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Embryonic stem cell culture medium and application thereof

A technology for embryonic stem cells and culture medium, applied in the field of embryonic stem cell culture medium, can solve the problems of unrepeatable experimental results, difficulty in obtaining mouse embryonic stem cell effects, slow growth, etc.

Inactive Publication Date: 2012-10-17
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing avian embryonic stem cell culture system is based on the imitation of the mouse embryonic stem cell culture system, most of which have the disadvantages of slow growth and poor stability, and most of the experimental results cannot be repeated
However, the special reproductive physiological system of poultry makes poultry embryonic stem cells far from the model organism mouse from acquisition to culture, making it difficult to apply the mature embryonic stem cell culture system in mice to the development of poultry embryonic stem cell culture system
Not only poultry, but also the research on embryonic stem cells of other species is difficult to obtain the effect of mouse embryonic stem cells. It can be seen that the existing mouse mature embryonic stem cell culture system is difficult to apply to the embryonic stem cell culture of other animals
On the one hand, this is due to the gap between species, and on the other hand, it may be due to the fact that the inner cell mass cells and blastodisc cells as the source of embryonic stem cells are not suitable for exogenous cytokines in terms of cell characteristics and regulatory networks of pluripotency and self-renewal. Intervention

Method used

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  • Embryonic stem cell culture medium and application thereof
  • Embryonic stem cell culture medium and application thereof
  • Embryonic stem cell culture medium and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Embodiment 1, the preparation of recombinant plasmid

[0069] 1. Preparation of recombinant plasmid pGSPF9R-IRES-EGFP

[0070] Using the pIRES2-EGFP vector as the backbone vector, insert the coding gene of the chicken growth hormone signal peptide between its NheI and BglII restriction sites (the chicken growth hormone signal peptide is shown in sequence 1 of the sequence listing, and its coding gene is shown in the sequence listing shown in sequence 2 of the SmaI and BamHI restriction sites, the gene encoding the short arginine peptide is inserted between the restriction sites of SmaI and BamHI (the arginine short peptide is shown in sequence 3 of the sequence listing, and the coding gene is shown in sequence 4 of the sequence listing shown; abbreviated as 9×Arg), the recombinant plasmid pGSPF9R-IRES-EGFP was obtained. In the recombinant plasmid pGSPF9R-IRES-EGFP, there is a multiple cloning site between the coding gene of the chicken growth hormone signal peptide and...

Embodiment 2

[0080] Embodiment 2, preparation of embryonic stem cell subculture medium

[0081] One. The protein expression plasmids prepared in steps 2 to 5 of Example 1 were carried out as follows:

[0082] 1. Transfect the protein expression plasmid by calcium phosphate transfection method (references describing the calcium phosphate transfection method: NANCY HSIUNG, RAYMOND S. ROGINSKI, PAULA HENTHORN, OLIVER SMITHIES, RAJU KUCHERLAPATI, AND ARTHUR I. SKOULTCHI, Introduction and expression of a fetal human globin gene in mouse fibroblasts, MOLECULAR AND CELLULAR BIOLOGY, Apr.1982, p.401-411) was introduced into 293FT cells, and cells expressing EGFP were screened out by observing the fluorescence intensity after 72 hours.

[0083] 2. Culture the cells screened in step 1 at 37°C for 12 hours, and collect the cells by centrifugation;

[0084] 3. Ultrasonicize the cells collected in step 2 (450Hz, 7s and 9s off each time, 44 times), and then use a protein concentration column (Millipore...

Embodiment 3

[0098] Embodiment 3, the cultivation of chicken embryonic stem cells

[0099] 1. Acquisition of blastodisc cells

[0100] Choose freshly hatched eggs (Shouguang chicken) and get blastodisc cells.

[0101] 2. Culture of blastodisc cells

[0102] 1. Experimental group

[0103] Use the chicken embryonic stem cell subculture medium prepared in step 2 of Example 2 to culture blastodisc cells (P0 generation), observe the cell shape, and when the cells appear dense mountain-shaped cell clumps, digest and replace half of the new chicken embryonic stem cell subculture The base was subcultured to obtain the P1 generation. The above-mentioned method was adopted to continue to pass generations, followed by P2 generation, P3 generation, P4 generation, P5 generation, P6 generation, and P7 generation.

[0104] 2. Control group A

[0105] The experiment in Step 1 was carried out by using the control subculture medium A prepared in step 3 of Example 2 instead of the chicken embryonic stem...

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Abstract

The invention discloses an embryonic stem cell culture medium and application thereof. The embryonic stem cell culture medium comprises the following ingredients: a protein A containing an SOX2 protein, a protein B containing a POUV protein, a protein C containing a Nanog protein, a protein D containing a cLIN28 protein, a cell factor LIF, a cell factor SCF and a cell factor bFGF, wherein the SOX2 protein is specifically shown in Sequence 5; the POUV protein is specifically shown in Sequence 7; the Nanog protein is specifically shown in Sequence 9; and the cLIN28 protein is specifically shown in Sequence 11. When the embryonic stem cell culture medium provided by the invention is adopted to culture embryonic stem cells, the embryonic stem cells can be expanded in quantity for a long term and maintain good totipotency.

Description

technical field [0001] The invention relates to an embryonic stem cell culture medium and its application. Background technique [0002] The cultivation of embryonic stem cells has always occupied an extremely prominent position in biological medicine and agricultural biotechnology. Recently, due to the urgent needs of transgenic technology and tissue regenerative medicine, the research in this field has shown a vigorous and strong trend. Mouse and human embryonic stem cell lines have been established and perfected, and have been put into practical research and therapeutic applications. However, the livestock and poultry embryonic stem cell lines urgently needed by transgenic bioreactors have not been reported so far. [0003] Transgenic animal technology and transgenic animal pharmaceuticals are one of the most popular and fastest-growing fields in research in recent years. The foreign gene of interest is introduced into the animal body and integrated with the animal geno...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735
Inventor 韩红兵于淼瑛连正兴李宁
Owner CHINA AGRI UNIV
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