Method for screening methylation sensitive gene of DNA

Abstract

The invention discloses a method for screening a methylation sensitive gene of DNA, relating to the genetic engineering field. The method comprises the following steps: a low methylation cell model construction step, a genetic expression change detection step, a gene specific region methylation detection step and a methylation sensitive gene identification step. According to the method, an adult fibroblast cell system is easily obtained by virtue of a common laboratory, the expression of a DNMT1 gene for maintaining the methylation of DNA in the cell is continuously inhibited by virtue of in-vitro-transfected anti-DNMT1 small RNA, and 1-3 generations of the cell are continuously interfered according to the activity and proliferation expression of passage cells; and practices prove that anefficient low methylation cell model can be obtained and can be combined with common PCR or high-throughput sequencing according to different research purposes so as to judge whether a target gene issensitive to the methylation of DNA or screen methylation sensitive transcription sequences of DNA in large scale. The cell model established by the technique is simple, efficient and easy to acquireand operate.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for screening DNA methylation sensitive genes. Background technique [0002] DNA methylation is very important to life activities and is one of the means of gene regulation (mainly through methylation of CpG islands and their adjacent sequences located in the 5'-UTR region, promoter region and the first exon and other functional regulatory regions. It plays a vital role in maintaining the normal function of cells, transmitting genome imprinting, embryonic development, tumorigenesis, etc., and it is a hot spot in epigenetics research. [0003] Current understanding of the methylome remains limited. Like other epigenetic phenomena, the study of methylation levels is complicated by the need for different study modes in different cell types (Laird et al. 2010). At present, the main technical methods for screening DNA methylation-sensitive genes include: DNA methylation c...

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Problems solved by technology

The limitations of MeDIP technology include: the protein MBD2b or 5'-methylcytosine antibody that specifically binds to methylated DNA is required, the amount of cells used is large, the requirements for experimental techniques are high, and the enrichment of DNA fragments with low methylation Low, and still need different differentiated cell models or disease models (Parketal.2009, Bocketal.2010), these defects have a great impact on the lack of stem cells (such as pigs, there is still no mature stem cell culture system in vitro), lack of disease models and stem cell sources Especially for species lacking (such as rare animals)

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