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Method for producing cloned tigers by employing inter-species nuclear transplantation technique

A technology of nuclear receptors and oocytes, applied in the field of producing cloned tigers through interspecies nuclear transfer technology, can solve problems such as the production of wild animals with difficult technology applications

Inactive Publication Date: 2001-07-18
财团法人SEOUL大学校产学协力财团
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this paper is aimed at the application of livestock, for which much research work has been done, making it difficult to apply this technology to the production of wild animals

Method used

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  • Method for producing cloned tigers by employing inter-species nuclear transplantation technique
  • Method for producing cloned tigers by employing inter-species nuclear transplantation technique
  • Method for producing cloned tigers by employing inter-species nuclear transplantation technique

Examples

Experimental program
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Effect test

Embodiment 1

[0040] The present invention is now further illustrated by the following examples, which should not be construed as limiting the scope of the present invention. Example 1: Preparation of Donor Cells and Recipient Oocytes

[0041] To prepare donor cells, tissues from the tips of male adult saxieri were sterilized with ethanol and betaine after shaving the hair around the tissues. Harvest skin tissue (area 1 to 2 cm2) with a sterile instrument, transfer it into PBS (phosphate-buffered saline, Gibco's BRL, USA Life Technology) in a 50 ml test tube. Cartilage and hairy skin were then separated from the harvested skin tissue using sterile scissors and a scalpel to obtain the cartilage-lined skin inner tissue as a donor. The tissue was washed with PBS and pulverized to 100 mesh. Then at 39°C, 5% CO 2 Incubate the tissue in PBS containing 0.25% trypsin, 1 mM EDTA, and 1 mg / ml collagenase type II for 1 hour at ambient temperature. After the tissue is enzymatically digested, centri...

Embodiment 3

[0049] Replace the cutting tube mounted on the micromanipulator with an injection tube. Enucleated oocytes were washed three times with TCM199 wash medium and then pipetted into injection droplets. The donor cells are drawn into the syringe and then transferred into the injection droplet. Figure 4 Indicates the process of transferring a somatic cell into an enucleated oocyte. Such as Figure 4 As shown, enucleated oocytes were placed with their cleft orientation at 1 o'clock, held with a holding tube, and then injected into donor cells through the cleft using a syringe and hydraulic pressure to obtain a reconstructed embryo. Embryos were washed three times with TCM199 wash medium and then incubated in TCM199 wash medium. Example 3: Electrofusion and activation

[0050] The reconstructed embryos were electrofused using an electrocytomanipulator (USA, BTX, ECM2001), and then reactivated. Add 15 microliters of 0.28M mannitol, 0.5mM HEPES (pH7.2), 0.1mM MgSO to the TCM199 cu...

Embodiment 4

[0051] To activate electrofused embryos, embryos were incubated for 4 minutes in the dark in a solution of ionomycin (Sigma Chemical Co., USA), a TCM199 wash containing 5 [mu]M ionomycin and 1% BSA. The ionomycin stock solution was prepared by dissolving 1 mg of ionomycin in 1.34 ml of DMSO. Activated embryos were incubated for 5 minutes in 35 mm dishes containing TCM199 wash medium supplemented with 10% FBS to wash ionomycin from the embryos. Example 4: Post-activation and in vitro culture of electrofused embryos

[0052] Activated embryos were activated for 4 hours in 25 microliters of cycloheximide (Sigma Chemical Co., USA) solution added by adding Cycloheximide stock solutions (10 mg / ml in ethanol) were prepared to a final concentration of 10 μg / ml. Embryos were then screened, and the selected embryos were placed at a temperature of 39 °C, 5% CO 2 Incubate for 7 days under ambient conditions.

[0053] Based on the above method, the present inventors respectively used Kor...

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Abstract

The present invention provides a method for producing cloned tigers by employing inter-species nuclear transplantation technique. The method for producing cloned tigers of the invention comprises the steps of: preparing donor somatic cell lines collected from tiger; maturing oocytes collected from ovary of cow or cat in vitro; removing the cumulus cells surrounding the oocytes; cutting a portion of zona pellucida of the matured oocytes to make a slit, and squeezing out a portion of cytoplasm including the first polar body through the slit to give enucleated recipient oocytes; transferring a nucleus to the recipient oocyte by injection of the donor cells to the enucleated recipient oocytes, followed by the subsequent electrofusion and activation of the electrofused cells to give embryos; postactivating and culturing the embryos in vitro; and, transferring the cultured embryos into surrogate mothers to produce cloned tigers. The present invention can be widely applied in preservation of genetic characters of rare or endangered species permanently in the form of nuclear transferred embryos.

Description

[0001] Background Art of the Invention field of invention [0002] The present invention relates to a method for producing cloned tiger with interspecies nuclear transfer technology, more specifically, relates to a method for producing cloned tiger with interspecies nuclear transfer technology, the method will obtain the somatic cell nucleus from tiger tissue Transferred into oocytes of bovine or feline origin. The present invention also relates to the cloned tiger embryo and cloned tiger produced by the above method. Background of the invention [0003] The production of animals by fertilization, which involves male and female gametes, has long been considered. However, great efforts have been made to produce cloned animals with the same appearance and the same genetic characteristics. [0004] For 30 years it was known that zygote cloning was only possible in amphibians, but has now been successfully produced in mice by replacement of the pronucleus of a single-celled zy...

Claims

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Application Information

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IPC IPC(8): A01K67/027C12N5/10C12N15/02C12N15/873
CPCC12N15/873
Inventor 李炳千申泰英卢湘镐林正默朴钟任赵钟基金琪渊李殷松申秀晶金晟基宋吉永龙焕律安国俊玄尚桓李秉东黄禹锡
Owner 财团法人SEOUL大学校产学协力财团
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