Used Rna Interference To Inhibit The Gene Expression Of Ctcf In The Mcf-7 Cells

CCCTC-binding factor (CTCF) is a widely expressed 11-zinc finger (ZF) nuclear protein originally identified as a transcription factor that binds to the avian and mammalian MYC promoters. CTCF is multivalent eukaryotic transfection factor. It involves in many different aspects of gene regulation, such as repression, activation, silencing and insulation. It also participates in epigenetic regulation of gene inprinting and X-chromosome inactivation. Recent studies found that CTCF play a very important role in mediating both long-range intrachromosomal and interchromosomal interactions, these advances have also suggested that CCCTC binding factor (CTCF) is a well known chromatin insulator protein, may play a central role in mediating long-range chromatin interactions. Since RNAi has the function of gene knockout, using RNAi mechanism, the researchers can quickly and easily reduce a specific gene expression in cells, making the effect of "gene knockout" to analyze the impact of gene expression changes in cells and further study the unknown function of the target gene.This subjects using RNA interference to slience CTCF gene in cells, we established a CTCF gene silenced (knock-down) cell line, contributing to the later comparison of the CTCF-mediated chromosomal interaction before and after the CTCF gene silencing. Its technical route has been executed as follows. Firstly, we selected different CTCF gene mRNA targets and designed knock-down vectors (pSilencerl-4). Secondly, we obtained stable transfection cells by transfection and pressing screening. Finally, we detect the CTCF protein expression and mRNA expression levels of the stable transfected cells and wild-type cells using Western Blot and Real Time PCR. Thus we can analysis the efficiency of CTCF knockout using RNA interference. At the same time this subjects has also successfully built a rescue vector of CTCF, This work will built the foundation for the later CTCF rescue experimentAccording to the experimental data and results that we can compared with wild-type cells the stable transfected knockout vector cells both the CTCF protein levels and mRNA levels were all reduced, the expression of CTCF gene is down regulated but not totally silenced. The knock-down efficiency of the target site across intron is significantly higher than that of the target site located within exon. On these target sites within exon the more close to the 5'end the knockout Efficiency are the better.