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Method for inducing cloned cells of large-scale experimental animal into pluripotent stem cells

A technology of pluripotent stem cells and experimental animals, which is applied in the field of pluripotent stem cells of large experimental animals, can solve problems such as difficult reconciliation, and achieve the effects of improving cell quality, reducing costs, and stabilizing cell passage and growth

Inactive Publication Date: 2014-12-24
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI +1
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  • Abstract
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  • Application Information

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Problems solved by technology

However, in the traditional method, it is difficult to reconcile the contradiction between the relay-style research and the temporary collection of specimens. Therefore, the establishment and preservation of pluripotent stem cells in experimental animals can effectively carry out long-lasting research on the same research object. Continuity of Research

Method used

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  • Method for inducing cloned cells of large-scale experimental animal into pluripotent stem cells
  • Method for inducing cloned cells of large-scale experimental animal into pluripotent stem cells
  • Method for inducing cloned cells of large-scale experimental animal into pluripotent stem cells

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Experimental program
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Effect test

Embodiment 1

[0087] Embodiment 1 The cultivation of large experimental animal cloned cells

[0088] (1) Primary culture: Based on the suspension cell culture method, full RPMI1640 medium A is used for transformation and small amount of culture in 24-well plates: 78% basic RPMI1640+20% FBS (GIBCO special grade)+1% double antibody+1% L -Giutamine+0.2% Hepes (adjust the pH to 7.0); after the immortalized cells are transferred to the medium bottle, use the whole RPMI1640 medium B: 73% basic RPMI1640+15% FBS (BIOCHROM AG superior grade)+1% double antibody+1% L -Giutamine+0.2% Hepes (adjust pH to 7.0). Immortalized cells were transformed and cultured in a 37.5°C, 5% CO2 culture system.

[0089] (2) Subculture: Discard the culture medium in the culture flask in step (1), and carefully wash the residue in the culture flask twice with PBS preheated to 37°C to remove residual serum, exfoliated tissue pieces and dead cells . After digesting with trypsin, add 6-10ml of culture medium to stop the di...

Embodiment 2

[0099] Example 2 Induction of pluripotent stem cells from large experimental animals:

[0100] The three-factor feeder-free system was used to induce pluripotent stem cells from the cloned cells of large experimental animals cultured in Example 1. The methods and conclusions are as follows.

[0101] 1. Preparation of three-factor lentivirus

[0102] Method: The lentiviral vector containing three factors was transfected into 293T cells to prepare lentivirus.

[0103] Conclusion: if Figure 1-5 As shown, the transfection efficiencies of the three factors are all above 50%. As shown in the figure, the titers of the lentiviruses are all at 10 8 The above proves that the prepared three-factor lentivirus meets the requirements of this experiment.

[0104] Two, three-factor lentivirus infection of cloned cells of large experimental animals

[0105] Methods: The three-factor lentivirus was used to infect the cloned cells of large experimental animals at a ratio of 10:1 for 48 hour...

Embodiment 3

[0110] Example 3 Optimization of the culture system of large experimental animal pluripotent stem cells:

[0111] Methods: The culture system of pluripotent stem cells was optimized by adding factors such as LIF and bFGF to the culture system and using STO feeder layer.

[0112] Conclusion: Adding factors such as LIF and bFGF to the culture system can promote the proliferation of pluripotent stem cells in large experimental animals, and the use of STO feeder layer can achieve the same effect.

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Abstract

The invention relates to a method for obtaining pluripotent stem cells with high propagation capacity and high differentiation potential of a large-scale experimental animal by using cloned cells of the large-scale experimental animal as target cells to perform induction of the pluripotent stem cells, optimization of in vitro culture conditions and functional verification, to the field of interdiscipline of animal resource science and cell biology. The pluripotent stem cells cultured by the method is pollution-free, high in cell purity and stable in cell quality and passage growth, so that that pluripotent stem cells are suitable for large-scale culture. The pluripotent stem cells of the large-scale experimental animal can be used for effectively performing lasting and continuous study on the same object of study, provide an object of study with constant characteristics for life science study, can be used as standard cells in the study fields of cell engineering, tissue engineering and the like, and can be used in a plurality of fields of the life science study, so that the pluripotent stem cells have far-reaching significance for the life science study in China.

Description

technical field [0001] The invention relates to a method for inducing pluripotent stem cells, specifically, using cloned cells of large experimental animals as target cells to induce pluripotent stem cells, optimize in vitro culture conditions and verify functions, and finally obtain high value-added, high The pluripotent stem cells of large experimental animals with differentiation potential belong to the interdisciplinary discipline and field of animal resource science and cell biology. Background technique [0002] With the rapid development of medical and biological sciences, it is recognized that pollution problems have not only become major social issues such as food, population, and the elderly, but also involve the survival of animals living on the earth, such as industrial pollution, food pollution, and drug toxicity. etc., all directly affect human health, and the research on these problems must finally be clarified and solved through animal experiments (including ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/867C12N5/10
Inventor 胡鹏飞白春雨李向臣姜龙江李万哲刘春华关伟军
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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