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L. Talbot, P. Gonzalez, P. Liton, D.L. Epstein, P. Challa; Optimizing Delivery of Novel Anti–Sense Oligonucleotides to Trabecular Meshwork Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1874.
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© ARVO (1962-2015); The Authors (2016-present)
To assess and refine the delivery of highly specific, nuclease resistant non–anionic morpholine–backbone anti–sense oligonucleotides (morpholinos) to porcine trabecular meshwork (TM) cells for modulating gene expression.
Porcine TM cells were harvested, digested, and grown to confluence. A morpholino with a fluorescent red lissamine end–modification was administered to the cultured cells in two groups, with and without an Endo–porter delivery agent. Controls lacking either morpholino or Endo–Porter were also studied in two groups. Trabecular meshwork cells were qualitatively evaluated for distribution of intracellular fluorescence and quantitatively evaluated for amount of fluorescence by flow cytometry.
Sole delivery of lissamine–tagged morpholino resulted in TM cells developing a diffuse red fluorescence characteristic of ctyosolic distribution after 72 hours, compared to same–age untreated controls. Cellular toxicity was observed with the delivery of Endo–porter and varied depending on the concentration of morpholino and Endo–porter. After reaction optimization a significant number of TM cells successfully incorporated cytosolic morpholinos.
These data indicate that morpholinos can be effectively delivered intracellularly to porcine TM cells for use in anti–sense modulation of gene expression.
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