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P. Challa, F. A. Soto Leon, L. Talbot, P. B. Liton, C. Luna, D. L. Epstein, R. Maddala, P. Gonzalez; Novel Anti-Sense Oligonucleotide Delivery to Primary Lens Epithelial and Trabecular Meshwork Cell Cultures. Invest. Ophthalmol. Vis. Sci. 2007;48(13):4242.
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To modulate gene expression in primary lens epithelial (LEC) and trabecular meshwork cell (TMC)cultures using highly specific, nuclease resistant non-anionic morpholine-backbone anti-sense oligonucleotides (morpholinos).
Porcine LECs and TMCs were harvested, digested, and grown to confluence. A morpholino with a fluorescent red lissamine end-modification was administered to the cultured cells with an Endo-porter delivery agent. Controls lacking either morpholino or Endo-Porter were also studied in two groups. Cells were qualitatively evaluated for distribution of intracellular fluorescence and quantitatively evaluated for amount of fluorescence by flow cytometry.
Delivery of lissamine-tagged morpholino resulted in LECs and TMCs developing a diffuse red fluorescence characteristic of ctyosolic distribution after 48 to 72 hours, compared to same-age untreated controls. After reaction optimization a significant number of LECs and TMCs successfully incorporated cytosolic morpholinos.
These data indicate that morpholinos can be effectively delivered intracellulary to porcine primary cultures of lens and trabecular meshwork cells for use in anti-sense modulation of gene expression.
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