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E.A. Hoffman, S.M. Conley, B.S. McKay, W.D. Stamer; Role of DNase I in Transfection Efficiency of Human Trabecular Meshwork Cells . Invest. Ophthalmol. Vis. Sci. 2005;46(13):3692.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: The trabecular meshwork (TM) is a likely site of pathology in some forms of glaucoma. Studying the role of specific proteins in TM physiology has been difficult due to low transient transfection rates in cultured cells. Since DNA degradation is associated with the normal phagocytic properties of TM cells, the purpose of this study was to test the hypothesis that DNase I degradation of transfected plasmid DNA contributes to the low transfection rates observed in cultured cells. Methods: Human TM and human retinal pigment epithelial (RPE) cells were probed with anti–DNase I IgG to determine the expression and subcellular localization of DNase I. In parallel, cells were transfected with plasmid DNA encoding the GFP reporter gene in the presence and absence of aurintricarboxylic acid (ATA), a DNase I inhibitor, to ascertain the effect of DNase I on transfection efficiency. Results: DNase I was detected in the cytosol and nucleus of both TM and RPE cell cultures with higher intensity staining in TM cells. ATA inhibition of DNase I led to a two–fold increase in transfection efficiency of TM cultures (3.7% +/– 1.1% untreated vs. 7.3% +/– 1.2% ATA treated) while having no effect on transfection efficiency of RPE cells (5.4% +/– 1.9% untreated vs. 5.2% +/– 1.9% ATA treated). Conclusions: Our results indicate that DNase I mediated DNA degradation contributes to the low transfection rates typically observed in HTM cell cultures. Cell–type specificity of DNase I activity in culture may be attributed to the normal phagocytic responsibilities of the TM in the outflow pathway.
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