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A method for preparing pluripotent stem cells

A technology of pluripotent stem cells and fibroblasts, applied to cells modified by introducing foreign genetic material, using vectors to introduce foreign genetic material, recombinant DNA technology, etc., can solve low-leakage expression and cannot accurately reflect endogenous protein expression Situation and other issues, to achieve the effect of ease of use

Active Publication Date: 2017-04-05
CHINA AGRI UNIV
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Problems solved by technology

But this system has the following disadvantages, first, there is a low level of leakage expression; second, it needs to continuously express the additional element tTA or rtTA
At the same time, this system is regulated at the mRNA level and cannot accurately reflect the expression of endogenous proteins, so many aspects need to be improved.

Method used

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  • A method for preparing pluripotent stem cells
  • A method for preparing pluripotent stem cells
  • A method for preparing pluripotent stem cells

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Embodiment Construction

[0028] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0029] Materials and sources involved in the embodiments:

[0030] Oct4-GFP mouse embryonic fibroblast cell line (purchased from The Jackson Laboratory, JaxMicestrain name: B6; CBA-Tg(Pou5f1-EGFP) 2Mnn / J; stock number004654)), mouse ES cell line (purchased from American Standard Object Collection Center ATCC). The former can use the positive expression of GFP to indicate the activation of the endogenous Oct4 gene during the reprogramming process.

[0031] DMEM: available from GIBCO, Catalog no.11960.

[0032] FBS (serum): purchased from GIBCO, Catalog no.10099.

[0033] ES FBS (ES serum): purchased from GIBCO, Catalog no.16141.

[003...

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Abstract

The invention provides a method of preparing a pluripotency stem cell by utilization of a regulatory protein stabilizing system. The method includes adding a degradation area before a target carrier factor to be regulated. When the degradation area is added before exogenous OCT4, if TMP is added, the protein of the factor to be regulated can be stabilized and the induced pluripotency stem cell can be obtained; and if the TMP is not added, the pluripotency stem cell cloning cannot be obtained. By changing the addition amount and the addition time of the TMP, the expression measurement and an expression stage of the exogenous factor can be controlled, so that different pluripotency cell induction efficiencies can be obtained. The method provides a regulatory route for preparation of the induced pluripotency stem cell.

Description

technical field [0001] The present invention relates to a method for preparing pluripotent stem cells, in particular to a method for preparing pluripotent stem cells by utilizing an adjustable protein stabilization system. Background technique [0002] In 1981, two scientists, Evans and Matin, successfully isolated mouse ES cells at the same time, that is, embryonic stem cells. Due to their unlimited proliferation and potential differentiation ability, they are widely used in basic research and applications. In particular, in 1998, Thomason et al. established five human embryonic stem cell lines using in-vitro fertilization and embryo transfer (IVF-ET) embryos donated voluntarily clinically for the first time, setting off an upsurge in cell therapy. At the same time, combined with gene targeting technology, it has great application prospects in the fields of cell development mechanism research, cell replacement therapy, new drug preparation and screening, and organ transplan...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10
Inventor 胡晓湘隋丹丹李宁
Owner CHINA AGRI UNIV
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