WO2015068411A1 - Animal non humain génétiquement modifié - Google Patents

Animal non humain génétiquement modifié 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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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; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified 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; AVICULTURE; APICULTURE; PISCICULTURE; 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; AVICULTURE; APICULTURE; PISCICULTURE; 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; AVICULTURE; APICULTURE; PISCICULTURE; 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; AVICULTURE; APICULTURE; PISCICULTURE; 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.

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  • Biodiversity & Conservation Biology (AREA)
  • Toxicology (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

　La présente invention concerne un animal non humain présentant (i) une séquence de base codant pour une protéine du récepteur de l'ocytocine, (ii) une séquence IRES, et (iii) une séquence de base codant pour une enzyme recombinante spécifique d'un site sous le contrôle d'un promoteur du gène du récepteur de l'ocytocine au niveau d'au moins un locus de gène du récepteur de l'ocytocine.
PCT/JP2014/058342 2013-11-05 2014-03-25 Animal non humain génétiquement modifié WO2015068411A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022079082A1 (fr) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Constructions d'acides nucléiques améliorées pour activation de gènes simultanée

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BLOUET C. ET AL.: "TXNIP in Agrp neurons regulates adiposity, energy expenditure and central leptin sensitivity", J.NEUROSCI., vol. 32, no. 29, 2012, pages 9870 - 7 *
CUSULIN J.I.W. ET AL.: "Characterization of corticotropin-releasing hormone neurons in the paraventricular nucleus of the hypothalamus of Crh-IRES-Cre mutant mice", PLOS ONE, 8 May 2013 (2013-05-08), pages E64943 *
TANIGUCHI H. ET AL.: "A resource of Cre driver lines for genetic targeting of GABAergic neurons in cerebral cortex", NEURON, vol. 71, no. 6, 2011, pages 995 - 1013 *
YOSHIDA M. ET AL.: "Evidence that oxytocin exerts anxiolytic effects via oxytocin receptor expressed in serotonergic neurons in mice", J. NEUROSCI., vol. 29, no. 7, 2009, pages 2259 - 71 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022079082A1 (fr) 2020-10-15 2022-04-21 F. Hoffmann-La Roche Ag Constructions d'acides nucléiques améliorées pour activation de gènes simultanée

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