Oligo-mediated targeting reporter system.

<p>(a) Reporter consists of a HeLa cell line with two stably integrated copies of EGFP with a mutated TTG start codon and a second potential start codon (AAG) downstream, both shown in red. Each mutated start codon can be targeted by sense or antisense oligos. Representative oligos used in this study are highlighted, in duplex form, with the mismatch shown by an asterisk. Further detail can be found on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036697#pone-0036697-t001" target="_blank">Table 1</a>. (b) Sample flow cytometry dot plot of cells with no oligo (left) or transfected with F5-8 (right, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036697#pone-0036697-t001" target="_blank">Table 1</a>). The frequency of oligo-induced correction of a start codon can be estimated as the %EGFP+/Propidium Iodide- cells (c) %EGFP+ cells generated after different oligo transfections, with and without 20 mM thymidine, assayed 36 h post transfections. Oligo sequences are shown 5′ to 3′, and the PTO bonds are highlighted in gray. The control oligos F5-1 and F5-6 did not produce any significant proportion of EGFP+ cells, neither did F5-2, which is complementary to the transcribed strand and encodes an ATG-restoring mutation. The oligos targeting the first and second potential start codons on the non-transcribed strand, F5-3 and F5-5 respectively, did produce EGFP+ cells. Lipo = lipofectamine only control. n = 4.