WO2009110209A1 - Souris à cri muté - Google Patents

Souris à cri muté Download PDF

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WO2009110209A1
WO2009110209A1 PCT/JP2009/000924 JP2009000924W WO2009110209A1 WO 2009110209 A1 WO2009110209 A1 WO 2009110209A1 JP 2009000924 W JP2009000924 W JP 2009000924W WO 2009110209 A1 WO2009110209 A1 WO 2009110209A1
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mouse
mice
poldl
amino acid
mating
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PCT/JP2009/000924
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English (en)
Japanese (ja)
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八木健
内村有邦
日高裕子
古澤満
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株式会社ネオ・モルガン研究所
国立大学法人大阪大学
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Publication of WO2009110209A1 publication Critical patent/WO2009110209A1/fr

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • 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
    • 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
    • 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

Definitions

  • the present invention relates to a mouse that rings in a sound range that can be heard by humans.
  • mice are widely used as experimental models for mammals. However, there are many limitations to using mice as experimental models of human higher nervous activity. For example, it has been difficult to measure mouse emotions.
  • An object of the present invention is to provide a mouse that generates sound in a sound range that a human can hear.
  • An object of the present invention is to provide a mouse that can easily grasp the emotions of the mouse by voices emitted from the mouse.
  • the audible sound range of humans basically, a mouse (hereinafter referred to as “the audible sound range of humans”) by repeating the process of mating mice lacking the 3′-5 ′ exonuclease activity of DNA polymerase ⁇ . It is based on the knowledge that it is also possible to obtain “Twitter mutant mice”.
  • the first aspect of the present invention relates to a mouse that emits sound in the human audible range.
  • the first aspect of the present invention relates to a mouse having a higher volume of voice in the human audible sound range than the volume of human voice in the non-audible sound range.
  • the mouse of the present invention had a life span of 1 year or more and also had normal fertility. Therefore, according to the present invention, the emotion of the mouse can be easily grasped by the voice emitted by the mouse. In addition, such studies can be systematically performed using genetically homogeneous mouse strains.
  • a preferred embodiment of the first aspect relates to a mouse in which the voice volume in the sound range of 20 Hz to 10 kHz is greater than the voice volume in the low frequency region and the high frequency region.
  • a preferred embodiment of the first aspect relates to a mouse whose voice volume in the sound range of 20 Hz to 10 kHz is 1.5 times or more the voice volume in the high frequency range.
  • a preferred embodiment of the first aspect relates to a mouse obtained by mating a mouse having a protein in which the 400th aspartic acid of the amino acid sequence represented by SEQ ID NO: 1 is mutated to another amino acid. In this way, it is possible to obtain a mouse that has a higher volume of voice in the human audible sound range than the volume of human voice in the non-audible sound range.
  • a preferred embodiment of the first aspect relates to a mouse obtained by mating a mouse deficient in the 3'-5 'exonuclease activity of DNA polymerase ⁇ . That is, a mouse deficient in the 3'-5 'exonuclease activity of DNA polymerase ⁇ is bred.
  • a preferred embodiment of the first aspect is a mouse obtained by mating a mouse having a protein in which the 400th aspartic acid of the amino acid sequence represented by SEQ ID NO: 1 (amino acid sequence of DNA polymerase ⁇ ) is mutated to another amino acid. is there. That is, among the mice lacking the 3'-5 'exonuclease activity of DNA polymerase ⁇ , the above-mentioned mice shown in the Examples can be crossed to obtain twitter mutant mice. In addition, by crossing twitter mutant mice, it is possible to efficiently obtain a twitter mutant mouse strain.
  • the second aspect of the present invention relates to a method for producing a twitter mutant mouse.
  • the second aspect of the present invention includes, for example, a step of mating a mouse having a protein in which the 400th aspartic acid of the amino acid sequence represented by SEQ ID NO: 1 is mutated to another amino acid. This makes it possible to manufacture a mouse that emits sound in the human audible range.
  • a mouse can be manufactured in which the volume of human voice is higher than the volume of human voice.
  • Another embodiment of the second aspect of the present invention includes the step of mating a mouse deficient in the 3'-5 'exonuclease activity of DNA polymerase ⁇ . This makes it possible to manufacture a mouse that emits sound in the human audible range. As shown in the examples described later, in order to obtain the warp mutant mouse of the present invention, one of the mating mice is a mouse deficient in the 3′-5 ′ exonuclease activity of DNA polymerase ⁇ , and the other May be wild type.
  • a step of mating mice lacking the 3'-5 'exonuclease activity of DNA polymerase ⁇ is performed. This makes it possible to manufacture a mouse that emits sound in the human audible range. In addition, it is possible to create a mouse with a louder volume in the human audible range than in a human non-audible range.
  • the mouse to be mated is preferably a mouse having a protein in which the 400th aspartic acid of the amino acid sequence represented by SEQ ID NO: 1 is mutated to another amino acid.
  • the human volume can be efficiently compared with the volume in the human non-audible sound range.
  • a mouse system with a high volume in the audible sound range can be manufactured.
  • the present invention it is possible to provide a mouse in which the emotion of the mouse can be easily grasped by the sound emitted by the mouse.
  • the first aspect of the present invention relates to a mouse that emits sound in the human audible range.
  • the present invention relates to a mouse having a higher volume of voice in a human audible sound range than a volume of voice in a human non-audible sound range.
  • Non-Patent Document 1 a mouse that generates sound in a sound range of 30 kHz or higher was created. However, 30 kHz is not a human audible range. Therefore, even if a mouse created based on this document is used, humans cannot directly observe mouse calls. Therefore, with this mouse, it is difficult to understand mouse emotions and mouse language mechanisms.
  • the mouse of the present invention relates to a mouse that emits sound in the human audible range (a twitter mutant mouse). By using this mouse, it is possible to easily grasp the emotion of the mouse.
  • the mouse of the present invention relates to a mouse that emits sound in the normal human audible range. Normal means that the mouse is at rest.
  • the mouse of the present invention emits sound in the human audible range in normal times.
  • the mouse constantly (continuously) emits sound in the human audible range in normal times. Normally, mice do not produce human-sounding voices during normal times.
  • mice are considered to communicate using ultrasound in the non-audible range of humans.
  • the mouse of the present invention constantly emits sound in the human audible range. This is thought to be a shift in the frequency of voice for communication. Therefore, if the mouse of the present invention is used, emotions of the mouse can be accurately grasped. Thereby, it is considered that the mouse of the present invention is effective for development of new drugs related to the cranial nervous system. Moreover, the mouse
  • the mouse of the present invention did not appear to have a particularly serious disease. For this reason, the mouse of the present invention had a lifespan of 1 year or more, like a healthy mouse. Therefore, the mouse of the present invention is considered to be a normal mouse except that the voice is low. Furthermore, as mentioned earlier, mice that utter in the audible range due to disease or abnormality are generally short-lived and lack normal fertility. On the other hand, the mouse of the present invention has normal reproductive ability, and a mouse having an equivalent character can be easily obtained at an arbitrary time and number by a normal mating operation. That is, if the mouse of the present invention is used, emotions of a healthy mouse can be accurately grasped.
  • Such studies can also be done systematically using genetically homogeneous mouse strains.
  • the human audible sound range is 20 Hz or more and 20 kHz or less. Therefore, this mouse is a mouse that often sounds in a sound range of 20 Hz to 20 kHz.
  • a mouse lacking the 3'-5 'exonuclease activity of DNA polymerase ⁇ lacks the proofreading function during DNA replication possessed by DNA polymerase ⁇ . For this reason, the incidence of mutations during DNA replication is increased, and it is easy to suffer from tumors. Therefore, a mouse obtained by mating a mouse lacking the 3'-5 'exonuclease activity of DNA polymerase ⁇ is generally short-lived.
  • mice lacking the 3′-5 ′ exonuclease activity of DNA polymerase ⁇ can be mated with each other, so that a mouse strain lacking the 3′-5 ′ exonuclease activity of DNA polymerase ⁇ can be efficiently produced.
  • mutations accumulate at each mating, and thus, by repeating mating between the mice of the present invention, a mouse strain in which mutations are accumulated can be produced.
  • the following may be performed. Let a single mouse rest and record the sound for 1 minute. Measure the frequency distribution of the recorded voice. Add the voice volume for each frequency component at multiple times. Repeat this operation three times to obtain the average value. In this way, the range of the mouse can be evaluated.
  • the type of mouse in the present invention may be any type of mouse.
  • the rat is a kind of mouse and is included in the mouse of the present invention.
  • a preferred embodiment of the first aspect relates to a mouse in which the voice volume in the sound range of 20 Hz to 10 kHz is greater than the voice volume in the low frequency region and the high frequency region.
  • the low frequency region is a frequency region of 20 Hz or less. This low-frequency sound cannot be heard by normal humans.
  • the high frequency region means a frequency region higher than 20 kHz. This high-frequency sound cannot be heard by normal humans.
  • a mouse produced using the manufacturing method described below produces a loud cry, especially in the 10 kHz or lower sound range.
  • a preferred embodiment of the first aspect relates to a mouse in which the voice volume in the sound range of 20 Hz to 10 kHz is 1.5 times or more the voice volume in the high frequency range.
  • a preferred embodiment of the first aspect relates to a mouse obtained by mating a mouse deficient in the 3'-5 'exonuclease activity of DNA polymerase ⁇ . That is, a mouse deficient in the 3'-5 'exonuclease activity of DNA polymerase ⁇ is bred. In this way, a mouse that emits sound in the human audible range can be manufactured. In addition, it is possible to obtain a mouse whose voice volume in the human audible sound range is larger than that in the human non-audible sound range.
  • the obtained mouse may be a mouse whose voice volume in the sound range of 20 Hz to 10 kHz is large (for example, 1.5 times or more) in the high frequency area.
  • one of the mating mice is a mouse deficient in the 3′-5 ′ exonuclease activity of DNA polymerase ⁇ , and the other May be wild type.
  • the female is preferably a mouse deficient in the 3'-5 'exonuclease activity of DNA polymerase ⁇ .
  • female offspring born by mating a mouse deficient in the 3′-5 ′ exonuclease activity of the above DNA polymerase ⁇ with a wild-type mouse were treated with a wild-type or heterozygous mouse.
  • a “twice mutant mouse” can be obtained by crossing with a mouse of a type (deletion of one exonuclease activity of a homologous chromosome).
  • a “twist mutant mouse” having the 3′-5 ′ exonuclease activity of DNA polymerase ⁇ can be obtained.
  • DNA polymerase ⁇ having the 3′-5 ′ exonuclease activity of DNA polymerase ⁇ thus obtained, DNA polymerase ⁇ having the 3′-5 ′ exonuclease activity “ A “Twitter mutant mouse” strain can be generated.
  • a method for obtaining a mouse deficient in the 3'-5 'exonuclease activity of DNA polymerase ⁇ is known.
  • such a mouse may be obtained using the method disclosed in Japanese Patent Application Laid-Open No. 2007-143562.
  • Golsby et al. Goldsby, R. E., L. E. Hays, X. Chen, E. A. Olmsted, W. B. Slayton, G. J. Spancrude, and B. onD. 2002. High incident of epicial cancers in mime defensive for DNA polymerase delta proofreading. Proc Natl Acad Sci US A5 is based on this method.
  • mice deficient in the 3′-5 ′ exonuclease activity of DNA polymerase ⁇ a mouse in which an amino acid in the 3′-5 ′ exonuclease domain of DNA polymerase ⁇ (positions 128 to 475 of SEQ ID NO: 1) has been mutated is can give.
  • mice having a protein in which the 400th aspartic acid of the amino acid sequence represented by SEQ ID NO: 1 is mutated to another amino acid may be mated.
  • “Other amino acids” are preferably glycine, alanine, valine, leucine, isoleucine, lysine, arginine, and glutamic acid, and more preferably alanine.
  • the 1118th to 1120th sequence (GAC) of SEQ ID NO: 3 base sequence of DNA polymerase ⁇ (exon only)) (400th position of SEQ ID NO: 1)
  • the codon corresponding to the aspartic acid of GCT, GCC, GCA, or GCG is, among the mice lacking the 3′-5 ′ exonuclease activity of DNA polymerase ⁇ , the mutant mice can be obtained by mating the above-mentioned mice shown in the Examples. Furthermore, it is possible to efficiently generate a strain mutant mouse strain by repeatedly mating between the mutant strain mice.
  • mice having a protein in which the 400th aspartic acid of the amino acid sequence having a high homology with the amino acid sequence represented by SEQ ID NO: 1 is mutated to another amino acid may be mated.
  • sequences having high homology include sequences having 90% or more homology, and preferably sequences having 98% or more homology.
  • Mice having proteins in which the 400th aspartic acid of the sequence in which one or two amino acid residues are substituted from the amino acid sequence represented by SEQ ID NO: 1 are mutated to other amino acids may be crossed.
  • Mice having a protein in which the 400th aspartic acid in the sequence in which one or two amino acid residues are lost from the amino acid sequence shown in SEQ ID NO: 1 are mutated to other amino acids may be crossed.
  • mice having a protein in which one or two amino acid residues are added to the amino acid sequence shown in SEQ ID NO: 1 may be crossed.
  • Mice having a protein in which one or two amino acid residues are inserted into the amino acid sequence represented by SEQ ID NO: 1 may be crossed.
  • the second aspect of the present invention relates to a method for producing a twitter mutant mouse.
  • this method includes the step of mating mice lacking the 3'-5 'exonuclease activity of DNA polymerase ⁇ once or twice or more. This makes it possible to create a mouse that emits sound in the human audible range. In addition, this makes it possible to create a mouse that has a higher volume of voice in the human audible range than the volume of human voice in the non-audible range.
  • the mouse to be mated is preferably a mouse having a protein in which the 400th aspartic acid of the amino acid sequence represented by SEQ ID NO: 1 is mutated to another amino acid. As shown in the Examples, it is possible to efficiently generate a warp mutant mouse strain by crossing warp mutant mice.
  • the targeting vector was constructed by cloning four gene fragments into pBlueScriptII (Invitrogen).
  • the bacterial artificial chromosome (BAC) clone RP23-406H21 contains a region (SEQ ID NO: 2) encoding the mouse pold1 gene.
  • the 3 ′ fragment for homologous recombination (6.9 kb) is an intron (SEQ ID NO: 2: 4381) between exons 10 (SEQ ID NO: 4240-4380) and 11 (SEQ ID NO: 4578-4688). 4578) to exon 25 (SEQ ID NO: 10436-10533) were excised from BAC with EcoRI and SacI.
  • the 5 ′ fragment (1.6 kb) is an intron between exon 5 (SEQ ID NO: 2930 to 2098) to exon 10 (SEQ ID NO: 4240 to 4380) and 11 (SEQ ID NO: 2578 to 4688) ( SEQ ID NO: 2: 4381-4578) in which the codon corresponding to the 400th aspartic acid of SEQ ID NO: 1 is substituted with alanine.
  • the mutated gene fragment was prepared by repeating PCR using 5′-CAGAACTTT GCC CTCCCCATACCTC-3 ′ (codon for alanine is underlined) (SEQ ID NO: 4) and its complementary sequence oligonucleotide (primer).
  • a Flox fragment containing an 800 bp SV40 splicing / polyadenylation signal and a neomycin (G418) resistance gene cassette for positive selection (drug selection) is inserted between the 5 'and 3' sequences for homologous recombination with poldl Has been.
  • the diphtheria toxin A (DT-A) gene fragment for negative selection is located outside the 5 ′ sequence for homologous recombination.
  • the targeting vector was confirmed for its success by restriction enzyme treatment and sequencing (base sequencing).
  • a targeting vector linearized with SacI was introduced into embryonic stem cells (ES cells) of C57BL / 6J (standard mouse strain) by electroporation. Recombinant clone candidates were selected by growing in the presence of G418 (neomycin). Appropriate homologous recombinants were verified by PCR, Southern blot, and DNA sequencing. PCR was performed using primers P0 (5′-TTGACCCCCGCACTCATCAG-3 ′) (SEQ ID NO: 5) and P2 (5′-CACCAGACCAACTGGTAATGG-3 ′) (SEQ ID NO: 6), LA Taq polymerase.
  • Southern blotting was performed by completely digesting (cutting) genomic DNA with DraI and NheI, and using a [ 32 P] dCTP-labeled DNA fragment as a probe, as shown in FIG. 1 (B).
  • Recombinant ES cells were injected into Balb / c (mouse strain) blastula to prepare chimeric mice.
  • a heterozygous (pold1 +/ ⁇ ) mouse was produced by mating the chimeric mouse with a female C57BL / 6J mouse.
  • the poldl exo / + mice were produced by mating poldl +/- mice with Sycp1-Cre transgenic mice. All mutations used in this study were maintained on inbred breeding lines.
  • P1 (5′-GGAGTCCAGGTGTGCGTACTAC-3 ′) (SEQ ID NO: 7)
  • P2 5′-CACCAGACCAACTGGTAATGG-3 ′
  • P3 PCR was carried out using three primers (5′-CAGATTCCCCCTCTTGCATC-3 ′) (SEQ ID NO: 9).
  • PCR was performed using EX Taq polymerase by repeating 30 cycles of 94 ° C. for 1 minute, 94 ° C. for 1 minute, 60 ° C. for 30 seconds, and 72 ° C. for 40 seconds.
  • Pold1 exo / + Construction of ES cell lines Pold1 exo / + ES cell lines were introduced in the electroporation cyclic pCre-Pac plasmid Pold1 +/- ES cells.
  • Candidate ES cells were selected by temporary exposure to puromycin, and then subjected to subculture (temporary culture) using a normal medium for ES cells. The genotype of the recombinant via Cre was confirmed by PCR and Southern blot.
  • Sycp1-Cre transgenic mice Plasmids for construction of Sycp1-Cre transgenic mice were constructed from two gene fragments.
  • the first fragment is the promoter sequence of the sycp1 gene expressed in mouse male germ cells, and includes the base sequence from the 737th upstream to the 87th downstream of the transcription start point (SEQ ID NO: 16).
  • the other is a fragment obtained by adding a polyadenylation signal sequence (pA) (SEQ ID NO: 18) to a nuclear translocation signal sequence (NLS) and a sequence encoding a Cre recombinase (SEQ ID NO: 17). Both fragments were inserted between the SpeI and HindIII sites of pBluescriptII.
  • the plasmid was digested with SalI and NotI, and the purified fragment was microinjected into a fertilized egg of a C57BL / 6 mouse. From the obtained progeny, transgene was obtained by genomic PCR using a Cre-specific primer (5′-CTGAGAGTGATGAGGTCC-3 ′) (SEQ ID NO: 10) and (5′-CTAATCGCCCATCTTCAGCAG-3 ′) (SEQ ID NO: 11). Describing offspring with inserted.
  • cDNA was prepared by Superscript III Reverse Transcriptase (Invitrogen) using oligo (dT) 25 primer.
  • PCR was performed using the 5 ′ terminal primer of poldl (5′-GGCGTATCTTGTGGCGGGAA-3 ′ (SEQ ID NO: 12)) and the 3 ′ terminal primer (5′-CCTTGCCCGTGTCAGGTCA-3 ′ (SEQ ID NO: 13)).
  • 3′RACE was reverse-transcribed using an oligo dT primer with an adapter sequence, and then P4 (5′-TCATGGCCCTTCTCATTTC-3 ′ (SEQ ID NO: 14)), P5 (5′-TGGAGCTGCCAGCTGGAAAG-3 ′ (SEQ ID NO: 15) ) (FIG. 1 (G)) and PCR using two types of adapter primers was repeated.
  • the pold1 gene encodes the catalytic subunit p125 of the Pol ⁇ complex. This subunit contains a DNA polymerase domain and a 3′-5 ′ exonuclease domain.
  • a splicing / polyadenylation (poly (A)) signal from SV40 was inserted into the upstream region encoding the Pol ⁇ polymerase domain (FIG. 1 (A)) (SEQ ID NO: 3).
  • the D400A substitution is located in the exonuclease active site (ExoII) and has high tumor sensitivity in mice.
  • the mouse D400A mutation has been shown to cause inhibition of 3'-5 'exonuclease activity by in vitro biochemical analysis.
  • the poldl gene targeting vector was introduced into C57BL / 6J embryonic stem (ES) cells by electroporation.
  • ES embryonic stem
  • G418 resistant clones were screened by PCR (FIG. 1 (C)) and Southern blotting. Point mutation (D400A) was verified by DNA sequencing.
  • Targeting ES cell clones were microinjected into Balb / c blastocysts to create chimeric mice that carried the target allele by germline. Chimeras were bred to C57BL / 6J females to produce poldl +/- mice.
  • Pol ⁇ 3′-5 ′ exonuclease activity-deficient mice are prepared by mating pol1 +/ ⁇ mice with Cre-expressing transgenic animals containing a mouse Sycp1 gene promoter-Cre transgene that expresses Cre recombinase in germline cells. did. Both Pold1 +/ ⁇ and Pold1 exo / + mice were confirmed by PCR and Southern blotting (FIG. 1 (D), FIG. 1 (E)).
  • RNA of targeting ES cells was analyzed.
  • the poldl exo / + ES cells were obtained by introducing a circular Cre-pac plasmid into the poldl +/- ES cells by electroporation and selecting appropriate clones.
  • RT-PCR was transcribed from Pold1 +/- and Pold1 exo / + ES cells using two primers covering the full length poldl encoding region. By direct sequencing of these RT-PCR products, substituted allele (G A C ⁇ G C C ) is expressed in pold1 exo / + ES cells, not expressing the Pold1 +/- ES cells was shown (FIG.
  • Pold1 +/- cleaved fragments from ES cells contain sequence replaced (G C C), 3 'end stop inside the inserted SV40 poly (A) sequence, these cutting The fragment was found to consist of two unusual splicing types (FIG. 1 (G)).
  • Pold1 +/- full length fragment sequences from ES cells only contain an endogenous poly (A) signal sequence between Pold1 gene-derived sequences of the wild type (G C C).
  • A endogenous poly
  • the mRNA terminated normally and contained both displaced and wild-type sequences (2 and 3 out of 5 subclones, respectively).
  • Blastocyst culture and genotyping whole embryos were produced by natural mating. The day when the vaginal plug was detected was set as E0.5. The embryo recovered E3.5 or E4.5 by perfusing the uterus with M2 medium (Sigma). At the time of culture, the collected embryos were cultured in a complete ES medium containing a leukemia inhibitory factor.
  • each embryo was treated with 5 ⁇ l PCR Lysis buffer (10 mM Tris-HCl [pH 8.0], 50 mM KCl, 2 mM MgCl 2 , 0.45% NP-40, 0.45% Tween 20, 0.2 mg / mL). It was dissolved by incubation overnight at 55 ° C. in proteinase K).
  • each PCR was performed using the same three primers used for mouse genotyping: P1, P2, and P3. PCR was performed using LA Taq polymerase by repeating 35 cycles of 94 ° C for 1 minute, 94 ° C for 1 minute -58 ° C for 30 seconds -72 ° C for 50 seconds.
  • S (ni) is the survival frequency of the F (n) generation progeny when compared to normal error-free conditions; n indicates the number of generations passed, and i indicates the frequency of occurrence of lethal mutations.
  • F (0) is defined as the first mating of mutator mice.
  • S (n, i) is obtained by the following equation (when n> 2).
  • the Pold1 deficiency caused embryonic lethality before and after the implantation stage.
  • Heterozygous pol1 +/ ⁇ mice showed no detectable developmental abnormality beyond 18 months, and were able to reproduce normally in appearance. No abnormalities were observed in the growth of fetal fibroblasts from these animals (data not shown).
  • homozygous poldl ⁇ / ⁇ mice are not among the 256 born from cross fertilization with poldl +/ ⁇ mice (Table 1), and one functional poldl allele is an embryo. Although important for both postnatal development and inactivation of both alleles, it has been shown to lead to embryonic lethality.
  • E3.5 blastocysts derived from poldl +/- cross fertilization were collected, cultured for several days in vitro, photographed, and then subjected to PCR Was genotyped.
  • poldl + / + and poldl +/- blastocysts after culturing for 3 days, the trophoblasts hatched from the zona pellucida and spread on the culture dish, and an inner cell mass (ICM) grew on the trophoblast layer.
  • ICM inner cell mass
  • Pold1-null blastocysts cultured in vitro for 1 day corresponded to about E4.5 in vivo, and no obvious difference was observed between wild-type and heterozygous embryos.
  • Pold1 ⁇ / ⁇ blastocysts cultured for 3 days corresponded to about E6.5, and no cell proliferation was observed.
  • Embryos were washed with phosphate buffered saline (PBS) containing 1.5% bovine serum albumin (BSA) and fixed in 4% paraformaldehyde-PBS at 4 ° C for 30 minutes. Then, it was permeated for 20 minutes at room temperature in PBS containing 0.3% Triton X-100 and 1.5% BSA. To treat the embryos with Brd-U, they were permeabilized with 0.25N HCl and 0.5% Triton X-100 for 20 minutes at room temperature to denature the DNA and washed thoroughly with PBS containing 1.5% BSA. Embryos were incubated overnight at 4 ° C. with specific primary antibodies.
  • PBS phosphate buffered saline
  • BSA bovine serum albumin
  • the primary antibodies used in this study were mouse anti-Brd-U (Dako Cytomation) and rabbit anti-phosphohistone H3 (Ser-10) (Cell Singaling). Fluorescently labeled secondary antibodies were purchased from molecular probes. To detect apoptotic cells, TUNEL (terminal deoxynucleotransferase-mediated dUTP-biotin nick end labeling) assay was performed using In Situ Cell Death Detection Kit (Roche).
  • Pold1 deficiency causes DNA synthesis deficiency and frequent occurrence of apoptosis Apoptotic assay of bromodeoxyuridine (BrdU) uptake to examine whether the growth defect of cultured Pold1 ⁇ / ⁇ blastocysts is due to inhibition of DNA synthesis caused by Pol ⁇ disruption DNA synthesis in cultured blastocysts was evaluated.
  • Blastocysts were cultured in normal ES medium for 1 or 3 days, and then cultured in the presence of BrdU for 3 hours. These cultured blastocysts were immunostained with an antibody specific for BrdU.
  • the 1 ′ deficient pol1 exo allele of 3′-5 ′ exonuclease is capable of embryonic development but increases tumor susceptibility.
  • the effect of its destruction on the development of mice was examined.
  • mice lacking 3′-5 ′ exonuclease activity of Pol ⁇ it has been shown that mutant alleles do not affect embryo viability and that homozygous mutant mice are fertile. However, recessive mutants are prone to cancer and die. It was found that poldl exo / exo and poldl exo / + mice can grow and reproduce normally (Table 2).
  • poldl exo / exo and poldl exo / -mice are normal in development, they die frequently from 3 to 8 months due to abnormal thymic hypertrophy (4/10 pol1 exo / exo animals, poldl exo) / ⁇ 4 out of 12 animals) (FIG. 4A). Many surviving mice without thymic hypertrophy developed an aneurysm in the tail 12 months later (FIG. 4B). These observations are consistent with typical cancer symptoms due to the lack of Pol ⁇ 3′-5 ′ exonuclease activity.
  • Histological analysis of thymic hypertrophy revealed a homogeneous population of cells with typical morphologic features of lymphoblastic lymphoma with a high nuclear-to-cytoplasmic ratio and a high frequency of mitosis ( Data not shown).
  • the poldl exo / + mice and wild type mice did not express such phenotypes in the hypertrophy or tail (more than 20 animals were observed for each phenotype).
  • the loss of Pol ⁇ 3′-5 ′ exonuclease activity also increased the cancer susceptibility of C57BL / 6 mice, but not mice that had been mated with C57BL / 6J and 129SvJ as previously seen. .
  • poldl exo / -mice developed similar tumors.
  • Pold1 exo / exo Preparation Polderuta mutation accumulation model using mice are essential for DNA replication in vivo mammalian, 3'-5 'Pol ⁇ exonuclease activity has been deleted is a frequent DNA replication errors result, mouse It has been suggested that cancer susceptibility is increased.
  • poldl exo / exo mice are fertile and many of the mice can survive long enough to be passaged to the next generation.
  • poldl exo / exo mice were produced using the C57BL / 6 strain.
  • the G1 generation mice were backcrossed again to the G0 generation mice to obtain G2 generation mice (FIG. 7E). Probabilistically, if the number of genetic mutations related to the phenotype is up to 4 and the penetrance is almost complete, some of the 24 G2 mice will have similar phenotypes. Was expected to show. If that was not the case, we determined that there was a non-inherited phenotype. When inheritance was observed in the phenotype, in order to identify the genetic mutation by linkage analysis between C57BL / 6J and DBA2 polymorphism, a new mutant mouse line was established by mating with a DBA2 line mouse.
  • phenotypes including two inherited phenotypes.
  • One of the hereditary phenotypes is a “chirping mutant” that produces a clear cry in the human audible frequency band, like a bird.
  • Analysis of the song recording showed that the song was composed of a series of occurrences (FIG. 6A).
  • Observation of offspring heritability showed that this phenotype is a recessive inheritance, possibly on the X chromosome, independent of Pol ⁇ modification (FIGS. 6 (B) -6 (E)).
  • the obtained mouse was a mouse with a higher volume in the human audible range than in the human non-audible range.
  • the mouse had a high voice volume in the sound range of 20 Hz to 10 kHz shown in FIG.
  • Another hereditary phenotype was a foot variant, with the number of feet decreasing from 5 (Fig. 7 (D)) to 4 (Figs. 7 (A) to 7 (C)).
  • FIG. 7 (E) 0 out of 9 G1 generation mice and 5 out of 18 G2 generation mice developed an abnormal paw phenotype. This indicates that the foot mutant is a recessive inheritance involving two independent mutations.
  • the hair color phenotype Fig. 8 (A)
  • the inferior chin phenotype Fig. 8 (B)
  • the phenotype of rotating behavior Fig. 8 (C)
  • Other mutant genotypes including were obtained. The genetic pattern of these mice has not been evaluated.
  • mice The effects of mutation accumulation in the mouse germline and the development of five mouse lines that independently accumulated natural mutations derived from a process- specific genetic background (C57BL / 6) continued to breed. As a control, the wild-type C57BL / 6 mouse line was similarly bred. All mouse genetic DNA has been stored at -80 ° C in order to investigate the development of germline mutations and the process of wandering from ancestors to offspring. Mutation-accumulating mice have passed 5 generations since their first offspring from cross fertilization.
  • the present invention can be suitably used in industries such as laboratory equipment for manufacturing laboratory mice. Moreover, since the mouse of the present invention is useful for drug development, it can be suitably used in the pharmaceutical industry.
  • FIG. 1 shows a method for producing two types of pol1 mutant mice by a gene targeting strategy.
  • FIG. 1 (A) is a schematic diagram showing the Pold1 domain structure and the position of mutation in the poldl gene targeting method.
  • FIG. 1 (B) shows a partial restriction enzyme map of the mouse pold1 locus before and after homologous recombination with the targeting vector and Cre-mediated recombination. Exons are represented by vertical black squares and introns by horizontal lines in between. * Indicates D400A substitution. The gene fragment used as a probe for Southern blotting is indicated by a horizontal line below the diagram showing exons and introns at the Pold1 locus. Restriction enzyme: D indicates DraI; N indicates NheI.
  • FIG. 1 (C) is a photograph replacing a drawing of an agarose gel showing the results of PCR screening of homologous recombinants. The 2430 bp band is amplified by a combination of P0-P2 primers and is specific to homologous recombinants. Lanes 1-3 are separate homologous recombinant ES cells; lane 4 is a wild type ES cell.
  • FIG. 1 (D) is a photograph replacing a drawing of an agarose gel showing the result of PCR genotyping of mouse tail DNA.
  • FIG. 1 (E) is a photograph in place of a drawing showing the results of Southern blotting analysis performed to identify each poldl allele using mouse tail DNA.
  • the 15.9 kb fragment corresponds to the wild type allele
  • the 16.0 kb fragment corresponds to the poldl exo allele (two duplicates)
  • the 6.4 kb fragment corresponds to pol ⁇ Corresponds to an allele.
  • FIG. 1 (F) shows the results of DNA sequencing of RT-PCT products obtained from heterozygous poldl +/- and poldl exo / + ES cells. In the poldl exo allele, amino acid D is replaced with amino acid A.
  • FIG. 1 (G) shows a schematic diagram of the results of nucleotide sequencing of the 3 ′ RACE product obtained from the Pold1 ⁇ allele in poldl +/ ⁇ . Primers P4 and P5 were used in 3 ′ RACE with repeated amplification.
  • FIG. 1 (H) is a photograph replacing a drawing showing the results of immunoblotting of two pairs of extracts extracted from the whole embryo of E12.5. One pair compares poldl +/- and poldl + / + of littermate embryos. The other pair compares poldl +/- and poldl + / + embryos. Anti-Plod1 antibody and Anti- ⁇ -actin (control) antibody were used as antibodies.
  • FIG. 2 is a photograph in place of a drawing showing morphological analysis of blastocysts and blastocyst outgrowth.
  • FIG. 2 (A) is a photograph replacing the drawing showing the appearance of the mutant embryo.
  • FIG. 2 (B) is a photograph in place of a drawing showing the growth disorder of the growth of the Pold1-deficient blastocyst in vitro. Blastocysts were collected at E3.5 and cultured in vitro for several days, after which genotyping was performed by PCR. The black horizontal bar indicates 50 ⁇ m.
  • FIG. 3 is a photograph in place of a drawing showing DNA synthesis failure and spontaneous apoptosis in a Pold1-deficient embryo.
  • FIG. 3 (B) are photographs in place of drawings showing the results of measuring the BrdU incorporation of blastocysts cultured in vitro and observing DNA synthesis.
  • E3.5 embryos were cultured for 24 hours and then for 3 hours in the presence of BrdU. Indirect immunofluorescence was performed using an anti-BrdU antibody and an anti-pHH antibody, and after counterstaining with DAPI, the embryos were genotyped by PCR (FIG. 3 (A)). E3.5 embryos were cultured for 3 days, and cultured for 3 hours in the presence of BrdU, followed by treatment for immunostaining for BrdU and DAPI counterstaining. Thereafter, each embryo was genotyped (FIG. 3B). A white horizontal bar indicates 100 ⁇ m.
  • FIG. 3 (C) shows the result of analysis of spontaneous apoptosis by TUNEL assay. It is the photograph replaced with drawing in which a TUNEL positive cell represents the increase of a pold1-null blastocyst. After photography, the genotype of each embryo was examined by PCR. A white horizontal bar indicates 100 ⁇ m.
  • FIG. 4 is a photograph replacing a drawing showing a tumor caused by a deficiency of 3′-5 ′ exonuclease activity of Pol ⁇ .
  • FIG. 4 (A) is a photograph of swollen thymus of 4-month-old poldl exo / ⁇ mice. Th indicates the thymus and H indicates the heart.
  • FIG. 4 is a photograph replacing a drawing showing a tumor caused by a deficiency of 3′-5 ′ exonuclease activity of Pol ⁇ .
  • FIG. 4 (A) is a photograph of swollen thymus of 4-month-old
  • FIG. 4 (B) is a photograph of the tail on which a nodule of 14 month old poll1 exo / ⁇ mouse was formed.
  • FIG. 5 is a schematic view of a mutation accumulation experimental mouse. Mutant mice effectively accumulate many non-lethal mutations, including neutral mutations, as generations repeat, because mice with lethal mutations cannot transmit DNA to their offspring.
  • FIG. 6 shows a chirping mutant.
  • FIG. 6 (A) shows a representative sonogram of a sound emitted by a chirping mutant. The vertical axis indicates the audible frequency (kHz), the horizontal axis indicates time (s), and the dot density indicates the sound intensity.
  • FIGS. 6B-6E characterize the genetic traits of the chirping phenotype.
  • FIG. 7 shows a foot variant.
  • FIG. 7 (A) to FIG. 7 (C) are photographs in place of drawings of mutant feet.
  • FIG. 7D is a photograph replacing a drawing of a wild-type foot.
  • FIG. 7 (E) is a systematic diagram showing the genetic traits of the foot phenotype. Circles indicate females, squares indicate males, and black bars indicate mice that have developed a paw phenotype. “Loss” in FIG. 7E shows the number of offspring whose birth index has decreased.
  • FIG. 8 is a photograph replacing a visual phenotype drawing.
  • FIG. 8A is a photograph replacing a drawing of a mouse whose hair color is partly brown.
  • FIG. 8B is a photograph replacing a drawing of a mouse with an immature mandible.
  • FIG. 8C is a photograph in place of a drawing of a mouse that rotates abnormally: continuously to the right.
  • FIG. 8D is a photograph replacing a drawing of a mouse with a tail wound.
  • FIG. 8 (E) is a photograph replacing a drawing of a mouse having a shorter tail (upper) than the normal tail (lower) of the control.
  • FIG. 9 shows a graph obtained by the theoretical model.
  • FIG. 9 (A) shows the relationship between the spontaneous lethal mutation rate, that is, the number of generations after the first mating set and the death frequency among the offspring.
  • FIG. 9B shows the relationship between the spontaneous lethal mutation rate after infinite generations and the death frequency among offspring.

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Abstract

Cette invention concerne une souris capable d'émettre des sons dans la gamme des sons audibles par l'homme. L'invention concerne également une souris dont les émotions peuvent être faciles à comprendre en fonction des sons qu'elle émet. Une souris à voix puissante située dans la gamme des sons audibles par l'homme par rapport à la voix dans la gamme des sons non audibles par l'homme peut être obtenue en croisant plusieurs fois des souris dépourvues de l'activité exonucléase 3'-5' de l'ADN polymérase δ les unes avec les autres. Comme souris dépourvue de l'activité exonucléase 3'-5' de l'ADN polymérase δ décrite ci-dessus, on peut citer une souris obtenue en croisant les unes avec les autres des souris portant une protéine mutée, où l'acide aspartique en position 400 dans la séquence d'acides aminés représentée par la séquence SEQ ID NO:1 a été substitué par un autre acide aminé. En croisant ces souris les unes avec les autres comme décrit ci-dessus, il est possible d'obtenir une souris dont le cri est muté.
PCT/JP2009/000924 2008-03-03 2009-03-02 Souris à cri muté WO2009110209A1 (fr)

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