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J.L. Taylor, K. Wilcox, A. Isaac, S. Sheriff; Human corneal cells repress expression, but not uptake of ecotopic DNA . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3763.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Human cornea stromal cells are reported to be difficult to transfect. We hypothesize that these cells are capable of uptake of transfected DNA, but have one or more mechanisms for repressing expression from DNA introduced into cells. Methods: Stromal cells derived from donor corneas were transfected with luciferase–expressing reporter plasmid DNA using several transfection reagents. Gene expression was measured by chemiluminescence assay of luciferase. Corneal cells were compared to U2OS cells, human osteosarcoma cells that are "easily transfected". To quantitate DNA uptake into cells, 3H–labeled plasmid DNA was transfected. Repression of gene expression from cellular DNA has been correlated with formation of heterochromatin characterized by nucleosome association and histone hypoacetylation. Micrococcal nuclease digestion was used to determine the association of plasmid DNA with nucleosomes. Treatment with trichostatin A (TSA), an inhibitor of histone deacetylases, was used to assess the effects of histone acetylation on gene expression from plasmid DNA. Results: Cornea stromal cells and U2OS cells take up equivalent amounts of transfected DNA. The DNA taken into cells is present in the cytoplasm and the nucleus, and that in the nucleus is both soluble and insoluble. The distribution of plasmid DNA into these subcellular fractions is similar in corneal cells and U2OS cells. However, expression of the DNA taken into corneal cells is negligible compared to expression in U2OS cells. The DNA localized to the nucleus of each cell type is associated with nucleosomes. Expression of transfected DNA in both cell types is enhanced by treatment of cells with TSA, suggesting that in both cell types, histones present on the DNA are not hypoacetylated. However, the level of expression achieved following TSA treatment of corneal cells remains much lower than that in U2OS cells, indicating that there is an additional mechanism of repression in cornea cells beyond histone deacetylation.Conclusions: The inability to detect expression of DNA transfected into corneal cells is not due to a block in DNA uptake, but rather due to the repression of intranuclear tranfected DNA by deacetylation of histones associated with the DNA as well as an additional repressive mechanism.
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