Embryonic stem cell culture medium and application thereof

A technology of embryonic stem cells and chicken embryonic stem cells, which is applied to embryonic cells, germ cells, animal cells, etc., can solve problems such as inappropriateness, poor stability, and difficulty

Inactive Publication Date: 2014-03-19
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] Example 1. 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 chicken growth hormone signal peptide coding gene between its NheⅠ and BglⅡ restriction sites (the chicken growth hormone signal peptide is shown in sequence 1 of the sequence table, and its coding gene is shown in the sequence table The sequence 2 is shown in), the coding gene of arginine short peptide is inserted between SmaⅠ and BamHI restriction sites (the short arginine peptide is shown in sequence 3 of the sequence list, and the coding gene is shown in sequence 4 of the sequence table. Show; abbreviated as 9×Arg) to obtain the recombinant plasmid pGSPF9R-IRES-EGFP. In the recombinant plasmid pGSPF9R-IRES-EGFP, there are multiple cloning sites between the coding gene of chicken growth hormone signal peptide and the coding gene of short arginine peptide, which can be used for the insertion...

Embodiment 2

[0080] Example 2. Preparation of culture medium for embryonic stem cells

[0081] 1. Perform the following experiments on the protein expression plasmids prepared in steps 2 to 5 of Example 1:

[0082] 1. The protein expression plasmid was transfected by calcium phosphate (references describing calcium phosphate transfection: 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, 72 hours later, cells expressing EGFP were screened out by observing the fluorescence intensity.

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

[0084] 3. The cells collected in step 2 are sonicated (450Hz, each sonication is 7s, stop 9s, 44 times), and then filtered and concentrated using a protein concentration column (Mil...

Embodiment 3

[0098] Example 3. Cultivation of chicken embryonic stem cells

[0099] 1. Obtaining blastoderm cells

[0100] Choose freshly hatched eggs (Shouguang chicken), and take blastoderm cells.

[0101] 2. Culture of blastoderm cells

[0102] 1. Experimental group

[0103] Use the chicken embryonic stem cell subculture medium prepared in step 2 of Example 2 to culture blastoderm cells (P0 generation), observe the cell morphology, when the cells appear dense mountain cell clumps, digest and replace half of them with new chicken embryonic stem cells for subculture The P1 generation is obtained by passing the base. The above-mentioned method was used to continue the passage, followed by P2, P3, P4, P5, P6, P7.

[0104] 2. Control group A

[0105] The experiment of step 1 was performed with the control passage medium A prepared in step 3 of Example 2 instead of the passage medium for chicken embryonic stem cells.

[0106] 3. Control group B

[0107] The experiment of step 1 was performed with the con...

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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, research in this field has shown a vigorous trend. The mouse and human embryonic stem cell lines have been established and perfected, and they have been put into practical research and therapeutic applications. However, the livestock and poultry embryonic stem cell lines urgently needed by genetically modified bioreactors have not yet been reported. [0003] Transgenic animal technology and transgenic animal pharmacy are one of the hottest and fastest growing fields in recent years. The target exogenous gene is introduced into the animal body and integrated with the animal genome. With the division and prolife...

Claims

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Application Information

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Patent Type & Authority Patents(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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