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

A technology for cloning embryos and humans, applied in the production of human cloned embryos by internuclear transfer technology, in the field of human cloned embryos, which can solve the problems of unsatisfactory human tissues

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, it is clear that suitable materials with the same tissue specificity are not always available
Although recent cloning techniques using

Method used

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

Examples

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Embodiment 1

[0032] 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

[0033] To prepare donor cells, tissues collected from human skin were washed with PBS (phosphate-buffered saline, Gibco's BRL, Life Technology, USA), and then crushed 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, centrifuge at 1500 rpm for two minutes and suspend In DMEM (Dulbecco's Modified Eagle's Medium, Gibco's BRL, Life Technology USA) supplemented with 10% FBS, 1% NEAA (non-essential amino acids), and 1% penicillin-streptomycin. Transfer the suspension into a cell culture dish and incubate at 39 °C, 5% CO 2 environment to obtain somatic cell lines. Thereafter, the cells were treated with...

Embodiment 3

[0041] 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

[0042] 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

[0043] 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

[0044] The activated embryos were activated for 4 hours in 25 microliters of cycloheximide (Sigma Chemical Co., U.S.) solution, and the cycloheximide solution was added to the in vitro culture medium mTALP by adding cycloheximide stock solution ( 10 mg / ml in ethanol), to a final concentration of 10 μg / ml to prepare. 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. E...

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Abstract

The present invention provides a method for producing human cloned embryos by employing inter-species nuclear transplantation technique. The method for producing human cloned embryos of the invention comprises the steps of: preparing donor somatic cell lines collected from human; maturing oocytes collected from ovary of cow 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; and, postactivating and culturing the embryos in vitro. The human cloned embryos of the invention can be employed to obtain the human embryonic stem cells, which may be widely applied in biological and medical fields.

Description

[0001] Background Art of the Invention field of invention [0002] The present invention relates to a method for producing human cloned embryos by using interspecies nuclear transfer technology, more specifically, relates to a method for producing human cloned embryos by using interspecies nuclear transfer technology, the method takes somatic cells obtained from human tissues Nuclei are transferred into mature oocytes of bovine origin. The invention also relates to human cloned embryos 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] Due to the development of biotechnology and genetic engineering, various gene recombinant plants having desired useful crop characteristics have recently been successfully produced (s...

Claims

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Application Information

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IPC IPC(8): C12N15/09A01K67/027C12N5/07C12N5/071C12N5/10C12N15/00C12R1/91
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韩在容龙焕律崔伦僖高凤卿黄禹锡
Owner 财团法人SEOUL大学校产学协力财团
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