Method for cloning embryo by nuclear transfer of bovine somatic cells

A technology for cloning embryos and somatic cells, applied to cells modified by introducing foreign genetic material, etc., can solve problems that have not yet been seen, and achieve the effects of saving test time, simplifying test steps, and facilitating subsequent development

Inactive Publication Date: 2008-11-12
HUAZHONG AGRI UNIV
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  • Abstract
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Problems solved by technology

[0010] So far, there is no research on the use of donor cells without serum starvation and membrane rupture to produce bovine cloned embryos by direct injection of whole cells

Method used

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  • Method for cloning embryo by nuclear transfer of bovine somatic cells
  • Method for cloning embryo by nuclear transfer of bovine somatic cells
  • Method for cloning embryo by nuclear transfer of bovine somatic cells

Examples

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Embodiment 1

[0042] 1. Sampling: The healthy cattle ovaries collected from the Hongqi Beef Slaughterhouse in Wuhan City, Hubei Province were stored in DPBS at 39°C. The formula is shown in "Contents of the Invention" and brought back to the laboratory within 3 hours;

[0043] 2. Collection and cultivation of bovine oocytes: use a small amount of ovum-absorbing liquid, see the "Contents of the Invention" for the formula, and use a syringe (No. 18 needle) to absorb bovine cumulus cells-oocytes from the 3-8mm follicles on the surface of the bovine ovary Complexes (COCs), the COCs were washed 3 times with DPBS, washed 3 times with mature culture medium (recipe as above), transferred to 100ul mature culture medium micro-droplets and cultured for 22h (cultivation conditions: 39°C, relative 100% humidity, 5% CO 2 incubator);

[0044] 3. Judging the maturity of bovine oocytes: the bovine oocytes that have been cultured in the maturation medium for 22 hours are blown repeatedly for 3 minutes in 0....

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Abstract

The invention belongs to the mammal reproduction technology field, particularly relates to a method for obtaining cloned embryos with cow somatic cells and discloses a method for producing cow cloned embryos simply and efficiently. According to the research results that pig somatic cells dissolves and disappears automatically in thirty minutes after being injected into ooplasm, whole cells with sound membrane are extracted from cows as donors and are injected into the whole cell cytoplast directly to construct cow cloned embryos, simplifying the operational process of somatic cell nuclear transfer. The method is characterized in that: (1) selecting and cultivating cow ovarian cumulus- oocytes complex; (2) enucleating of the occytes; (3) preparing donor cells; (4) preparing co-cultivation cell monolayer; (5) constructing reconstructed fetus; (6) activating the reconstructed fetus; and (7) cultivating the reconstructed fetus. The method has the best feather of easy operation, simple steps, stable results, providing a new idea and method for the expansion and the further study of cow somatic cells nuclear transplanting technology.

Description

technical field [0001] The invention relates to the technical field of mammalian reproduction, in particular to a method for cloning embryos by bovine somatic cell nuclear transfer. Background technique [0002] Somatic cell nuclear transfer has made a breakthrough in the late 20th century. Since the birth of Dolly the sheep, more and more somatic cell cloned animals have been born, and the significance of somatic cell nuclear transfer has gradually been recognized by people. However, the development rate of the reconstructed embryos constructed by this technique is low, and in the technique of making reconstructed embryos, the pre-treatment of nucleated cells and the operation of micronucleation and nucleation are the key limiting steps. [0003] At present, there are mainly serum starvation method (Campbell et al., 1996) and contact inhibition method (Kasinathan et al., 2001) for the pretreatment of donor cells in order to achieve the purpose of cell synchronization. Othe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10
Inventor 于孟飞易建明高国龙杨利国
Owner HUAZHONG AGRI UNIV
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