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Z. Li, X. Jiao, S. Chakrabarty, Q. Nguyen, B. Liu, J. F. Hejtmancik, R. B. Nussenblatt; Comparative Study on DNA Methylation of IL-10 Gene Promoter in Human Immune Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3749.
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To investigate DNA methylation pattern of human IL-10 gene promoter in immune cells
Primary human immune cells including CD4+CD45RA+CD5RO- naïve T cells, CD14+ monocytes, CD56+ NK cells and CD22+ B cells were purified by either magnetic beads or FACS sorting from isolated human peripheral blood mononuclear cells (PBMCs). Bisulfite treatment to facilitate genomic DNA cytosine (C) to thymine (T) conversion was performed using the EZ DNA Methylation Direct kit. Bisulfite sequencing PCR (BSP), Methylation specific PCR (MSP), methylation sensitive restriction digestion and quantitative PCR were used to analyze potential CpG dinucleotide methylation. DNA methylation was confirmed by sequencing data. To characterize clonal differential methylation, BSP products were also analyzed by TA cloning and subsequent DNA sequencing for individual clones.
We show that there is a differential methylation in the1.3 kb proximal promoter region for the human IL-10 gene with 2 regions being methylated most of the time but one region with great variability in CpG methylation. There also appears to be a differential methylation pattern among those developmentally distinct populations of immune cells. BSP is by far the most accurate and reliable method to determine DNA methylation but also tedious and time-consuming. Methylation specific PCR (MSP), methylation sensitive restriction digestion and quantitative PCR are fast, relatively easy and efficient, therefore are useful complementary methods for screening purposes with great potential for clinical application.
We have compared several strategies to qualitatively and quantitatively analyze DNA methylation focusing on human IL-10 gene promoter in immune cells. Our data reveal an interesting pattern of DNA methylation in this region and among different immune cells. However, the functional implication of this differential methylation pattern in human IL-10 proximal promoter region remains to be determined. We suggest that DNA methylation can be a very dynamic and complex process that may be implicated in the regulation of gene expression.
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