Method for researching reprogramming process regulated by DNA methylation

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...

AI Technical Summary

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