WO1997017843A1 - Procede pour realiser des animaux transgeniques - Google Patents

Procede pour realiser des animaux transgeniques Download PDF

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Publication number
WO1997017843A1
WO1997017843A1 PCT/JP1996/003359 JP9603359W WO9717843A1 WO 1997017843 A1 WO1997017843 A1 WO 1997017843A1 JP 9603359 W JP9603359 W JP 9603359W WO 9717843 A1 WO9717843 A1 WO 9717843A1
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WO
WIPO (PCT)
Prior art keywords
gene
hvj
gene transfer
vector
present
Prior art date
Application number
PCT/JP1996/003359
Other languages
English (en)
Japanese (ja)
Inventor
Ryuichi Morishita
Yasushi Kaneda
Toshio Ogiwara
Tsuneaki Sakata
Mamoru Hasegawa
Original Assignee
Dnavec Research Inc.
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 Dnavec Research Inc. filed Critical Dnavec Research Inc.
Priority to AU75879/96A priority Critical patent/AU7587996A/en
Publication of WO1997017843A1 publication Critical patent/WO1997017843A1/fr

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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
    • A01K67/0276—Knock-out vertebrates
    • 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
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, 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
    • A01K2217/05—Animals comprising random inserted nucleic acids (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
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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
    • 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
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription

Definitions

  • the present invention relates to a technique for producing transgenic animals.
  • transgenic animals In developmental biology and molecular biology, techniques for creating transgenic animals include: (1) analysis of the expression of genetic information that accompanies temporal changes such as gene development, differentiation, growth, and aging in vivo; Analysis of the gene structure involved in the expression of tissue-specific genetic information, 3 Analysis of the physiological and pathological roles of gene products at the individual level, ⁇ Creation of disease model animals, 5 Occurred by the introduction of foreign genes It has been applied to the analysis of unknown genes using insertion mutations, etc., and ⁇ ⁇ the direct function evaluation of unknown genes.
  • a knockout animal in which a specific gene is inactivated by using a technology obtained by applying and developing this technology, it is also possible to identify the role of the gene at the developmental stage or the function in vivo. it can.
  • transgenic technology has greatly contributed to the development of biotechnology.
  • this technology to livestock, it can be used to produce useful bioactive substances and improve varieties.
  • the most commonly used method for producing transgenic animals by a conventional method is to inject the isolated gene into the pronucleus of a fertilized egg through a glass capillary attached to the tip of a micromanipulator. I have. After injection, survived recipients The sperm is transplanted into the oviduct of the foster mother mouse. In other words, basically, three operations are performed: collection of a fertilized egg, injection of a gene, and transplantation of a fertilized egg into a fallopian tube. These operations require a high level of skill. Further, the number of transgenic mice finally obtained by the conventional method is about 1 to 3 on average when starting from 100 fertilized eggs, and the efficiency is very low. Furthermore, since the conventional method uses a fertilized egg, there is a disadvantage that it is impossible to knock out essential genes involved in the early stage of development.
  • An object of the present invention is to overcome the technical difficulties of the conventional technology and to easily produce a transgenic mammal. It is another object of the present invention to introduce a gene in a stage-specific and site-specific manner at various stages of development, which was impossible with the prior art.
  • the present inventors have found that a composition containing a compound having an affinity for both DNA and cells and a vector for gene transfer can be efficiently contacted with the epidermis of a mammal including living epidermis cells directly.
  • the present inventors have found that gene transfer can be performed and completed the present invention. More specifically, the present inventors have found that, when a gene transfer vector is introduced into fetal amniotic fluid, the gene is transferred in a body surface-specific manner over the entire body surface, thus completing the present invention. did.
  • the present invention provides a method for producing a transgenic mammal other than a human, Regarding the composition for gene transfer used in the method, specifically,
  • a method for producing a transgenic mammal other than a human which comprises injecting a gene transfer composition comprising a compound having affinity and a gene transfer vector.
  • composition for gene transfer contains an inactivated Sendai virus (HV J);
  • composition for gene transfer which is used in the method according to any one of (1) to (3),
  • Examples of the “compound having an affinity for both DNA and the body surface of a mammalian embryo or fetus” in the “gene transfer composition” of the present invention include cationic ribosomes (PLFelgner et al., Pro Natl. Acad. Sci. USA, 84, 7413 (1987), CM Corma et al., Ol. Cell. Biol., 2, 1044 (1982), N. Haga, K. Yagi, J. Clin. Biochem. Nutr.
  • the “gene transfer vector” in the “gene transfer composition” of the present invention basically all gene transfer vectors are used.
  • retrovirus vectors for gene transfer such as adeno-associated virus vectors.
  • the “gene introduction composition” of the present invention As a method for injecting the “gene introduction composition” of the present invention into amniotic fluid, there is a method of injecting directly into amniotic fluid in the uterus using an injector or the like. Further, as the genes to be guided by the “gene transfer vector” of the present invention, all types of genes such as development stage-specific genes, organ-specific, particularly skin-specific shoalers, and other genes for treating various diseases are included. No.
  • the range of animals to which the method of the present invention can be applied or the range of animals to which the composition of the present invention can be administered includes all mammals such as mice, puppies, puppies, and monkeys except humans. It is possible.
  • the fertilized egg which is a pre-implantation embryo for embryo development, as well as the 2-cell stage embryo, the 4-cell stage embryo, and the 8-cell stage Genes can be introduced into embryos at any stage, including embryos, morulae, blastocysts, late blastocysts, early ovarian embryos, ovarian embryos, and late ovarian embryos.
  • the gene can also be introduced into the embryo after implantation.
  • Such "introduction of a stage-specific gene" according to the present invention is considered to give very important findings to embryology research.
  • the role of the gene at each stage of development can be inferred by suppressing genes that act stage-specifically, such as by introducing antisense.
  • the gene by introducing the gene into each site at each developmental time, the future fate of the development at that site and the fate of the cell can be observed.
  • introduction of the gene after a certain stage of development can avoid toxicity due to overexpression of the gene.
  • Fig. 1 (A)-Fig. 1 (B) are photomicrographs showing the distribution of FITC-labeled 0DN introduced into fetal rat amniotic fluid by IS-injector after 2 hours.
  • (A) is a micrograph observed at 40 ⁇ and
  • (B) is a micrograph observed at 100 ⁇ .
  • Figure 2 is a photomicrograph showing the distribution of FITC-labeled 0DN 5 days after introduction into fetal rat amniotic fluid by direct injection.
  • FIG. 3 shows the structure of plasmid pSV40Gal expressing ⁇ -galactosidase gene.
  • FIG. 4 (A) -FIG. 4 (C) are photographs of rat fetal skin on day 5 after transfusion.
  • (A) and (B) relate to rats transfected with the S-galactosidase vector and control vector, respectively, and (C) relates to untreated rats.
  • FIG. 5 (A) -FIG. 5 (C) are photographs showing histological analysis of rats on day 5 after transfusion.
  • (A) and (B) relate to a rat transfected with one galactosidase vector and a control vector, respectively, and (C) relates to an untreated rat.
  • FIG. 6 (A) -FIG. 6 (C) are photographs showing histological analysis of rats 10 days after transfection.
  • (A) and (B) relate to the rat transfected with the -galactosidase vector and the control vector, respectively, and (C) relates to the untreated rat.
  • Phosphatidylserine, phosphatidylcholine, and cholesterol were mixed at a weight ratio of 1: 4.8: 2.
  • the dried lipid was dissolved in 200 1 BSS solution (137 mM NaCl, 5.4 mM C1, 10 mM Tris-HCl, pH 7.6) containing plasmid DNA or FITC-labeled oligonucleotide (0DN).
  • the ribosome was prepared by shaking and ultrasound (see “Liposome Experiments in Life Sciences”, Springer 1. Pharaak Tokyo (1992), p. 282-287).
  • HVJ strain Z
  • UV irradiation 110 erg / nun 2 / sec
  • the ribosome solution described above (10 mg liposome / 0.5 ml solution) was mixed with 10,000 hemagglutination units of HVJ and diluted with BSS solution to a final volume of 4 ml. The mixed solution was kept at 4 for 5 minutes, and then gently shaken at 37 for 30 minutes. Unreacted HVJ and liposomes were removed from the HVJ-ribosome by a sucrose density gradient.
  • FITC-labeled oligonucleotide (0DN)
  • the oligonucleotide (0DN) was labeled with FITC at the 5 'end and 3' end (Antisense Res. Develop. Vol. 2, 27-39 (1992)).
  • the HVJ-ribosome containing the labeled ODN was prepared by the method of Example 1.
  • the final concentration of the FITC-labeled 0DN injected into the amniotic fluid was 3 M (concentration of 0DN relative to the total ribosome solution).
  • 101 ⁇ l of HVJ-ribosome containing labeled 0DN prepared by the method of Example 1 was injected through a 30-gauge injection needle. .
  • HVJ ribosomes containing no FITC-labeled 0DN prepared by the method of Example 1 were injected. All rats were sacrificed 2 hours or 5 days later, and the extracted tissues were fixed in a 4% paraformaldehyde solution according to a conventional method, and the sections were stained with erythrochrome black and observed with a fluorescence microscope.
  • pSV40Gal a plasmid that expresses the ⁇ -peroxidase gene, was constructed as follows. Plasmid DNA pAct-c-myb containing the 370 base pair 5 'promoter overnight region and the 900 base pair primary 1-xon region of the nitrile / S-actin promoter to remove the cmb region Cleavage with restriction enzymes Ncol and Xbal was followed by ligation with Sail linker. 3. One kilobase pair of E. coli; the 3-galactosidase gene was excised from the plasmid DN ApMC1871 using the restriction enzyme Sail and bound to the Sail site on the plasmid thigh (Fig. 3).
  • a plasmid DNA pAct-CV containing no E. coli yS-galactosidase gene was simultaneously prepared.
  • HVJ-ribosomes containing the 9-galactosidase gene plasmid pSV40Gal or control plasmid DNA pAct-CV were prepared.
  • HMG-1 protein was simultaneously encapsulated in HVJ-ribosomes.
  • a syringe was injected directly into the amniotic fluid from the uterine wall, and 10 ⁇ 1 of HVJ-ribosome prepared by the method of Example 2 was injected.
  • transgenic animals can be created more easily than in the past.
  • Transgenic animals in which genes have been specifically introduced into the entire body are particularly useful for studies of skin cells, including fibroblasts. Specifically, (1) the expression of genetic information associated with temporal changes such as the development, differentiation, growth, and aging of skin-specific genes; (2) the analysis of the gene structure involved in the expression of skin-specific genetic information; and (3) the skin. It can be applied to research such as creation of disease model animals.
  • the present invention can be applied to the production of useful livestock with altered skin characteristics.
  • knockout of essential genes and introduction of lethal genes have become possible, and the range of transgenic animals that can be produced has been expanded.
  • “stage-specific gene transfer” has become possible using the present invention.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • General Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Environmental Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Animal Behavior & Ethology (AREA)
  • Molecular Biology (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Biochemistry (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

L'invention concerne un procédé pour réaliser aisément des mammifères transgéniques, consistant à injecter dans le liquide amniotique d'un mammifère une composition permettant l'introduction d'un gène contenant un composé ayant des affinités pour l'ADN et l'embryon du mammifère ou la surface du corps foetal de celui-ci, ainsi qu'un vecteur permettant l'introduction du gène. Ce procédé autorise une introduction du gène spécifique du stade, à divers stades du développement, ou bien une introduction sito-spécifique du gène, lesquelles ne peuvent pas être réalisées par les techniques classiques.
PCT/JP1996/003359 1995-11-16 1996-11-15 Procede pour realiser des animaux transgeniques WO1997017843A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU75879/96A AU7587996A (en) 1995-11-16 1996-11-15 Process for constructing transgenic animals

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP29871395 1995-11-16
JP7/298713 1995-11-16
JP8/114260 1996-04-11
JP11426096 1996-04-11

Publications (1)

Publication Number Publication Date
WO1997017843A1 true WO1997017843A1 (fr) 1997-05-22

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Application Number Title Priority Date Filing Date
PCT/JP1996/003359 WO1997017843A1 (fr) 1995-11-16 1996-11-15 Procede pour realiser des animaux transgeniques

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AU (1) AU7587996A (fr)
WO (1) WO1997017843A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7576080B2 (en) 2004-12-23 2009-08-18 Memory Pharmaceuticals Corporation Certain thienopyrimidine derivatives as phosphodiesterase 10 inhibitors

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
BLOOD, Vol. 78(4), 1991, WADE CLAPP et al., pages 1132-1139. *
GENE THERAPY, Vol. 2, July 1995, PITT et al., pages 344-350. *
GENE, Vol. 149, 1994, MORISHITA et al., pages 13-19. *
J. BIOL. CHEM., Vol. 264, 1989, KANEDA et al., pages 12126-12129. *
J. CLIN. INVEST., Vol. 95, June 1995, McCRAY et al., pages 2620-2632. *
NATURE SCIENCE, Vol. 1(11), 10 November 1995, HARMANJATINDER et al., pages 1201-1203. *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7576080B2 (en) 2004-12-23 2009-08-18 Memory Pharmaceuticals Corporation Certain thienopyrimidine derivatives as phosphodiesterase 10 inhibitors

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AU7587996A (en) 1997-06-05

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