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WO1997018703A1 - Procede de creation d'animaux transgeniques - Google Patents

Procede de creation d'animaux transgeniques Download PDF

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Publication number
WO1997018703A1
WO1997018703A1 PCT/JP1996/003375 JP9603375W WO9718703A1 WO 1997018703 A1 WO1997018703 A1 WO 1997018703A1 JP 9603375 W JP9603375 W JP 9603375W WO 9718703 A1 WO9718703 A1 WO 9718703A1
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WO
WIPO (PCT)
Prior art keywords
animal
adenovirus
egg
recombinant adenovirus
transgenic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/JP1996/003375
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English (en)
Japanese (ja)
Inventor
Yutaka Toyoda
Izumu Saito
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sumitomo Pharma Co Ltd
Original Assignee
Sumitomo Pharmaceuticals Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sumitomo Pharmaceuticals Co Ltd filed Critical Sumitomo Pharmaceuticals Co Ltd
Priority to AU75881/96A priority Critical patent/AU7588196A/en
Publication of WO1997018703A1 publication Critical patent/WO1997018703A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A01K2217/05—Animals comprising random inserted nucleic acids (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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a transgenic animal obtained by introducing a foreign gene into a genome using a non-replicating recombinant adenovirus, and a method for producing the transgenic animal.
  • non-propagating (E1A deficient) recombinant adenovirus is hardly integrated into the host genome (Rosenfeld et al., Science—252, 43 1-434 (1991)).
  • Various studies focusing on the fact that it has been used for gene regulation research and gene expression for the development of somatic cell gene therapy (Akli et al., Nat. Genet. _3, 224-228 (1993)).
  • a non-replicating adenovirus containing the LacZ gene could infect pronuclear-stage non-enveloping mouse eggs and was expressed without any adverse effects in the cleavage-stage preimplantation embryo. (Tsukui et al., J. Ma Rat Ova. Res., 10 (1), (1993, 4) 150-151).
  • a first object of the present invention is to provide a simple and efficient method for producing a transgenic animal in which a foreign gene has been introduced into the genome. Further, a second object of the present invention is to provide a transgenic animal produced by the method of the present invention.
  • the gist of the present invention is:
  • a transgenic animal characterized in that a non-replicating recombinant adenovirus carrying a foreign gene is infected into a fertilized egg from which the egg membrane has been removed, and then the embryo is transplanted into the uterus of a parent animal.
  • FIG. 1 is a photograph of an electrophoresis showing the identification of integration of a non-replicating adenovirus DNA by Southern analysis.
  • Ec Genomic DNA isolated from the tails of 27 animals. The digest was digested with a shakupobi ⁇ ! IndIII, and the cosmid DNA of pAxCANLacZ, which has 98% homology with the AxCANLacZ sequence, was used as a probe.
  • a EcoRV digestion data
  • b is Hind1 [1 digestion data. Open circles are end fragments of adenovirus. The asterisk indicates the connected fragment of the virus-cell DNA after the integration event.
  • FIG. 2 shows adenovirus DNA integrated into the genome of three transgenic mice.
  • the thick line shows the restriction enzyme map of the recombinant adenovirus AxCANLacZ.
  • FIG. 3 is a photograph of electrophoresis showing germline inheritance in F1 progeny and expression of the adenovirus transgene on F1.
  • LacZ expressing transgenic mice AC L9.23
  • Genomic DNA isolated from the 10 placentas was subjected to Southern analysis according to a procedure similar to that shown in FIG. Lanes 1, 3, 4, 7, 8, and 10 indicate that there was transmission of the adenovirus transgene, and lanes 2, 5, 6, and 9 indicate that there was no such transmission.
  • the asterisk indicates the ligated fragment of virus-one cell DNA after the integration event.
  • the proliferative adenovirus used in the present invention was Hiromi Kanegae, Shizuko Harada and Izumi Saito "Experimental Medicine Supplement” Bio Manual Series 4 "Gene Transfer and Expression ⁇ Analysis Method” 4 page 3-58 "Adenovirus was used. Gene Transfer ”, 1989. A non-propagating adenovirus into which a foreign gene has been introduced can be prepared.
  • the non-competent adenovirus used in the present invention has several features.
  • adenoviruses have a very wide host range, and secondly, allow the introduction of recombinant genes into non-dividing cells.
  • the non-replicating recombinant adenovirus has such properties and is expected to be a vector for fertilized animal eggs in the pronuclear phase.
  • Adex4SRLacZL containing the E. coli LacZ gene under the control of SRa Promote, a non-reproductive adenovirus vector. Attempts to introduce a gene into the fertilized egg at the pronucleus stage failed to detect expression in 13.5-day embryos that grew from infected eggs (Tsukui, T., et al. Ol.
  • a promoter into an adenovirus vector in order to enable expression of a foreign gene after infecting animal cells.
  • Any of those that have been used for gene transfer using E. coli can be used, and in particular, CAG promoter (Araki et al., Proc.
  • a nuclear localization signal sequence may be incorporated together with the foreign gene.
  • the nuclear localization signal sequence is expressed by the adenovirus vector in the nucleus of infected cells and promotes the translocation of foreign gene products secreted outside the nucleus into the nucleus again (Daniel Kalderon et al., Cell 39, 499- 509 (1984)).
  • Ax C ANL ac Z is expressed by a foreign gene (L ac Z) —CAG used for universal expression in transgenic mice to identify galactosidase ( ⁇ -ga 1) activity Includes nuclear-targeted LacZ gene under the control of the promoter.
  • the preparation of this vector can be performed as follows. For example,
  • the foreign gene used in the present invention is not particularly limited. From the viewpoint of usefulness, nucleotides having a base sequence encoding a bioactive protein, an antibody, a chromogenic protein as a marker gene, a drug-resistant protein as a marker gene and the like can be mentioned.
  • bioactive proteins include cytokins (eg, interleukin 1-1-15, interferon-1, or a, tumor necrosis factor or granulocyte colony stimulating factor, erythropoietin, growth hormone, insulin, Insulin-like growth hormone, etc.), neurotrophic factors, ⁇ -1 antitribcine, blood coagulation factor 8, blood coagulation factor 9 and ⁇ -galactosidase.
  • cytokins eg, interleukin 1-1-15, interferon-1, or a, tumor necrosis factor or granulocyte colony stimulating factor, erythropoietin, growth hormone, insulin, Insulin-like growth hormone, etc.
  • neurotrophic factors eg
  • a transgenic animal refers to a non-human mammal containing a foreign gene introduced into the reproductive system.
  • mouse rat, Usagi, guinea pig, catcher formic, Hijji, pigs, mice below c where ⁇ sheet etc.
  • FIG. 1 illustrates an exemplary method of creating Bok Ransuji Nick animals I do.
  • superovulation is induced by intraperitoneally injecting PMSG (human chorionic gonadotropin) and hCG (human chorionic gonadotropin) into female mice.
  • PMSG human chorionic gonadotropin
  • hCG human chorionic gonadotropin
  • non-proliferating adenoviruses such as AxCANL ac Z
  • the virus is infected at a dose of 10 to 10 a pfu Zm 1, preferably 5 ⁇ 10 7 to 10 ′′ pfu / m 1, and the reaction time with the virus is 2 to 8 hours.
  • the infected embryo is transferred to the uterus of the parent mouse.
  • the transgene is stably transmitted, and the recombinant non-proliferating adenovirus Confirm that the derived foreign gene is expressed.
  • the following properties are observed in the transgenic mouse obtained as described above.
  • proviral integration can occur independently in one or more cells. Therefore, germline heritability is generally low due to mosaicism (Jaenisch et al., Cell 24, 519 529 (1981)).
  • mice One-cell eggs of mice were infected with 1 ⁇ 10 8 pfu / ml of Ax CANL ac Z (an adenovirus having a nuclear translocation signal) with or without the egg membrane.
  • the infected eggs were cultured and stained at the 2-cell stage 24 hours after infection.
  • Galactosidase activity was detected by histochemical (X—Ga1) staining (Beddington et al., Development 106, 37-46 (1989)).
  • Example 2 Determining Whether the Adenovirus Genome Was Incorporated into the Mouse Egg Genome
  • infected embryos in b stocyst (4 days after culture) were implanted into the uterus of the borrowing parent.
  • Genomic DNA isolated from tail vein blood of 27 born mice was digested with restriction enzymes Eco RV and Hind III.
  • Southern blot analysis revealed that three of the animals contained adenovirus DNA, as shown in Figure 1. Each of lanes 2 to 4 was identified as an internal fragment of the virus. Then, asterisks are put on putative fragments of the virus-one-cell DNA generated by integration of the adenovirus genome into the host genome.
  • mice All three mice (ACL9.!, 9.3 and 9.23) carried one adenovirus genome, and the above analysis indicated that the adenovirus genome was randomly integrated into the mouse genome.
  • Ec mouse genomic DNA (10 g). Shaku or! After digestion with indill, electrophoresis was performed on a 1.0% agarose gel, blotted on a nylon membrane Hybond-1N (manufactured by Amersham), and hybridized with a randomly digoxigenin-labeled probe, followed by Southern analysis.
  • the viral transgenes of the other two transgenic mice also transmitted to progeny F1 (11 out of 2 and 7 out of 13, respectively) ). Therefore, it can be said that the adenovirus transgene was confirmed to be stably inherited in F1 progeny.
  • the transfer of the adenovirus transgene to the germ line was according to Mendel's law, and no translocation was observed (Figure 3).
  • fetuses were stained with X-ga1 to examine / 3-ga1 activity by a modification of the method of Allen et al. (Allen, ND et al., Nature 333, 852-855 (1988)). Briefly, fetuses were placed in phosphate buffer (pH 7.7) containing 0.5% formaldehyde, 2.5% glutaraldehyde, and 0.05% NP40 for 30 minutes at 4 ° C. Dipped and fixed. A second fixation was performed for another 15 minutes.
  • phosphate buffer pH 7.7
  • Example 6 Incubation at 37 ° C for 4 hours in l ⁇ mI phosphate buffer containing X-ga1 c
  • Ax CANL ac Z was infected, cultured to b1 astocyst in vitro, and then returned to the borrower's uterus. Indeed, transplanted infected embryos 1 X 1 0 7, 5 X 1 0 7 and 1 X 1 0 8 (pfu / m 1), 5 5, 3 2, and 3 6 animals individuals respectively obtained was. Table 1 shows the results of Southern blot analysis of these individuals using the LacZ gene as a probe.
  • transgenic animals can be easily and efficiently produced.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • Animal Behavior & Ethology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Biodiversity & Conservation Biology (AREA)
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Abstract

L'invention porte sur un procédé pratique et efficace de création d'animaux transgéniques consistant à infecter un oeuf d'animal fécondé, dont la membrane a été éliminée, avec de l'ADN de recombinaison non reproducteur dans lequel a été introduit un gène étranger, puis à transférer l'embryon dans l'utérus de l'hôte.
PCT/JP1996/003375 1995-11-20 1996-11-18 Procede de creation d'animaux transgeniques Ceased WO1997018703A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU75881/96A AU7588196A (en) 1995-11-20 1996-11-18 Method for constructing transgenic animals

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP32653395A JP3799426B2 (ja) 1995-11-20 1995-11-20 トランスジェニック動物の作成方法
JP7/326533 1995-11-20

Publications (1)

Publication Number Publication Date
WO1997018703A1 true WO1997018703A1 (fr) 1997-05-29

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PCT/JP1996/003375 Ceased WO1997018703A1 (fr) 1995-11-20 1996-11-18 Procede de creation d'animaux transgeniques

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JP (1) JP3799426B2 (fr)
AU (1) AU7588196A (fr)
WO (1) WO1997018703A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7129084B2 (en) 2000-08-03 2006-10-31 Therapeutic Human Polyclonals, Inc. Production of humanized antibodies in transgenic animals
US11230697B2 (en) 2006-09-01 2022-01-25 Therapeutic Human Polyclonals Inc. Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CELL, 39, DANIEL KALDERON et al., pages 499-509, (1984). *
J. MAMM. OVA. RES., 10(1), TSUKUI et al., pages 150-151 (1993). *
MOL. REPROD. DEV., 42, TSUKUI et al., pages 291-297, (1995). *
PROC. NATL. ACAD. SCI. U.S.A., 92, ARAI et al., pages 160-164 (1995). *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7129084B2 (en) 2000-08-03 2006-10-31 Therapeutic Human Polyclonals, Inc. Production of humanized antibodies in transgenic animals
US11230697B2 (en) 2006-09-01 2022-01-25 Therapeutic Human Polyclonals Inc. Enhanced expression of human or humanized immunoglobulin in non-human transgenic animals

Also Published As

Publication number Publication date
JPH09140292A (ja) 1997-06-03
AU7588196A (en) 1997-06-11
JP3799426B2 (ja) 2006-07-19

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