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Method for producing cloned dog

A technology of nuclear transfer and embryos, applied in biochemical equipment and methods, botanical equipment and methods, special equipment for doors/windows, etc., can solve problems such as low cloning efficiency, difficulty in cloning dogs, and difficulty in practical application. Achieve the effects of improving cloning efficiency and optimizing electrofusion conditions

Inactive Publication Date: 2008-07-23
SEOUL NAT UNIV R&DB FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, due to the species-specific reproductive characteristics of dogs, it is difficult to clone dogs by somatic cell nuclear transfer
The researchers of this invention have successfully cloned dogs for the first time, but because the cloning efficiency is too low, it is difficult to put it into practical application

Method used

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  • Method for producing cloned dog

Examples

Experimental program
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Effect test

Embodiment 1

[0063] Extraction of dog recipient oocytes

[0064] Twenty-three female mongrel dogs aged 1-5 years were used as oocyte donors. These dogs are cultivated in accordance with the standards approved and established by the Animal Culture Laboratory of Seoul National University. The ovulation period of the dog is determined by daily vaginal smear test and serum progesterone level analysis of dogs in natural estrus, and mature oocytes are obtained 48-72 hours after ovulation. The obtained oocytes include immature oocytes, mature oocytes, slightly aged oocytes, moderately aged oocytes and severely aged oocytes. The morphology of these oocytes is shown in Figure 2.

[0065] In order to measure the serum progesterone level, 3-5ml of blood is extracted every day, the serum is obtained through a centrifuge, and then the DSL-3900 Active Progesterone Coated-Tube Radioimmunoassay Kit (DSL-3900 ACTIVE Progesterone Coated-Tube Radioimmunoassay Kit) is used (diagnostics) System Laboratories Inc....

Embodiment 2

[0073] Enucleation of recipient oocytes

[0074] Without Ca 2+ Add Hepes buffer to the CR2 medium to make hCR2aa medium (see Table 2), and then add 0.1% (v / v) hyaluronidase (Sigma, USA) (Charles Rosenkrans 2; Rosenkrans et al., Biol. Reprod) .49, 459-462, 1993). Then, the cumulus cells of the oocytes obtained in Example 1 were removed by repeatedly using a pipette in the medium prepared above. The oocytes were stained with 5 μg / mL benzimine (Hoechst 33342) for 5 minutes, and then observed with an inverted epi-fluorescence microscope at 200 times magnification to select the oocytes containing the first polar body. Add 10% (v / v) FBS and 5μg / ml cytochalasin B to the hCR2aa medium (see Table 3), and then use a micromanipulator (Narishige, Tokyo, Japan) in the culture medium to enucleate the selected oocytes . That is, the oocytes are placed in a micropipette (internal diameter 150μm), and then the first polar body, adjacent cytoplasm (less than 5%) and the oocyte nucleus are removed...

Embodiment 3

[0082] Donor cell production

[0083] Extract adult fibroblasts from dogs as nucleus donor cells. To this end, the ear skin of a male Afghan hound (2 months old) was first isolated for biopsy. Some small fragments in the ear tissue fragments were washed 3 times with DPBS (Dulbecco's phosphate buffered saline) and then minced with a surgical scalpel. The minced tissue was placed in Dulbecco's modified Eagle's medium containing 0.25% (w / v) insulin and 1 mM EDTA and dissociated at 37°C for 1 hour. Put the enzyme-hydrolyzed cells in Ca-free 2+ And Mg 2+ -Wash once in DPBS, centrifuge at 300xg for 2 minutes, and then plant in a 60-mm plastic petri dish (Becton Dickinson, Lincoln Park, NJ). The seeded cells were then put into the supplemented with 10% (v / v) FBS, 1mM glutamate, 25mM NaHCO 3 And 1% (v / v) minimal essential medium (MEM) non-essential amino acid solution (Invitrogen, CA) in DMEM at 39°C, 5% CO 2 Cultivate for 6-8 days under the condition of 95% air. After removing the non-...

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Abstract

The invention provides a method for cloning dog, comprising: producing the enucleated oocytes by enucleating the oocytes; transplanting the somatic cell of dog into the enucleated oocytes; producing the nuclear transfer embryos by electrofusion at the optimum condition; and transplanting the nuclear transfer embryos into surrogate maternal body. The said method has high efficiency and great contribution to animal medicine, anthropology and medicine, such as propagation of good breed, heterogeneity and animal in illness model. In addition, the method successfully clones female dog for the first time and can more accurately research the propagation features of the cloning dog.

Description

Technical field [0001] The invention relates to a method for producing cloned dogs. In particular, it is a method of enucleating a dog’s oocyte to form an enucleated oocyte, transplanting a somatic cell of the dog into the enucleated oocyte, and then producing a nucleus by electrofusion under optimal conditions A method of producing cloned dogs by transferring embryos and then transferring the nuclear transfer embryos into the fallopian tubes of the surrogate mother. [0002] This application claims the priority of the Korean patent application filed on January 17, 2007 with the application number 20070005350, and the content of the patent application is quoted in this article in its entirety. Background technique [0003] At present, animal cloning can be achieved through somatic cell nuclear transfer technology of cell fusion or intracytoplasmic cell injection. [0004] Somatic cell nuclear transfer technology, so that the birth of offspring does not need to undergo meiosis and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/06A61D19/04C12N15/873
CPCA61D19/04C12N15/873A01K67/027A01K2227/00E05Y2800/406E06B1/56E06B7/28
Inventor 李柄千姜成根金大暎金敏奎張龟吴炫周M·萨敏·何塞因费班托·羽达金惠珍洪少君朴正恩金正柱
Owner SEOUL NAT UNIV R&DB FOUND
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