WO2002011529A1 - Procede de construction d'un animal par manipulation genetique et animal clone - Google Patents
Procede de construction d'un animal par manipulation genetique et animal clone Download PDFInfo
- Publication number
- WO2002011529A1 WO2002011529A1 PCT/JP2000/005326 JP0005326W WO0211529A1 WO 2002011529 A1 WO2002011529 A1 WO 2002011529A1 JP 0005326 W JP0005326 W JP 0005326W WO 0211529 A1 WO0211529 A1 WO 0211529A1
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- WO
- WIPO (PCT)
- Prior art keywords
- animal
- cells
- nucleus
- metaphase
- cell cycle
- Prior art date
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- GBOGMAARMMDZGR-TYHYBEHESA-N cytochalasin B Chemical compound C([C@H]1[C@@H]2[C@@H](C([C@@H](O)[C@@H]3/C=C/C[C@H](C)CCC[C@@H](O)/C=C/C(=O)O[C@@]23C(=O)N1)=C)C)C1=CC=CC=C1 GBOGMAARMMDZGR-TYHYBEHESA-N 0.000 description 3
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- 210000002459 blastocyst Anatomy 0.000 description 1
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Classifications
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/873—Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
-
- 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
Definitions
- the present invention relates to a method for producing a genetically modified animal and a method for producing a cloned animal (a genetically identical animal population). Animals created by these methods are useful as model animals for various diseases and the like. Conventional technology
- Known methods for producing genetically engineered animals include a method of injecting a gene into the nucleus of a fertilized egg and a method of introducing a gene into embryonic stem cells (ES cells) using homologous recombination. Also requires a great deal of labor and time. In particular, when a gene is introduced into embryonic stem cells, the target genetically engineered animal cannot be obtained directly, but can be obtained in the next generation through a reproductive chimera individual, so that the labor and time are greater. Embryonic stem cells from which reproductive chimeras can be efficiently produced are limited to a specific lineage, so if you want to obtain a genetically engineered animal of a lineage other than that lineage, backcross to the target lineage Need to be repeated.
- somatic cloned animals have been produced in horses, sheep, goats and mice.
- cells whose cell cycle has been synchronized to the G0 / G1 phase by serum starvation culture or the like or cells cultured without synchronized culture are usually used.
- Wakayama T. et al. Reported a method using cells synchronized in metaphase (M phase) (Wakayama T. et al., Proc. Natl. Acad. Sci. USA, 1999, 26, 14984-14989).
- Metaphase cells used in this method were selected using only cell size as an index.
- the nuclear transfer operation is performed only once.
- a first object of the present invention is to solve the problems of the conventional method for producing a somatic cell cloned animal and to provide a means for more efficiently producing a somatic cell cloned animal.
- a second object of the present invention is to solve the above-mentioned problems concerning the production of a genetically modified animal and to provide means for producing a genetically modified animal in a short time. Means for solving the problem
- the present inventors have conducted intensive studies in order to solve the above problems, and as a result, have obtained the following knowledge.
- genetically engineered animals can be created in a shorter time than conventional methods.
- embryonic stem cell nuclei as the donor nucleus can significantly reduce the mortality during the growth of the cloned animals produced.
- the present invention has been completed based on the above findings.
- the first aspect of the present invention relates to a method for producing a genetically modified animal, comprising the following steps.
- a second aspect of the present invention relates to a method for producing a genetically modified animal, comprising the following steps.
- the third aspect of the present invention relates to a method for producing a cloned animal, comprising the following steps.
- the fourth aspect of the present invention relates to a method for producing a cloned animal, comprising the following steps.
- the method for producing a genetically modified animal of the present invention includes the following steps (A) to (D) and (F), and optionally includes step (E).
- step (A) genetic manipulation is performed on animal cells.
- Any animal cells may be used here, and for example, fibroblasts, embryonic stem cells, cumulus cells, Sertoli cells and the like can be used, but embryonic stem cells are preferably used.
- the use of embryonic stem cells can reduce the mortality during the growth of the resulting individual.
- Animal cells are not particularly limited as long as they are derived from animals other than humans, and cells derived from mice, mice, pigs, sheep, monkeys and the like can be used.
- Examples of the gene manipulation include gene introduction, gene disruption (gene targeting), and gene replacement (knock-in).
- the cell cycle of the animal cell is synchronized with the metaphase.
- Entrainment to metaphase can be achieved with appropriate reagents, such as Nocodazole, Colcemid, and the like.
- step (C) the nucleus of the cell whose cell cycle has been synchronized to metaphase is transferred to the cytoplasm of an enucleated unfertilized egg.
- the enucleation of an unfertilized egg is performed, for example, by aspirating and removing a slightly transparent portion of the cytoplasm of the unfertilized egg in an operation solution containing cytochalasin B, a cytoskeleton polymerization inhibitor.
- Nuclear transfer can be performed by cell fusion using the Sendai virus or electrical stimulation or intracytoplasmic injection.
- step (D) the nucleus is activated.
- the activation treatment include a method using strontium, ethanol, electrical stimulation and the like. The activated nucleus starts fissioning.
- step (E) the activated nucleus is transplanted to an enucleated fertilized egg.
- Step (E) is not essential, and step (F) may be immediately shifted to step (F).
- Enucleation and nuclear transfer can be performed in the same manner as in step (C).
- the fertilized eggs are preferably used 8 to 12 hours after insemination.
- an operation for obtaining a transplanted nucleus-derived newborn is performed. Specifically, operations such as culturing activated eggs and transplanting them into the uterus of a foster parent are performed. These operations can be performed according to a conventional method.
- the method for producing a cloned animal of the present invention comprises the following steps (B) ′ to (D) and (F) And (E) if necessary.
- Steps (C) to (F) are the same as the above-described method for producing a genetically modified animal.
- (B) ′ is the same as the above-described method for producing a genetically engineered animal, except that embryonic stem cells are essential as animal cells.
- the genetically engineered embryonic stem cell line TT2 was seeded on a single feeder cell layer to form embryonic stem cell colonies.
- the genetically engineered TT2 strain was obtained from Kyoto University's Virus Research Institute and Assistant Prof. Yoichi Makai. The gene is in a state of heterogeneity).
- As the feeder cells fibroblasts obtained by shredding a 1-day-old fetus of a CD-1 mouse with scissors and the like and then treating with trypsin were cultured and treated with mitmycin. After sufficient growth of the embryonic stem cells, the cells were treated with trypsin-EDTA, detached from the culture dish together with one feeder cell, and seeded on another culture dish. One hour after the seeding, the cell suspension in the culture dish was collected.
- the time required for the embryonic stem cells and feeder cells to adhere to the culture dish varies, and within 1 hour after seeding, the feeder cells adhere to the bottom of the culture dish, but the embryonic stem cells float in the culture medium It is in a state. Therefore, the cell suspension collected by the above operation contains only embryonic stem cells. After centrifugation, the cell suspension was suspended in a cryopreservation solution (70% DMEM, 10MEMDMS0, 20% FBS) at a concentration of 1 ⁇ 10 5 cells / 0.5 ml per tube, and cryopreserved in liquid nitrogen.
- a cryopreservation solution 70% DMEM, 10MEMDMS0, 20% FBS
- cryopreserved embryonic stem cells pre-gelatinized in co one-diameter and 60 thigh 2xl0 4 cell seeded in culture dishes coating, and cultured for 3 days 15% FBS added ES culture. Since this culture dish does not contain feeder cells, subsequent embryonic stem cells are not contaminated with feeder cells.
- a culture solution containing the same amount of nocodazole was added except for half of the culture solution (final concentration of nocodazole: O ⁇ g / ml). Synchronization treatment was performed on metaphase cells. If the time of nocodazole treatment was prolonged, damage to the cells was observed, so the treatment time was set to 2 hours in this example.
- Metaphase cells are weakly adherent and are suspended in the culture medium, so collecting the culture medium allows recovery of metaphase cells.
- Metaphase cells have a round morphology and are about 1.5 times larger in diameter than cells in the interphase cell cycle, and the proportion of metaphase cells in recovered cells was determined using this morphology as an index. . As a result, about 80 cells were metaphase cells.
- HCG was administered to B6CBF1 female mice, and unfertilized eggs at the second metaphase were collected 14 hours later.
- a cut of about 1 to 4 was made with a glass knife as a micro tool.
- a M2 solution containing cytochalasin B 5 Mg / ml
- enucleation of the unfertilized egg was performed by confirming that the chromosome was inside.
- the first nuclear transfer was performed using the unfertilized eggs thus enucleated as the recipient cytoplasm and the nuclei of the embryonic stem cells as donor nuclei.
- the nuclear transfer procedure is equipped with a Hoffman modulation contrast device in an egg manipulation solution (solution using M2 medium as a basic medium) containing cytochalasin B (5 g / ml) and nocodazole (0.4 zg / ml). This was performed under a microscope.
- the remaining eggs were subjected to a second nuclear transfer.
- the nucleus of the constructed egg 8 to 12 hours after the activation treatment was used as the donor nucleus
- the enucleated fertilized egg (F1 X F1) 8 to 12 hours after the insemination was used as the recipient cytoplasm. Went as.
- Enucleation and nuclear transfer operations were performed as described above. The operation after nuclear transfer was performed in the same manner as above. Table 1 shows the status of each transplant operation.
- Table 1 As shown in Table 1, 51.1% of eggs obtained by a single nuclear transfer operation developed to transferable embryos, and finally 3.7% (20) of newborns Grew up. Of the eggs obtained by the two transfer operations, 68.0% developed to transferable embryos and 2.6 2. (7) developed to neonates. Of these newborns, 17 and 6 are growing normally, respectively.
- the present invention provides a novel method for producing genetically engineered animals and cloned animals.
- the method for producing a genetically modified animal of the present invention since the target genetically modified cells can be selected at the stage of cell culture, all the individuals produced are genetically modified individuals, and the genetically modified animals can be obtained efficiently. .
- this method makes it possible to directly obtain a genetically engineered animal without using a reproductive chimera individual. By performing genetic manipulation on the target line, backcrossing to the target line is unnecessary. Therefore, in this method, genetically engineered animals can be produced in a short period of time.
- a cloned animal can be obtained efficiently because the mortality of the produced cloned animal in the growth process is low.
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Environmental Sciences (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Developmental Biology & Embryology (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Biophysics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
Claims
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP00953424A EP1308092A4 (en) | 2000-08-09 | 2000-08-09 | METHODS FOR THE PRODUCTION OF A GENE-manipulated ANIMAL AND A CLONED ANIMAL |
KR1020037001866A KR100544922B1 (ko) | 2000-08-09 | 2000-08-09 | 유전자 조작 동물 및 클론 동물의 생성 방법 |
PCT/JP2000/005326 WO2002011529A1 (fr) | 2000-08-09 | 2000-08-09 | Procede de construction d'un animal par manipulation genetique et animal clone |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/JP2000/005326 WO2002011529A1 (fr) | 2000-08-09 | 2000-08-09 | Procede de construction d'un animal par manipulation genetique et animal clone |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2002011529A1 true WO2002011529A1 (fr) | 2002-02-14 |
Family
ID=11736338
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2000/005326 WO2002011529A1 (fr) | 2000-08-09 | 2000-08-09 | Procede de construction d'un animal par manipulation genetique et animal clone |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP1308092A4 (ja) |
KR (1) | KR100544922B1 (ja) |
WO (1) | WO2002011529A1 (ja) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2000228929A (ja) * | 1999-02-10 | 2000-08-22 | Jitsuken Doubutsu Chuo Kenkyusho | 体細胞クローン動物の作製法 |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IL150025A0 (en) * | 1999-12-20 | 2002-12-01 | A method to produce cloned embryos and adults from cultured cells |
-
2000
- 2000-08-09 EP EP00953424A patent/EP1308092A4/en not_active Withdrawn
- 2000-08-09 WO PCT/JP2000/005326 patent/WO2002011529A1/ja not_active Application Discontinuation
- 2000-08-09 KR KR1020037001866A patent/KR100544922B1/ko not_active IP Right Cessation
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2000228929A (ja) * | 1999-02-10 | 2000-08-22 | Jitsuken Doubutsu Chuo Kenkyusho | 体細胞クローン動物の作製法 |
Non-Patent Citations (5)
Title |
---|
H. TAKANO ET AL., THERIOGENOLOGY, vol. 47, 1997, pages 1365 - 1373, XP002933296 * |
K.H.S. CAMPBELL ET AL., NATURE, vol. 380, 1996, pages 64 - 66, XP002933297 * |
See also references of EP1308092A4 * |
T. WAKAYAMA ET AL., PROC. NATL. ACAD. SCI. USA, vol. 96, no. 26, 1999, pages 14984 - 14989, XP002933295 * |
W.M. RIDEOUT ET AL., NATURE GENETICS, vol. 24, no. 2, February 2000 (2000-02-01), pages 109 - 110, XP002933294 * |
Also Published As
Publication number | Publication date |
---|---|
EP1308092A1 (en) | 2003-05-07 |
KR100544922B1 (ko) | 2006-01-24 |
KR20030027948A (ko) | 2003-04-07 |
EP1308092A4 (en) | 2004-05-26 |
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