplanted into a uterus of a foster parent mouse pseudo-pregnant by hormonal treatment to be mated with a male mouse having no fertility, and a chimeric mouse was obtained by giving birth. The character of the born mouse was confirmed by the color of the hair of the mouse.
  • Oxtr-Cre-Neo (+/-) mice were obtained by mating with chimeric mice using C57BL / 6J as a partner.
  • Mice with heterogeneous genotype obtained by mating C57BL / 6J with chimeric mice have a neomycin resistance gene (neo) inserted downstream of the strong promoter PGK. Yes. Due to these effects, it was predicted that the original Oxtr gene expression effect could not be maintained, and the Oxtr gene expression decreased.
  • Oxtr-Cre-Neo (+/-) mice were mated with Flippase mice expressing Flippase enzyme throughout the body, and heterozygous mice Oxtr-Cre (+/-) completely lacking PGK-Neo were obtained. Produced.
  • FIG. 2 shows the results of Southern blot hybridization analysis of the genotypes of Oxtr-Cre-Neo (+/ ⁇ ), Oxtr-Cre (+/ ⁇ ), and wild type mice.
  • Oxtr-Cre-Neo (+/-), Oxtr-Cre (+/-) when extracted from the tail DNA of wild-type mice and digested with the restriction enzyme XbaI, 3 'probe is used for detection
  • Wild type (WT) mice have a band at 13.5 kbp
  • Oxtr-Cre-Neo (+/-) and Oxtr-Cre (+/-) mice have bands at 13.5 kbp and 4.7 kbp.
  • FIG. 2A shows the results of Southern blot hybridization analysis of the genotypes of Oxtr-Cre-Neo (+/ ⁇ ), Oxtr-Cre (+/ ⁇ ), and wild type mice.
  • Oxtr-Cre (+ / + mice obtained by mating Oxtr-Cre (+/-), the expression of OXTR and CRE is considered to be derived from the completely inserted Oxtr gene and Cre recombinase gene. . From the inventor's knowledge, it has been shown that newborn mice born from Oxtr-/-female mice die 100% within 24 hours because milk is not ejected from Oxtr-/-female mice (Takayanagi Y., Nishimori K. et .al. PNAS 16096-16101 (2005)).
  • Oxtr-Cre (+ / +) mice expressed the Oxtr gene the milk ejection ability of female parent mice was examined.
  • OTXR Immunostaining with anti-HA antibody
  • OTXR is known to be expressed in the uterus.
  • the sample was washed 3 times with 1 ⁇ PBS for 5 minutes, then reacted with 0.5% TritonX-100 / 1 ⁇ PBS for 30 minutes at room temperature, and then washed with 1 ⁇ PBS for 10 minutes. Thereafter, blocking was performed with 5% Normal Horse Serum / 0.3% Triton X-100 / 1 ⁇ PBS (blocking buffer) at room temperature for 30 minutes. Thereafter, the primary antibody anti- ⁇ gal mouse antibody (1: 300) was reacted at 4 ° C. for 18 to 24 hours. After washing 3 times for 5 minutes with 1 ⁇ PBST, the secondary antibody Alexa 488 conjugated goat anti-mouse IgG antibody (1: 500) was reacted at room temperature for 2 hours.
  • X-gal solution (5 mM K3Fe (CN) 6, 5 mM K4Fe (CN) 6, 2 M MgCl2 dissolved in 1 ⁇ PB and adjusted to pH 7.4, where X-gal was 1 mg / ml And incubated at 37 ° C. overnight.
  • the sections were dehydrated with ethanol / xylene and sealed with Fisher® Scientific® Permount® Mounting® Medium.
  • blue staining derived from the expression of Cre recombinase was observed in the hippocampus, Dorsal endopiriform nucleus (Fig. 6). 5 and 6, it was confirmed that the OXTR-Cre knock-in mouse expressed Cre recombinase retaining the activity.

Abstract

　The present invention provides a non-human animal having (i) a base sequence encoding an oxytocin receptor protein, (ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombinant enzyme under the control of an oxytocin receptor gene promoter at at least one oxytocin receptor gene locus.

Description

遺伝子組み換え非ヒト動物Genetically modified non-human animals
　［関連出願の相互参照］
　本出願は、２０１３年１１月５日に出願された、日本国特許出願第２０１３－２２９３１５号明細書（その開示全体が参照により本明細書中に援用される）に基づく優先権を主張する。 [Cross-reference of related applications]
This application claims priority based on Japanese Patent Application No. 2013-229315 filed on Nov. 5, 2013, the entire disclosure of which is incorporated herein by reference.
　本発明は、主に特定の塩基配列を有する非ヒト動物に関する。 The present invention mainly relates to non-human animals having a specific base sequence.
　オキシトシン（Oxytocin、OXT）は、９つのアミノ酸からなる神経ペプチドホルモンであり、オキシトシン受容体（Oxytocin receptor、OTXR）を介して様々な生理作用、動物の生殖行動などに寄与すると考えられている。これまでにオキシトシン受容体のノックアウトマウスが作成され、乳汁射出が異常となる表現型を示すことが明らかにされている（特許文献１、非特許文献１）。さらに、ノックアウトマウスは、社会行動障害の表現型をも示すことが明らかにされている。しかしながら、オキシトシン－オキシトシン受容体の分子メカニズムについては、さらなる研究が必要である。 Oxytocin (Oxytocin, OXT) is a 9-amino acid neuropeptide hormone that is thought to contribute to various physiological actions and reproductive behavior of animals through the oxytocin receptor (OTXR). So far, oxytocin receptor knockout mice have been created and have been shown to exhibit a phenotype that causes abnormal milk ejection (Patent Document 1, Non-Patent Document 1). In addition, knockout mice have been shown to exhibit a social behavioral disorder phenotype. However, further research is needed on the molecular mechanism of the oxytocin-oxytocin receptor.
　また、近年は、オキシトシンがオキシトシン受容体を介して、抗鬱作用、抗自閉症作用、抗統合失調作用を表すことが着目されている（非特許文献２～４）。 In recent years, attention has been focused on that oxytocin exhibits an antidepressant action, an autism action, and an anti-schizophrenia action via an oxytocin receptor (Non-Patent Documents 2 to 4).
　斯かる状況下、オキシトシン－オキシトシン受容体の系が関与する生理作用等のメカニズム解明、オキシトシンの薬理作用に基づく新規な医薬の開発や薬理試験に用いることができる手段が求められている。 Under such circumstances, there is a need for means that can be used for elucidation of mechanisms such as physiological actions involving the oxytocin-oxytocin receptor system, development of new drugs based on the pharmacological action of oxytocin, and pharmacological tests.
　ここで、OTXRノックアウトマウスは、ハプロ不全の表現型を示すことが知られている。ハプロ不全とは姉妹染色体同士のうち一方に変異が起った場合、作られるタンパク質の量が不足するために片方の染色体だけではまかないきれずその表現型が優性的に遺伝する現象をいう。オキシトシン受容体のノックアウトマウスは社会認識能の低下、攻撃性上昇、母性行動低下などの行動異常を示す。オキシトシン受容体のハプロタイプのノックアウトマウス（染色体の一方が野生型、もう一方がノックアウト型）は攻撃性については野生型と同様であるが、社会認識能がノックアウトマウスと同様に低下することから社会行動の一部はハプロ不全を示すとされる（非特許文献５）。そのため、OTXR遺伝子を、ノックアウトを含む遺伝子組み換えにより操作する場合、ハプロ不全の影響が問題となる。 Here, OTXR knockout mice are known to exhibit a haploinsufficient phenotype. Haploinsufficiency is a phenomenon in which when one of the sister chromosomes is mutated, the amount of protein produced is insufficient, so that one chromosomal chromosome cannot be used alone and the phenotype is inherited dominantly. Oxytocin receptor knockout mice exhibit behavioral abnormalities such as reduced social cognitive ability, increased aggression, and decreased maternal behavior. Oxytocin receptor haplotype knockout mice (one of the chromosomes is wild type and the other is knockout type) have the same aggressiveness as wild type, but the social recognition ability is reduced in the same way as knockout mice. Is said to show haploinsufficiency (Non-Patent Document 5). Therefore, when the OTXR gene is manipulated by genetic recombination including knockout, the effect of haploinsufficiency becomes a problem.
　本発明は、オキシトシン－オキシトシン受容体の系が関与する生理作用等のメカニズム解明、オキシトシンの薬理作用に基づく新規な医薬の開発や薬理試験に用いることができる非ヒト動物を提供することを主な課題とする。 The main object of the present invention is to provide a non-human animal that can be used for elucidation of mechanisms such as physiological actions involving the oxytocin-oxytocin receptor system, development of new drugs based on the pharmacological action of oxytocin, and pharmacological tests. Let it be an issue.
　本発明者は、鋭意検討の結果、オキシトシン受容体遺伝子座の少なくとも１つにおいて、オキシトシン受容体遺伝子のプロモーターの制御下に（i）オキシトシン受容体蛋白質をコードする塩基配列、（ii）IRES配列、及び（iii）部位特異的組み換え酵素をコードする塩基配列を有する、非ヒト動物が上記課題を解決できることを見出した。 As a result of intensive studies, the present inventor has made (i) a base sequence encoding an oxytocin receptor protein under the control of the promoter of the oxytocin receptor gene at at least one of the oxytocin receptor loci, (ii) an IRES sequence, And (iii) it has been found that a non-human animal having a base sequence encoding a site-specific recombinase can solve the above problem.
　すなわち、本願発明は以下の態様を包含する。 That is, the present invention includes the following aspects.
　項１、オキシトシン受容体遺伝子座の少なくとも１つにおいて、オキシトシン受容体遺伝子のプロモーターの制御下に
（i）オキシトシン受容体蛋白質をコードする塩基配列、
（ii）IRES配列、及び
（iii）部位特異的組み換え酵素をコードする塩基配列
を有する、非ヒト動物。 Item 1, at least one of the oxytocin receptor loci, (i) a base sequence encoding an oxytocin receptor protein under the control of a promoter of the oxytocin receptor gene,
A non-human animal having (ii) an IRES sequence and (iii) a base sequence encoding a site-specific recombinase.
　項２、IRES配列が、ヒトeIF4G遺伝子に由来するIRES配列である、項１に記載の非ヒト動物。 Item 2. The non-human animal according to Item 1, wherein the IRES sequence is an IRES sequence derived from a human eIF4G gene.
　項３、オキシトシン受容体遺伝子座の第３エクソンのコード領域が、
（i）オキシトシン受容体蛋白質をコードする塩基配列、
（ii）IRES配列、及び
（iii）部位特異的組み換え酵素をコードする塩基配列
を含む塩基配列で置換された、項１又は２に記載の非ヒト動物。 Item 3, the coding region of exon 3 of the oxytocin receptor locus is
(I) a base sequence encoding an oxytocin receptor protein,
Item 3. The non-human animal according to Item 1 or 2, which is substituted with a base sequence comprising an IRES sequence and (iii) a base sequence encoding a site-specific recombinase.
　項４、部位特異的組み換え酵素が、Creである、項１～３のいずれか１項に記載の非ヒト動物。 Item 4. The non-human animal according to any one of Items 1 to 3, wherein the site-specific recombinase is Cre.
　項５、オキシトシン受容体蛋白質が、Ｃ末端側にタグ配列を有する、項１～４のいずれか１項に記載の非ヒト動物。 Item 5. The non-human animal according to any one of Items 1 to 4, wherein the oxytocin receptor protein has a tag sequence on the C-terminal side.
　項６、前記部位特異的組み換え酵素により、遺伝子機能が改変され得る塩基配列をさらに有する、項１～５のいずれか１項に記載の非ヒト動物。 Item 6. The non-human animal according to any one of Items 1 to 5, further comprising a base sequence whose gene function can be modified by the site-specific recombinant enzyme.
　項７、（I）オキシトシン受容体遺伝子座の少なくとも１つにおいて、オキシトシン受容体遺伝子のプロモーターの制御下に
（i）オキシトシン受容体蛋白質をコードする塩基配列、
（ii）IRES配列、及び
（iii）部位特異的組み換え酵素をコードする塩基配列、並びに、
　（II）前記部位特異的組み換え酵素により、遺伝子機能が改変され得る塩基配列
を有する非ヒト動物を用いることを特徴とする、オキシトシン受容体及び／又は前記機能が改変され得る遺伝子の機能解析方法。 Item 7, (I) a base sequence encoding an oxytocin receptor protein under the control of a promoter of the oxytocin receptor gene in at least one of the oxytocin receptor gene loci,
(Ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombinase, and
(II) A method for analyzing the function of an oxytocin receptor and / or a gene whose function can be altered, comprising using a non-human animal having a base sequence whose gene function can be altered by the site-specific recombinant enzyme.
　項８、下記の塩基配列を含むベクター：
（i）オキシトシン受容体蛋白質をコードする塩基配列、
（ii）IRES配列、及び
（iii）部位特異的組み換え酵素をコードする塩基配列
（iv）オキシトシン受容体遺伝子座の塩基配列と相同組み替え可能な塩基配列。 Item 8. A vector comprising the following base sequence:
(I) a base sequence encoding an oxytocin receptor protein,
(Ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombination enzyme (iv) a base sequence that can be recombined with the base sequence of the oxytocin receptor locus.
　本発明により、オキシトシン－オキシトシン受容体の系が関与する生理作用等のメカニズム解明やオキシトシンの薬理作用に基づく新規な医薬の開発や薬理試験に用いることができる新規なツールとなりうる非ヒト動物が提供される。 The present invention provides a non-human animal that can be used as a new tool that can be used for elucidation of mechanisms such as physiological actions involved in the oxytocin-oxytocin receptor system, development of new drugs based on the pharmacological action of oxytocin, and pharmacological tests. Is done.
実施例で使用したノックインベクターの構造、及び相同組み換えにより得られる遺伝子の構造を示す。The structure of the knock-in vector used in the examples and the structure of the gene obtained by homologous recombination are shown. サザンブロットハイブリダイゼーションにより解析した結果を示す。１：Oxtr-Cre+/-マウス、２：Oxtr-Cre-Neo+/-マウス、３：野生型（Wt）マウス。(A)３’プローブ、(B)５’プローブ、(C)Neo プローブ。The result analyzed by Southern blot hybridization is shown. 1: Oxtr-Cre +/- mouse, 2: Oxtr-Cre-Neo +/- mouse, 3: wild type (Wt) mouse. (A) 3 'probe, (B) 5' probe, (C) Neo probe. 乳汁射出能の解析結果を示す。具体的には、分娩及び母性行動に対する遺伝型の効果（Effect of  genotype on parturition and maternal behavior）を示す。野生型（Wt, Oxtr+/+）の雄（Male）とOxtr+/+、Oxtr-/-及びOxtr-Cre+/+の各遺伝子型の雌（Female）との交配（Pairing）における、妊娠した雌マウスの数（No. of pregnant female）、産子数の平均値（Average of litter size）、生存動物の平均値（Average of survivor）及び出生数の合計に対する出生後生存数の割合（％）（Postnatal survivors per total birth %）を示す。The analysis result of milk injection ability is shown. Specifically, the effect of genotype on parturition and maternal behavior is shown. Pregnancy in mating (Pairing) of wild type (Wt, Oxtr + / + ) male (Male) with female of Oxtr + / + , Oxtr -/- and Oxtr-Cre + / + genotypes No. of pregnant females, average of litter size, average of survivor, and ratio of survival after birth to total number of live births (% ) (Postnatal survivors per total birth%). 子宮切片における抗HA抗体による免疫染色の結果を示す。The result of the immunostaining by the anti-HA antibody in a uterine section is shown. Oxtr-Cre+/-:ROSA26+/-マウス（雄）の脳におけるβ-ガラクトシダーゼ抗体による免疫染色像を示す。（Ａ）海馬（Hippocampus）ＣＡ３領域、（Ｂ）DEn (Dorsal endopiriform nucleus)での染色をそれぞれ示す。右図は、それぞれ左図の拡大図（Enlarged view）を示す。The immuno-staining image by the beta-galactosidase antibody in the brain of Oxtr-Cre +/-: ROSA26 +/- mouse (male) is shown. (A) Hippocampus CA3 region and (B) DEn） (Dorsal endopiriform nucleus) staining are shown. The right figure shows the enlarged view (Enlargedlargeview) of the left figure. Oxtr-Cre+/-:ROSA26+/-マウス（雄）の脳におけるX-gal染色の結果を示す。（Ａ）外側中隔（lateral septum（LS））、（Ｂ）海馬（Hippocampus）ＣＡ１及びＣＡ２領域、（Ｃ）海馬ＣＡ３領域及びDEn（Dorsal endopiriform nucleus）、（Ｄ）DEn（Dorsal endopiriform nucleus）での染色をそれぞれ示す。The result of X-gal staining in the brain of Oxtr-Cre +/−: ROSA26 +/− mouse (male) is shown. (A) Lateral septum (LS), (B) Hippocampus CA1 and CA2 region, (C) Hippocampal CA3 region and DEn (Dorsal endopiriform nucleus), (D) DEn (Dorsal endopiriform nucleus) Each staining is shown. ノックインコンストラクトの配列を示す。オキシトシン受容体（Oxytocin receptor）、HAタグ（HA tag）、ヒトeIF4G IRES配列（Human eIF4G IRES sequence）及びCreリコンビナーゼ（Cre recombinase）の塩基配列を含む。The sequence of the knock-in construct is shown. It includes the base sequences of oxytocin receptor (Oxytocin receptor), HA tag (HA tag), human eIF4G IRES sequence (Human eIF4G IRES sequence) and Cre recombinase. ノックインコンストラクトの配列を示す（続き）。FRT配列（FRT site）及びPGK-neoの塩基配列を含む。The sequence of the knock-in construct is shown (continued). Includes the FRT sequence (FRT site) and the base sequence of PGK-neo. AAV-loxP-WGAウイルスの構造を示す。矢印（Coding）は、コード領域の向きを示す。The structure of AAV-loxP-WGA virus is shown. An arrow (Coding) indicates the direction of the code area.
　１．非ヒト動物
　本発明の非ヒト動物は、オキシトシン受容体遺伝子座の少なくとも１つにおいて、オキシトシン受容体遺伝子のプロモーターの制御下に
（i）オキシトシン受容体蛋白質をコードする塩基配列、
（ii）IRES配列、及び
（iii）部位特異的組み換え酵素をコードする塩基配列
を有することを主な特徴とする。 1. Non-human animal The non-human animal of the present invention comprises (i) a base sequence encoding an oxytocin receptor protein under the control of a promoter of the oxytocin receptor gene at at least one oxytocin receptor gene locus,
The main features are (ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombinase.
　非ヒト動物
　本発明の非ヒト動物は、オキシトシン受容体遺伝子座を有するものである限り、いかなる種の非ヒト動物でもよい。ヒトにおけるオキシトシン－オキシトシン受容体の系のモデル系として用いる観点からは、ヒトに近縁なヒト以外の哺乳類であることが好ましい。このような哺乳類としては、サルなどの類人猿、齧歯類（ネズミ目）、ウサギ、ネコ、イヌ、ウマ、ウシ等が挙げられる。実験動物としての取り扱いが容易であるとの観点から、マウス、ラット、モルモット、ハムスター等の齧歯類が好ましく、遺伝子組み換え操作を容易に行えるとの観点からマウスがより好ましい。マウスとしては、C56BL/6、C57BL/6、BALB/c等の近交系化された系統に属するマウスが特に好ましい例として挙げられる。 Non-human animal The non-human animal of the present invention may be any kind of non-human animal as long as it has an oxytocin receptor locus. From the viewpoint of use as a model system for the oxytocin-oxytocin receptor system in humans, mammals other than humans that are closely related to humans are preferred. Examples of such mammals include apes such as monkeys, rodents (murines), rabbits, cats, dogs, horses, cows and the like. Rodents such as mice, rats, guinea pigs and hamsters are preferable from the viewpoint of easy handling as experimental animals, and mice are more preferable from the viewpoint of easy genetic recombination operations. As a mouse, a mouse belonging to an inbred strain such as C56BL / 6, C57BL / 6, BALB / c is particularly preferable.
　本発明の非ヒト動物は、オキシトシン受容体遺伝子座の少なくとも１つにおいて、オキシトシン受容体遺伝子のプロモーターの制御下に、上記（i）～（iii）の塩基配列を有する。すなわち、本発明の非ヒト動物は、遺伝子組み換え非ヒト動物（トランスジェニック非ヒト動物）である。上記（i）～（iii）がノックイインされた非ヒト動物であるとも言える。本明細書において、「本発明のノックイン動物」と記載する場合がある。 The non-human animal of the present invention has the above base sequences (i) to (iii) under the control of the promoter of the oxytocin receptor gene at at least one of the oxytocin receptor loci. That is, the non-human animal of the present invention is a genetically modified non-human animal (transgenic non-human animal). It can be said that the above (i) to (iii) are knocked-in non-human animals. In this specification, it may be described as “the knock-in animal of the present invention”.
　本発明の非ヒト動物は、内在性のオキシトシン受容体遺伝子座のうち1つにおいて上記（i）～（iii）の塩基配列を有するものであっても（ヘテロ体）、内在性のオキシトシン受容体遺伝子座の両方が上記（i）～（iii）の塩基配列を有するもの（ホモ体）であってもよい。 The non-human animal of the present invention may have an endogenous oxytocin receptor even if it has the base sequences of (i) to (iii) above (hetero) at one of the endogenous oxytocin receptor loci. Both of the loci may be those having the base sequences (i) to (iii) (homo).
　オキシトシン遺伝子及びオキシトシン遺伝子座
　オキシトシン受容体遺伝子は、公知の遺伝子である。オキシトシン受容体遺伝子がコードするオキシトシン受容体タンパク質は、オキシトシンをリガンドとする、７回膜貫通型のＧＴＰ結合タンパク質（Ｇタンパク質）共役受容体である。オキシトシン受容体遺伝子は、魚類（例えば、ゼブラフィッシュ。）、鳥類（例えば、ニワトリ。）、哺乳類（例えば、ヒト、ウシ、マウス。）にかけて高度に保存されている。 The oxytocin gene and the oxytocin locus oxytocin receptor gene are known genes. The oxytocin receptor protein encoded by the oxytocin receptor gene is a seven-transmembrane GTP-binding protein (G protein) -coupled receptor having oxytocin as a ligand. The oxytocin receptor gene is highly conserved in fish (eg, zebrafish), birds (eg, chickens) and mammals (eg, humans, cows, mice).
　オキシトシン受容体遺伝子のmRNAの塩基配列及びオキシトシン受容体タンパク質のアミノ酸配列は、米国生物工学情報センター（NCBI; National Center for Biotechnology Information）が提供するGenBankに登録がされている。例えば、マウスのものは、下記のアクセッション番号で登録されている（複数のリビジョン（revision）が登録されている場合、最新のリビジョンを指すと理解される。）：
マウスオキシトシン受容体mRNA：NM_001081147
マウスオキシトシン受容体タンパク質：NP_001074616
　オキシトシン受容体をコードするオキシトシン受容体遺伝子座は、例えばマウスでは、第6染色体上に存在する。マウスのオキシトシン受容体遺伝子座は、４つのエクソンと３つのイントロンから構成され、オキシトシン受容体タンパク質をコードする配列は第３エクソンと第4エクソンに存在する。 The nucleotide sequence of the oxytocin receptor gene mRNA and the amino acid sequence of the oxytocin receptor protein are registered in GenBank provided by the National Center for Biotechnology Information (NCBI). For example, the mouse is registered with the following accession number (if multiple revisions are registered, it is understood to refer to the latest revision):
Mouse oxytocin receptor mRNA: NM_001081147
Mouse oxytocin receptor protein: NP_001074616
The oxytocin receptor locus encoding the oxytocin receptor is present on chromosome 6 in mice, for example. The mouse oxytocin receptor locus is composed of four exons and three introns, and sequences encoding oxytocin receptor proteins are present in the third and fourth exons.
　（i）オキシトシン受容体蛋白質をコードする塩基配列
　オキシトシン受容体タンパク質をコードする塩基配列としては、オキシトシン受容体のcDNA配列が好適な例として挙げられる。別の態様においては、天然の若しくは人工のイントロン配列（例えば、オキシトシン受容体の第３イントロン）で中断された塩基配列であってもよい。マウスのオキシトシン受容体をコードする塩基配列を配列番号１に例示する。 (I) Base Sequence Encoding Oxytocin Receptor Protein As a base sequence encoding oxytocin receptor protein, a cDNA sequence of oxytocin receptor can be mentioned as a suitable example. In another embodiment, the nucleotide sequence may be interrupted by a natural or artificial intron sequence (for example, the third intron of the oxytocin receptor). The base sequence encoding the mouse oxytocin receptor is exemplified in SEQ ID NO: 1.
　オキシトシン遺伝子の機能を十分に相補するとの観点から、上記（i）の塩基配列がコードするオキシトシン受容体は、それが導入される非ヒト動物由来のものであることが好ましいが、これに限定されない。 From the viewpoint of sufficiently complementing the function of the oxytocin gene, the oxytocin receptor encoded by the base sequence (i) is preferably derived from the non-human animal into which it is introduced, but is not limited thereto. .
　当該塩基配列がコードするオキシトシン受容体は、（x）天然のオキシトシン受容体のアミノ酸配列を有するもの、（y）天然のオキシトシン受容体のアミノ酸配列において１個又は複数（例えば、20個以下程度；10個以下程度；9、8、7、6、5、4、3、2個程度）アミノ酸が置換、付加若しくは欠失されたものであってオキシトシン受容体としての機能を有するものを包含する。 The oxytocin receptor encoded by the base sequence is (x) one having an amino acid sequence of a natural oxytocin receptor, (y) one or more (for example, about 20 or less in the amino acid sequence of a natural oxytocin receptor; About 10 or less; about 9, 8, 7, 6, 5, 4, 3, 2) including amino acids substituted, added or deleted and having a function as an oxytocin receptor.
　オキシトシン受容体タンパク質は、その末端（アミノ末端（N末端）又はカルボキシ末端（C末端）、好ましくはC末端）にタグ配列を有していてもよい。タグ配列を有する場合、発現するオキシトシン受容体タンパク質の局在を、抗体染色などの手法により可視化をすることができる。タグ配列は特に限定されるものではないが、HAタグ（YPYDVPDYA）（配列番号２）、FLAGタグ（DYKDDDDK）（配列番号３)、Mycタグ（EQKLISEEDL）（配列番号４）、V5タグ（GKPIPNPLLGLDST）（配列番号５）などが例示される。 The oxytocin receptor protein may have a tag sequence at its terminal (amino terminal (N terminal) or carboxy terminal (C terminal), preferably C terminal). When it has a tag sequence, the localization of the expressed oxytocin receptor protein can be visualized by a technique such as antibody staining. Tag sequence is not particularly limited, but HA tag (YPYDVPDYA) (SEQ ID NO: 2), FLAG tag (DYKDDDDK) (SEQ ID NO: 3), Myc tag (EQKLISEEDL) (SEQ ID NO: 4), V5 tag (GKPIPNPLLGLDST) (SEQ ID NO: 5) and the like are exemplified.
　（ii）IRES配列
　IRES（Internal Ribosome Entry Sequence）配列は、mRNAのCAP構造に依存しない翻訳開始を可能とする配列を指す。本発明の非ヒト動物はIRES配列を有することで、IRESの下流のタンパク質をコードする塩基配列も効率的に翻訳される。 (Ii) IRES sequence An IRES (Internal Ribosome Entry Sequence) sequence refers to a sequence that allows translation initiation independent of the CAP structure of mRNA. Since the non-human animal of the present invention has an IRES sequence, a base sequence encoding a protein downstream of IRES is also efficiently translated.
　IRES配列としては、公知のものを使用することができる。例えば、ピコルナウイルスに由来するIRES配列、eIF4G（eukaryotic initiation factor-4G）遺伝子、PDGF2（platelet-derived growth factor 2）遺伝子、VEGF（vascular endothelial growth factor）遺伝子、IGF-II（insulin-like growth factor II）遺伝子などの遺伝子に由来するIRES配列などが例示される。なかでも、ヒトeIF4G遺伝子に由来するIRES配列は、上記（i）の塩基配列がコードするオキシトシン受容体タンパク質の発現量が、野生型のオキシトシン受容体タンパク質の発現量と同程度になるとの観点から好ましい。ヒトeIF4G遺伝子に由来するIRES配列の塩基配列を、配列番号６に示す。 As the IRES sequence, a known one can be used. For example, IRES sequence derived from picornavirus, eIF4G (eukaryotic initiation factor-4G) gene, PDGF2 (platelet-derived growth factor 2) gene, VEGF (vascular endothelial growth factor) gene, IGF-II (insulin-like growth factor) II) Examples include IRES sequences derived from genes such as genes. In particular, the IRES sequence derived from the human eIF4G gene has a viewpoint that the expression level of the oxytocin receptor protein encoded by the base sequence (i) is comparable to the expression level of the wild-type oxytocin receptor protein. preferable. SEQ ID NO: 6 shows the base sequence of the IRES sequence derived from the human eIF4G gene.
　（iii）部位特異的組み換え酵素をコードする塩基配列
　部位特異的組み換え酵素とは、特定の認識配列（塩基配列）において若しくは特定の認識配列間で、特異的なDNAの組み換えを引き起こす酵素を指す。部位特異的組み換え酵素の具体例としては、Creリコンビナーゼ、FLPリコンビナーゼなどが例示される。 (Iii) Base sequence encoding site-specific recombination enzyme The site-specific recombination enzyme refers to an enzyme that causes recombination of specific DNA at a specific recognition sequence (base sequence) or between specific recognition sequences. Specific examples of the site-specific recombinase include Cre recombinase and FLP recombinase.
　なお、Creリコンビナーゼの認識配列としてはloxP配列、FLPリコンビナーゼの認識配列としてはFRT配列が挙げられる。 The recognition sequence for Cre recombinase includes the loxP sequence, and the recognition sequence for FLP recombinase includes the FRT sequence.
　部位特異的組み換え酵素のアミノ酸配列及びこれをコードする塩基配列は公知である。一例としてCreリコンビナーゼをコードする塩基配列を配列番号７に例示する。 The amino acid sequence of the site-specific recombination enzyme and the base sequence encoding it are known. As an example, the base sequence encoding Cre recombinase is exemplified in SEQ ID NO: 7.
　配置
　本発明の非ヒト動物において、上記（i）～（iii）の塩基配列は、オキシトシン受容体遺伝子のプロモーターの制御下に配置されている。すなわち、野生型のオキシトシン受容体遺伝子と同様の発現パターン（発現時期、発現細胞、発現強度など。）で、上記（i）～（iii）の塩基配列を含む転写産物が発現されるように配置がされていればよい。 Arrangement In the non-human animal of the present invention, the base sequences (i) to (iii) are arranged under the control of the promoter of the oxytocin receptor gene. That is, the transcription product containing the nucleotide sequences (i) to (iii) described above is expressed with the same expression pattern (expression time, expression cell, expression intensity, etc.) as the wild-type oxytocin receptor gene. It only has to be done.
　本発明において、上記（i）～（iii）の塩基配列は一体として同一の転写産物に転写されること、すなわちポリシストロニック（例えば、バイシストロニック。）な態様で転写されることが好ましい。また、本発明において、上記（i）がコードするオキシトシン受容体蛋白質と上記（iii）がコードする部位特異的組み換え酵素とは、別個のタンパク質として発現すことが好ましい。この場合、上記（i）及び上記（iii）は、それぞれ別個に終始コドンを有する。 In the present invention, it is preferable that the base sequences (i) to (iii) are integrally transferred to the same transcription product, that is, transferred in a polycistronic (for example, bicistronic) manner. In the present invention, the oxytocin receptor protein encoded by (i) and the site-specific recombinant enzyme encoded by (iii) are preferably expressed as separate proteins. In this case, the above (i) and (iii) each have a termination codon separately.
　本発明の非ヒト動物は、上記（i）～（iii）の塩基配列をこの順番で有することが好ましいが、これに限定されない。例えば、（iii）、（ii）、（i）の順番で配置されていてもよい。 The non-human animal of the present invention preferably has the base sequences (i) to (iii) in this order, but is not limited thereto. For example, you may arrange | position in order of (iii), (ii), (i).
　本発明の好ましい態様の１つにおいて、オキシトシン受容体遺伝子座の第３エクソンのコード領域が上記（i）～（iii）で置換されている。 In one preferred embodiment of the present invention, the coding region of the third exon of the oxytocin receptor locus is substituted with the above (i) to (iii).
　その他の構成
　本発明の非ヒト動物は、さらに上記部位特異的組み換え酵素により、遺伝子機能が改変され得る塩基配列をさらに有していてもよい。遺伝子機能が改変され得る塩基配列の典型例としては、ゲノム上の機能が改変される遺伝子をコードする塩基配列の全部又は一部が、２つの上記部位特異的組み換え酵素の認識配列により挟まれているものが例示される。 Other Configurations The non-human animal of the present invention may further have a base sequence whose gene function can be modified by the site-specific recombinant enzyme. As a typical example of a base sequence whose gene function can be modified, all or part of the base sequence encoding a gene whose function on the genome is modified is sandwiched between the recognition sequences of the two site-specific recombinant enzymes. What is present is exemplified.
　例えば上記部位特異的組み換え酵素がCreリコンビナーゼである場合、機能が改変される遺伝子をコードする塩基配列の全部又は一部が、loxP配列に挟まれている。 For example, when the site-specific recombination enzyme is Cre recombinase, all or part of the base sequence encoding the gene whose function is modified is sandwiched between loxP sequences.
　作製方法
　本発明の非ヒト動物は、公知の遺伝子組み換え技術を用いて作製することができる。一例として、上記（i）～（iii）がオキシトシン受容体遺伝子のプロモーターの制御下にノックインされた胚性幹細胞（ES細胞）を胚盤胞に導入して得られるキメラ胚を、対象動物の雌の子宮に着床させることにより、キメラ動物を得る方法が例示される。 Production Method The non-human animal of the present invention can be produced using a known genetic recombination technique. As an example, a chimeric embryo obtained by introducing an embryonic stem cell (ES cell) in which the above (i) to (iii) are knocked in under the control of the promoter of the oxytocin receptor gene into a blastocyst is obtained from a female of the subject animal. An example is a method of obtaining a chimeric animal by implantation in the uterus.
　上記ノックインがされたES細胞は、例えば下記の配列を有するベクターを用いて作製することができる：
（i）オキシトシン受容体蛋白質をコードする塩基配列、
（ii）IRES配列、及び
（iii）部位特異的組み換え酵素をコードする塩基配列
（iv）オキシトシン受容体遺伝子座の塩基配列と相同組み替え可能な塩基配列。 The knocked-in ES cell can be prepared using, for example, a vector having the following sequence:
(I) a base sequence encoding an oxytocin receptor protein,
(Ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombination enzyme (iv) a base sequence that can be recombined with the base sequence of the oxytocin receptor locus.
　オキシトシン受容体遺伝子座の塩基配列と相同組み替え可能な塩基配列は、好適には、挿入位置の5’側の塩基配列及び3’側の塩基配列を指す。 The base sequence capable of homologous recombination with the base sequence of the oxytocin receptor gene locus preferably refers to the 5'-side base sequence and the 3'-side base sequence of the insertion position.
　上記ベクターは、その他に、ネガティブセレクションを行うためのマーカーポジティブセレクション（例えば、ネオマイシン耐性遺伝子などの薬剤耐性遺伝子。）、ポジティブセレクションを行うためのマーカー遺伝子（例えば、HSV（単純ヘルペスウイルス）のチミジンキナーゼ（tk）遺伝子、ジフテリアトキシン（DT）遺伝子など。）、大腸菌内で維持及び複製をするために必要な構成（複製開式起点、薬剤耐性遺伝子など。）を必要に応じて有する。 In addition to the above, the above-mentioned vector includes marker positive selection (eg, drug resistance gene such as neomycin resistance gene) for negative selection, and marker gene (eg, HSV (herpes simplex virus) thymidine kinase) for positive selection. (Tk) gene, diphtheria toxin (DT) gene, etc.), and the necessary components for maintaining and replicating in E. coli (replication open origin, drug resistance gene, etc.).
　上記マーカー遺伝子は、FLPリコンビナーゼ、Creリコンビナーゼなどの部位特異的組み換え酵素により除去ができるように、該酵素の認識配列で挟まれていることが好ましい。 The marker gene is preferably sandwiched between recognition sequences of the enzyme so that it can be removed by site-specific recombinant enzymes such as FLP recombinase and Cre recombinase.
　本発明の非ヒト動物が、上記部位特異的組み換え酵素により、遺伝子機能が改変され得る塩基配列を有している場合、例えば、得られた上記（i）～（iii）の塩基配列を烏有する非ヒト動物と、遺伝子機能が改変され得る塩基配列を有している非ヒト動物とを交配することによって得ることができる。 When the non-human animal of the present invention has a base sequence whose gene function can be modified by the site-specific recombination enzyme, for example, it has the obtained base sequences (i) to (iii). It can be obtained by mating a non-human animal with a non-human animal having a base sequence whose gene function can be modified.
　２．遺伝子の機能解析方法
　本発明はまた、下記（I）及び（II）を有する非ヒト動物を用いることを特徴とする、オキシトシン受容体及び／又は前記機能が改変され得る遺伝子の機能解析方法をも提供する：
　（I）オキシトシン受容体遺伝子座の少なくとも１つにおいて、オキシトシン受容体遺伝子のプロモーターの制御下に
（i）オキシトシン受容体蛋白質をコードする塩基配列、
（ii）IRES配列、及び
（iii）部位特異的組み換え酵素をコードする塩基配列、並びに、
　（II）前記部位特異的組み換え酵素により、遺伝子機能が改変され得る塩基配列。 2. Gene Function Analysis Method The present invention also provides a function analysis method for an oxytocin receptor and / or a gene whose function can be altered, characterized by using a non-human animal having the following (I) and (II): provide:
(I) at least one of the oxytocin receptor gene loci, under the control of the promoter of the oxytocin receptor gene (i) a base sequence encoding an oxytocin receptor protein,
(Ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombinase, and
(II) A base sequence whose gene function can be modified by the site-specific recombinant enzyme.
　上記（I）及び（II）を有する非ヒト動物においては、オキシトシン受容体が発現する細胞で、特異的に、所定の遺伝子の機能が改変される。典型的は、オキシトシン受容体が発現する細胞で、当該遺伝子の機能が喪失される。 In the non-human animal having the above (I) and (II), the function of a predetermined gene is specifically altered in cells in which the oxytocin receptor is expressed. Typically, the function of the gene is lost in cells in which the oxytocin receptor is expressed.
　このような構成を有する非ヒト動物を用いることで、オキシトシン受容体及び／又は前記機能が改変される遺伝子の機能解析、特にオキシトシン受容体と前記機能が改変される遺伝子との相互作用の機能解析が可能となる。 Functional analysis of the oxytocin receptor and / or the gene whose function is modified by using a non-human animal having such a configuration, particularly the functional analysis of the interaction between the oxytocin receptor and the gene whose function is modified Is possible.
　特に限定されるものではないが、本発明の方法の具体例を以下に挙げる。１つ態様においては、上記「１．」欄に記載の本発明のノックイン動物に、図９に示すAAV-loxP-WGAウイルスを感染させる（特に、脳内などのオキシトシン受容体の発現部位が好ましい。）ことで、上記（I）及び（II）を有する非ヒト動物を作製する。 Although not particularly limited, specific examples of the method of the present invention are listed below. In one embodiment, the knock-in animal of the present invention described in the column “1.” is infected with the AAV-loxP-WGA virus shown in FIG. 9 (particularly, an oxytocin receptor expression site such as in the brain is preferable). Thus, a non-human animal having the above (I) and (II) is produced.
　図９に示すAAV-loxP-WGAウイルスは、目的タンパク質をコードする塩基配列を、プロモーターの向きに対して逆向きに有するため、通常は目的タンパク質が発現しない。ここではWGA（wheat germ agglutinin）タンパク質を一例としてあげるが、これに限定されるものではない。また、目的タンパク質は１種のタンパク質、又は２種以上のタンパク質の融合タンパク質であってもよい。融合タンパク質である場合、融合タンパク質を構成する一例として、蛍光タンパク質が挙げられる。 Since the AAV-loxP-WGA virus shown in FIG. 9 has a base sequence encoding the target protein in the opposite direction to the direction of the promoter, the target protein is usually not expressed. Here, WGA (wheat germ agglutinin) protein is given as an example, but the present invention is not limited to this. The target protein may be a single protein or a fusion protein of two or more proteins. In the case of a fusion protein, a fluorescent protein is an example of the fusion protein.
　また、図９に示すAAV-loxP-WGAウイルスにおいて、目的タンパク質をコードする塩基配列は、1対の部位特異的組み換え酵素の認識配列（ここでは、loxP配列を一例としてあげるが、これに限定されるものではない。また、当該loxP配列は、loxPJTZ17とloxP71の組み合わせなどの不可逆反転型であることが好ましい。）で挟まれている。 In addition, in the AAV-loxP-WGA virus shown in FIG. 9, the base sequence encoding the target protein is a recognition sequence of a pair of site-specific recombinant enzymes (here, the loxP sequence is taken as an example, but is not limited thereto). In addition, the loxP sequence is preferably sandwiched between irreversible inversion types such as a combination of loxPJTZ17 and loxP71.
　感染部位で記部位特異的組み換え酵素（ここでは、Creリコンビナーゼを一例としてあげるが、これに限定されるものではない。）が発現している場合、前記部位特異的組み換え酵素が認識配列の対の間での組み換え反応を起こすことにより、目的タンパク質をコードする塩基配列の向きが反転し、目的タンパク質を発現するようになる。 When the site-specific recombinase is expressed at the site of infection (here, Cre recombinase is taken as an example, but is not limited thereto), the site-specific recombinase is used as a recognition sequence pair. By causing a recombination reaction between them, the direction of the base sequence encoding the target protein is reversed, and the target protein is expressed.
　図９に示すAAV-loxP-WGAを用いる場合、Creリコンビナーゼが発現している細胞（すなわち、オキシトシン受容体を発現している細胞。）でのみ組み換えが起こり、WGA-TdTomatoが発現する。なお、TdTomato は蛍光タンパク質である。WGAタンパク質は神経細胞間を順行性に移動するため、オキシトシン受容体を発現する神経細胞の投射先に移動する。例えば、WGAタンパク質を抗体染色によって可視化することにより、投射先の神経細胞を明らかにし、オキシトシン受容体を発現する神経細胞が形成するネットワークの情報が得ることができる。あるいは、蛍光タンパク質によっても可視化を行うことができる。 When AAV-loxP-WGA shown in FIG. 9 is used, recombination occurs only in cells expressing Cre recombinase (ie, cells expressing oxytocin receptor), and WGA-TdTomato is expressed. TdTomato is a fluorescent protein. Since the WGA protein moves anterogradely between neurons, it moves to the target of neurons expressing the oxytocin receptor. For example, by visualizing the WGA protein by antibody staining, the target neuron can be identified and information on the network formed by the neuron expressing the oxytocin receptor can be obtained. Alternatively, visualization can also be performed with a fluorescent protein.
　目的タンパク質は、当業者が解析目的に応じて適宜選択することができる。例えば、WGAタンパク質に替えて狂犬病ウイルスを用いることで、その逆行性から神経細胞の投射元を可視化できる。別の態様においては、目的タンパク質としてコレラトキシン、チミジンカイネースなどの細胞毒性を示すタンパク質を用いることで、オキシトシン受容体を発現する神経を特異的に除去することができる。 The target protein can be appropriately selected by those skilled in the art according to the purpose of analysis. For example, by using a rabies virus instead of the WGA protein, it is possible to visualize the projection source of a neuron from its retrograde nature. In another embodiment, nerves expressing the oxytocin receptor can be specifically removed by using a cytotoxic protein such as cholera toxin or thymidine kinase as the target protein.
　さらに別の態様においては、目的タンパク質としてチャネルロドプシン（活性性）、ハロロドプシン（抑制性）などの光受容体分子を用いることで、オキシトシン受容体を発現する神経細胞において光受容体分子を発現させることができる。この場合、例えば、固有の光刺激をあたえることによりオキシトシン受容体を発現する神経細胞のみで、神経細胞の活性化または抑制を行うことが可能となる。このような系をもちいて、社会行動解析を行い、社会行動におけるオキシトシン受容体のより詳細な機能を解明する事が可能となる。 In yet another embodiment, a photoreceptor molecule such as channelrhodopsin (active) or halorhodopsin (inhibitory) is used as a target protein to express the photoreceptor molecule in a neuron that expresses the oxytocin receptor. be able to. In this case, for example, it becomes possible to activate or suppress the nerve cell only by the nerve cell that expresses the oxytocin receptor by giving a specific light stimulus. Using such a system, it is possible to analyze social behavior and elucidate more detailed functions of oxytocin receptors in social behavior.
　以下、本発明を実施例を用いてより詳細に説明する。
　（Oxtr-Creノックインベクターの作製）
　オキシトシン受容体（Oxtr）遺伝子のORF（Open Reading Frame；タンパク質をコードする領域）開始点が存在するエクソン３の領域にOxtr cDNA、Cre recinbinase cDNAを組み込みOxtr遺伝子の発現制御によりOxtrとCre recinbinaseを発現することを可能とするOxtr-Creノックインベクターの構築した。 Hereinafter, the present invention will be described in more detail with reference to examples.
(Preparation of Oxtr-Cre knock-in vector)
Oxtr cDNA and Cre recinbinase cDNA are inserted into the exon 3 region where the ORF (Open Reading Frame; protein coding region) start point of the oxytocin receptor (Oxtr) gene is present. Oxtr and Cre recinbinase are expressed by controlling the expression of the Oxtr gene. An Oxtr-Cre knock-in vector was constructed.
　Oxtr cDNA、キャップ非依存性翻訳開始に必要なeIF4G由来のIRES、Cre recinbinase cDNA, エクソン２及び３、ポシティブ選択のためのネオマイシン耐性遺伝子、相同領域の５’ａｒｍ及び３’ａｒｍ、大腸菌への形質転換に必要なアンピシリン耐性遺伝子、ポジティブ選別のために必要な遺伝子として薬剤耐性遺伝子とそのプロモーター(pgk-neo), ノックインマウスができた際にpgk-neoを取り除くためのFRT配列、ネガティブ選別のための遺伝子として核酸アナログを細胞毒性のある物質に変化させて細胞を死滅させる性質を持つＨＳＶ（単純ヘルペスウイルス）のチミジンキナーゼ（ｔｋ）遺伝子（ｍｃ１ｔｋ）等のパーツからなる２０ｋｂのノックインベクターを作製した。 Oxtr cDNA, IRES derived from eIF4G required for cap-independent translation initiation, Cre recinbinase cDNA, exons 2 and 3, neomycin resistance gene for positive selection, homologous regions 5'arm and 3'arm, traits to E. coli Ampicillin resistance gene required for conversion, drug resistance gene and its promoter (pgk-neo) necessary for positive selection, FRT sequence to remove pgk-neo when knock-in mice are made, for negative selection A 20 kb knock-in vector consisting of parts such as the thymidine kinase (tk) gene (mc1tk) of HSV (herpes simplex virus) having a property of killing cells by changing a nucleic acid analog into a cytotoxic substance as a gene of .
　（ＥＳ細胞へのノックインベクターの導入）
　ノックインベクターを精製し、制限酵素SalI消化によって直鎖状にした。その後、ＥＳ細胞株Ｅ１４ＴＧ２ａに、電気穿孔法の手法を用いて、上記のノックインベクターを導入した。次に薬剤選択により相同組み換えの起こったＥＳ細胞株コロニーを単離、培養しサザンブロットハイブリダイゼーションの手法を用いて、相同性組み換えの起こったコロニーのスクリーニングを行った。 (Introduction of knock-in vector into ES cells)
The knock-in vector was purified and linearized by restriction enzyme SalI digestion. Thereafter, the above knock-in vector was introduced into the ES cell line E14TG2a using the electroporation technique. Next, ES cell line colonies in which homologous recombination occurred were isolated and cultured by drug selection, and colonies in which homologous recombination occurred were screened using the Southern blot hybridization technique.
　（サザンブロッティング）
　図１に、使用したノックインベクターの構造、及び相同組み換えにより得られる遺伝子の構造を示す。図１において、一番上に野生型遺伝子の構造、二段目にノックインベクターの構造、一番下に相同組み換えを起こした結果得られる構造を示す。図１において、Ｅ１はエクソン１、Ｅ２はエクソン２、Ｅ３はエクソン３を、それぞれ示す。ＸｂａＩはＸｂａＩ消化部位を、XhoIはXhoI消化部位を、SphIはＳphＩ消化部位を、それぞれ示す。 (Southern blotting)
FIG. 1 shows the structure of the used knock-in vector and the structure of the gene obtained by homologous recombination. In FIG. 1, the structure of the wild-type gene is shown at the top, the structure of the knock-in vector at the second stage, and the structure obtained as a result of homologous recombination at the bottom. In FIG. 1, E1 represents exon 1, E2 represents exon 2, and E3 represents exon 3. XbaI represents an XbaI digestion site, XhoI represents an XhoI digestion site, and SphI represents an SphI digestion site.
　ノックインベクターの配列を配列番号８及び図７に示す。 The sequence of the knock-in vector is shown in SEQ ID NO: 8 and FIG.
　（キメラマウスの作製）
マイクロインジェクションの技術を用いて、相同組み換えを正しく起こしたＥＳ細胞を、マウス胚盤胞に導入した。生殖能を有しない雄マウスと交配させるホルモン処理により擬制妊娠させた仮親マウスの子宮内に、ＥＳ細胞を導入したBL6系統マウス由来胚盤胞を移植し、出産させてキメラマウスを得た。マウスの毛の色により、産まれたマウスの形質を確認した。 (Production of chimeric mice)
Using microinjection technology, ES cells that had undergone homologous recombination were introduced into mouse blastocysts. A blastocyst derived from a BL6 strain mouse into which ES cells were introduced was transplanted into a uterus of a foster parent mouse pseudo-pregnant by hormonal treatment to be mated with a male mouse having no fertility, and a chimeric mouse was obtained by giving birth. The character of the born mouse was confirmed by the color of the hair of the mouse.
　（ノックインマウスの取得のための交配）
　目的とするノックインマウスの取得のため以下の交配を行った。ここでも、毛の色とサザンブロットハイブリダイゼーションにより、産まれたマウスの形質を確認した。Ｃ５７ＢＬ／６Ｊを相手としてキメラマウスと交配させて、Oxtr-Cre-Neo(+/-)マウスを得た。Ｃ５７ＢＬ／６Ｊとキメラマウスを交配することによって得たヘテロの遺伝子型を有するマウス(Oxtr-Cre-Neo(+/-))は強力なプロモーターPGKの下流にネオマイシン耐性遺伝子（ｎｅｏ）が挿入されている。これらの影響で本来のOxtr遺伝子の発現効果が維持できずにOxtr遺伝子の発現の低下が起こることが予想された。よってOxtr-Cre-Neo(+/-)マウスと全身でFlippase酵素を発現しているFlippaseマウスと交配させて、完全にPGK-Neoが欠損しているヘテロマウスOxtr-Cre(+/-)を作製した。 (Mating for obtaining knock-in mice)
The following mating was performed to obtain the desired knock-in mouse. Again, the traits of the born mice were confirmed by hair color and Southern blot hybridization. Oxtr-Cre-Neo (+/-) mice were obtained by mating with chimeric mice using C57BL / 6J as a partner. Mice with heterogeneous genotype (Oxtr-Cre-Neo (+/-)) obtained by mating C57BL / 6J with chimeric mice have a neomycin resistance gene (neo) inserted downstream of the strong promoter PGK. Yes. Due to these effects, it was predicted that the original Oxtr gene expression effect could not be maintained, and the Oxtr gene expression decreased. Therefore, Oxtr-Cre-Neo (+/-) mice were mated with Flippase mice expressing Flippase enzyme throughout the body, and heterozygous mice Oxtr-Cre (+/-) completely lacking PGK-Neo were obtained. Produced.
　（ノックインマウスゲノムのサザンブロッティング解析）
　Oxtr-Cre-Neo(+/-)、Oxtr-Cre(+/-)、野生型マウスの遺伝子型をサザンブロットハイブリダイゼーションにより解析した結果を、図２に示す。Oxtr-Cre-Neo(+/-)、Oxtr-Cre(+/-)、野生型マウスのTail DNAからゲノムを抽出し制限酵素XbaIで消化したものに対し、３’プローブを検出に使用した場合野生型（ＷＴ）マウスにおいては１３．５ｋｂｐの位置にバンドが認められOxtr-Cre-Neo(+/-)、Oxtr-Cre(+/-)マウスにおいて１３．５ｋｂｐと４．７ｋｂｐにバンドが認められた（図２Ａ）。一方、５’プローブを検出に使用した場合、野生型（ＷＴ）マウスにおいては１３．５ｋｂｐ、Oxtr-Cre-Neo(+/-)、Oxtr-Cre(+/-)マウスにおいて１３．５ｋｂｐと９．８ｋｂｐにバンドが認められた（図２B）。Neo プローブを用いた場合、Oxtr-Cre-Neo(+/-)は1．６ｋの位置にバンドが認められ野生型（ＷＴ）、Oxtr-Cre(+/-)においてはその位置にバンドが認められなかった（図２Ｃ）。 (Southern blotting analysis of knock-in mouse genome)
FIG. 2 shows the results of Southern blot hybridization analysis of the genotypes of Oxtr-Cre-Neo (+/−), Oxtr-Cre (+/−), and wild type mice. Oxtr-Cre-Neo (+/-), Oxtr-Cre (+/-), when extracted from the tail DNA of wild-type mice and digested with the restriction enzyme XbaI, 3 'probe is used for detection Wild type (WT) mice have a band at 13.5 kbp, and Oxtr-Cre-Neo (+/-) and Oxtr-Cre (+/-) mice have bands at 13.5 kbp and 4.7 kbp. (FIG. 2A). On the other hand, when the 5 ′ probe was used for detection, 13.5 kbp in wild type (WT) mice, 13.5 kbp and 9 in Oxtr-Cre-Neo (+/−), Oxtr-Cre (+/−) mice. A band was observed at .8 kbp (FIG. 2B). When using Neo probe, Oxtr-Cre-Neo (+/-) has a band at the position of 1.6k, wild type (WT), and Oxtr-Cre (+/-) has a band at that position. Not (FIG. 2C).
　（Oxtr-Cre（+／+）マウスの乳汁射出能の解析）
　Oxtr-Cre(+/-)同士を交配させて得られたOxtr-Cre（+/+）マウスにおいては、OXTR,CREの発現は完全に挿入したOxtr遺伝子,Cre recombinase遺伝子由来であると考えられる。発明者の知見からOxtr-/-雌親マウスより産まれた新生マウスはOxtr-/-雌親マウスから乳汁が射出されないため２４時間以内に１００％死亡することが示されている(Takayanagi Y., Nishimori K. et .al. PNAS 16096-16101 (2005))。そこでOxtr-Cre（+/+）マウスがOxtr遺伝子を発現していることを確認するために雌親マウスの乳汁射出能を調べた。Oxtr-Cre（+/+）雌マウスは新生マウスを出産後、乳汁射出が可能であり離乳期まで新生マウスを育てた。このことによりOxtr-Cre（+/+）はベクター由来のOXTR cDNAが機能していることが示された（図３）。 (Analysis of milk ejection ability of Oxtr-Cre (+ / +) mice)
In Oxtr-Cre (+ / +) mice obtained by mating Oxtr-Cre (+/-), the expression of OXTR and CRE is considered to be derived from the completely inserted Oxtr gene and Cre recombinase gene. . From the inventor's knowledge, it has been shown that newborn mice born from Oxtr-/-female mice die 100% within 24 hours because milk is not ejected from Oxtr-/-female mice (Takayanagi Y., Nishimori K. et .al. PNAS 16096-16101 (2005)). Therefore, in order to confirm that Oxtr-Cre (+ / +) mice expressed the Oxtr gene, the milk ejection ability of female parent mice was examined. Oxtr-Cre (+ / +) female mice were able to inject milk after giving birth to newborn mice and raised newborn mice until weaning. This indicates that Oxtr-Cre (+ / +) functions as a vector-derived OXTR cDNA (FIG. 3).
　（抗HA抗体による免疫染色）
　OTXRは子宮で発現することが知られている。Oxtr-Cre KI（ノックイン）マウスのOxtr-cDNAの3'末端に付加されているHA-tagを確認するために、子宮を用いた抗HA抗体を用いた免疫染色を行った。Oxtr-Creノックインマウスに麻酔をした後、4％paraformaidehydeを用いて灌流固定を行い子宮を取得した。子宮を4％paraformaldehyde (in 0.1M PB)で4℃にて一晩後固定を行った。後固定後の子宮を30％スクロース溶液(in 0.1M PB)に移し、二晩4℃で浸すことで30％スクロースへの置換を行った。十分に置換を行った子宮をドライアイス中に埋め凍結させた後、クライオスタットを用いて30μmの切片を作成した。作成した切片をマスコートに張り付け、室温で一晩乾燥した。サンプルは1×PBSで5分間3回洗浄した後、0.5％ TritonX-100/1×PBSで室温にて30分反応させ、その後1×PBSで10分間洗浄した。その後、5％ Normal Horse Serum/0.3% TritonX-100/1×PBS(ブロッキングバッファー)で室温にて30分ブロッキングを行った。その後、1次抗体 anti-HA rat antibody(1:100)を用いて4℃で18～24時間反応させた。1×PBSTで5分間3回洗浄した後、二次抗体 Alexa 594 conjugated goat anti-rat IgG antibody (1:500)を室温で二時間反応させた。1×PBSで5分2回洗浄した後、PermaFluorTM Mounting Medium (LVC)で封入した。その結果、子宮においてHA-tagが確認された（図４）。 (Immunostaining with anti-HA antibody)
OTXR is known to be expressed in the uterus. To confirm the HA-tag added to the 3 ′ end of Oxtr-Cre KI (knock-in) mouse Oxtr-cDNA, immunostaining using an anti-HA antibody using the uterus was performed. After anesthetizing the Oxtr-Cre knock-in mouse, the uterus was obtained by perfusion fixation using 4% paraformaidehyde. The uterus was fixed with 4% paraformaldehyde (in 0.1M PB) at 4 ° C overnight. The post-fixed uterus was transferred to a 30% sucrose solution (in 0.1 M PB) and immersed at 4 ° C. overnight for replacement with 30% sucrose. The fully replaced uterus was buried in dry ice and frozen, and then a 30 μm section was prepared using a cryostat. The prepared section was attached to a mass coat and dried overnight at room temperature. The sample was washed 3 times with 1 × PBS for 5 minutes, then reacted with 0.5% TritonX-100 / 1 × PBS for 30 minutes at room temperature, and then washed with 1 × PBS for 10 minutes. Thereafter, blocking was performed with 5% Normal Horse Serum / 0.3% Triton X-100 / 1 × PBS (blocking buffer) at room temperature for 30 minutes. Thereafter, the primary antibody anti-HA rat antibody (1: 100) was reacted at 4 ° C. for 18 to 24 hours. After washing with 1 × PBST three times for 5 minutes, the secondary antibody Alexa 594 conjugated goat anti-rat IgG antibody (1: 500) was reacted at room temperature for 2 hours. After washing twice with 1 × PBS for 5 minutes, it was sealed with PermaFluor ™ Mounting Medium (LVC). As a result, HA-tag was confirmed in the uterus (FIG. 4).
　(Oxtr- Cre +/-；Rosa+/-マウスの作成)
　OXTR-CreノックインマウスとRosa26レポーターマウスを交配することにより、Cre recombinaseを発現する細胞においてβガラクトシダーゼ遺伝子が発現するマウスを作製した。 (Create Oxtr- Cre +/-; Rosa +/- mouse)
By crossing an OXTR-Cre knock-in mouse and a Rosa26 reporter mouse, a mouse expressing a β-galactosidase gene in a cell expressing Cre recombinase was produced.
　（Cre recombinaseの発現の確認）
上記で作成したマウスに麻酔をした後、4％paraformaidehydeを用いて灌流固定し脳取得した。脳を4％paraformaldehyde (in 0.1M PB)で4℃にて一晩後固定を行った。後固定後の脳を30％スクロース溶液(in 0.1M PB)に移し、二晩4℃で浸すことで30％スクロースへの置換を行った。十分に置換を行った脳をドライアイス中に埋め凍結させた後、クライオスタット（Leika）を用いて30μmの切片を作成した。サンプルは1×PBSで5分間3回洗浄した後、0.5％TritonX-100/1×PBSで室温にて30分反応させ、その後1×PBSで10分間洗浄した。その後、5％ Normal Horse Serum/0.3% TritonX-100/1×PBS(ブロッキングバッファー)で室温にて30分ブロッキングを行った。その後、1次抗体 anti-βgal mouse antibody(1:300)を用いて4℃で18～24時間反応させた。1×PBSTで5分間3回洗浄した後、二次抗体 Alexa 488 conjugated goat anti-mouse IgG antibody (1:500)を室温で二時間反応させた。1×PBSで5分2回洗浄した後、ゼラチンコートしたスライドガラスに張りつけ、PermaFluorTM Mounting Medium (LVC)で封入し蛍光顕微鏡で観察した。その結果、海馬、Dorsal endopiriform nucleusでCre recombinaseの発現を確認した（図５）。 (Confirmation of Cre recombinase expression)
After anesthetizing the mouse prepared above, the brain was obtained by perfusion fixation using 4% paraformaidehyde. The brain was fixed with 4% paraformaldehyde (in 0.1M PB) at 4 ° C overnight. The brain after post-fixation was transferred to a 30% sucrose solution (in 0.1M PB) and immersed at 4 ° C. overnight for replacement with 30% sucrose. The fully substituted brain was buried in dry ice and frozen, and then a 30 μm section was prepared using a cryostat (Leika). The sample was washed 3 times with 1 × PBS for 5 minutes, then reacted with 0.5% TritonX-100 / 1 × PBS for 30 minutes at room temperature, and then washed with 1 × PBS for 10 minutes. Thereafter, blocking was performed with 5% Normal Horse Serum / 0.3% Triton X-100 / 1 × PBS (blocking buffer) at room temperature for 30 minutes. Thereafter, the primary antibody anti-βgal mouse antibody (1: 300) was reacted at 4 ° C. for 18 to 24 hours. After washing 3 times for 5 minutes with 1 × PBST, the secondary antibody Alexa 488 conjugated goat anti-mouse IgG antibody (1: 500) was reacted at room temperature for 2 hours. After washing twice with 1 × PBS for 5 minutes, it was attached to a gelatin-coated slide glass, sealed with PermaFluor ™ Mounting Medium (LVC), and observed with a fluorescence microscope. As a result, expression of Cre recombinase was confirmed in the hippocampus and Dorsal endopiriform nucleus (FIG. 5).
　同様に脳切片を調整しX-gal溶液（5mM K3Fe(CN)6、5mM K4Fe(CN)6、2 mM MgCl2を 1×PBに溶かし pH 7.4に合わせ、ここにX-galを1 mg/mlになるように加える）に移し37℃で一晩インキュベートした。切片はエタノール／キシレンで脱水後、Fisher Scientific Permount Mounting Mediumで封入した。その結果、海馬、Dorsal endopiriform nucleusでCre recombinaseの発現に由来する青い染色が認められた(図６)。図５，６よりOXTR-Creノックインマウスは活性を保持したCre recombinaseを発現していることを確認した。 Similarly, brain slices were prepared and X-gal solution (5 mM K3Fe (CN) 6, 5 mM K4Fe (CN) 6, 2 M MgCl2 dissolved in 1 × PB and adjusted to pH 7.4, where X-gal was 1 mg / ml And incubated at 37 ° C. overnight. The sections were dehydrated with ethanol / xylene and sealed with Fisher® Scientific® Permount® Mounting® Medium. As a result, blue staining derived from the expression of Cre recombinase was observed in the hippocampus, Dorsal endopiriform nucleus (Fig. 6). 5 and 6, it was confirmed that the OXTR-Cre knock-in mouse expressed Cre recombinase retaining the activity.

Claims (8)

  1. 　オキシトシン受容体遺伝子座の少なくとも１つにおいて、オキシトシン受容体遺伝子のプロモーターの制御下に
    （i）オキシトシン受容体蛋白質をコードする塩基配列、
    （ii）IRES配列、及び
    （iii）部位特異的組み換え酵素をコードする塩基配列
    を有する、非ヒト動物。     (I) a base sequence encoding an oxytocin receptor protein under the control of the promoter of the oxytocin receptor gene at at least one of the oxytocin receptor loci;
    A non-human animal having (ii) an IRES sequence and (iii) a base sequence encoding a site-specific recombinase.
  2. 　IRES配列が、ヒトeIF4G遺伝子に由来するIRES配列である、請求項１に記載の非ヒト動物。 The non-human animal according to claim 1, wherein the IRES sequence is an IRES sequence derived from a human eIF4G gene.
  3. 　オキシトシン受容体遺伝子座の第３エクソンのコード領域が、
    （i）オキシトシン受容体蛋白質をコードする塩基配列、
    （ii）IRES配列、及び
    （iii）部位特異的組み換え酵素をコードする塩基配列
    を含む塩基配列で置換された、請求項１又は２に記載の非ヒト動物。     The coding region of the third exon of the oxytocin receptor locus is
    (I) a base sequence encoding an oxytocin receptor protein,
    The non-human animal according to claim 1 or 2, which is substituted with a base sequence comprising (ii) an IRES sequence and (iii) a base sequence encoding a site-specific recombination enzyme.
  4. 　部位特異的組み換え酵素が、Creである、請求項１～３のいずれか１項に記載の非ヒト動物。 The non-human animal according to any one of claims 1 to 3, wherein the site-specific recombinase is Cre.
  5. 　オキシトシン受容体蛋白質が、Ｃ末端側にタグ配列を有する、請求項１～４のいずれか１項に記載の非ヒト動物。 The non-human animal according to any one of claims 1 to 4, wherein the oxytocin receptor protein has a tag sequence on the C-terminal side.
  6. 　前記部位特異的組み換え酵素により、遺伝子機能が改変され得る塩基配列をさらに有する、請求項１～５のいずれか１項に記載の非ヒト動物。 The non-human animal according to any one of claims 1 to 5, further comprising a base sequence whose gene function can be modified by the site-specific recombinant enzyme.
  7. 　（I）オキシトシン受容体遺伝子座の少なくとも１つにおいて、オキシトシン受容体遺伝子のプロモーターの制御下に
    （i）オキシトシン受容体蛋白質をコードする塩基配列、
    （ii）IRES配列、及び
    （iii）部位特異的組み換え酵素をコードする塩基配列、並びに、
    　（II）前記部位特異的組み換え酵素により、遺伝子機能が改変され得る塩基配列
    を有する非ヒト動物を用いることを特徴とする、オキシトシン受容体及び／又は前記機能が改変され得る遺伝子の機能解析方法。     (I) at least one of the oxytocin receptor gene loci, under the control of the promoter of the oxytocin receptor gene (i) a base sequence encoding an oxytocin receptor protein,
    (Ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombinase, and
    (II) A method for analyzing the function of an oxytocin receptor and / or a gene whose function can be altered, comprising using a non-human animal having a base sequence whose gene function can be altered by the site-specific recombinant enzyme.
  8. 　下記の塩基配列を含むベクター：
    （i）オキシトシン受容体蛋白質をコードする塩基配列、
    （ii）IRES配列、及び
    （iii）部位特異的組み換え酵素をコードする塩基配列
    （iv）オキシトシン受容体遺伝子座の塩基配列と相同組み替え可能な塩基配列。     A vector containing the following base sequence:
    (I) a base sequence encoding an oxytocin receptor protein,
    (Ii) an IRES sequence, and (iii) a base sequence encoding a site-specific recombination enzyme (iv) a base sequence that can be recombined with the base sequence of the oxytocin receptor locus.
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