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Method for researching reprogramming process regulated by DNA methylation

A methylation and reprogramming technology, applied in biochemical equipment and methods, preparation of test samples, determination/inspection of microorganisms, etc., can solve the problems that epigenetic factors need to be mined, regulatory factors have not been mined, etc. To achieve the effect of clear experimental ideas, strong applicability and ductility, and high feasibility

Pending Publication Date: 2021-08-20
YANGZHOU UNIV
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Problems solved by technology

However, it only studies the factors of somatic cell reprogramming from the perspective of transcription factors, and a large number of regulatory factors have not yet been discovered
Studies have shown that the addition of histone deacetylase inhibitor VPA significantly improves the reprogramming process, and there are still quite a few epigenetic factors to be discovered

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0016] The present invention will be further described below in conjunction with specific embodiments. But the scope of protection of the present invention is not limited to this:

[0017] 1. DNA methylation inhibitor combined with Yamanaka factor to induce reprogramming;

[0018] Retrovirus-encapsulated Oct4, Sox2, Klf4 and c-Myc overexpression vectors combined with DNA methylase inhibitor 5'aza were used to induce somatic cells, and the cell state was observed at different induction days, and photographed and recorded at the same time.

[0019] 2. Identification of ips cells;

[0020] When clonoid cells were observed, the cells were collected for identification.

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Abstract

The invention relates to a method for researching reprogramming process regulated by DNA methylation. The method includes the following steps: step (1), adding a DNA methylase inhibitor 5'aza based on the induction of Yamanaka factors Oct4, Sox2, Klf4 and c-Myc, inducing somatic cells according to an inducing method provided by Shinya Yamanaka, and observing cellular morphology at different inducing days; step (2), when cell clone is observed, collecting cells to perform fluorescence quantitative detection, indirect immunofluorescence detection, alkaline phosphatase staining, karyotype analysis, and edu proliferation assay; and step (3), after determining an effect of the 5'aza, respectively combining four factors and the 5'aza to treat the somatic cells to perform induction, detecting the formation of ips clone, and determining the most effective combination form. Through the induction of different combinations of the Yamanaka factors and the DNA methylase inhibitor 5'aza, an effect of DNA methylation for inducing the reprogramming of the somatic cells can be verified.

Description

technical field [0001] The invention relates to a method for studying the reprogramming process regulated by DNA methylation, which belongs to the field of biotechnology. Background technique [0002] Cell reprogramming is a process of reverse differentiation. Fully differentiated somatic cells can be reprogrammed into pluripotent cells through somatic cell nuclear transfer, transcription factor induction, cell fusion, and cytoplasmic incubation. In somatic cell nuclear transfer, somatic cell nuclei are collected and injected into enucleated oocytes, and the fused cells are cultured in vitro to embryos and then implanted into mothers to obtain cloned animals. This method is effective and can actually produce offspring, but it violates ethics to a certain extent and cannot be used on humans. Cell fusion is mainly used in plant breeding and preparation of monoclonal antibodies, but it still involves ethical and moral issues in animals. Since Shinya Yamanaka discovered in 200...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851G01N33/68G01N33/58G01N21/64G01N1/30G01N21/84
CPCC12Q1/6851G01N33/68G01N33/582G01N21/64G01N1/30G01N21/84C12Q2531/113C12Q2521/107C12Q2563/107
Inventor 李碧春张晨左其生张亚妮孙红艳
Owner YANGZHOU UNIV
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