Figure 1. Loss-of-silencing (LOS) screen.

A triple reporter cassette carrying puromycin-resistance (PUR), luciferase (LUC), and emerald GFP (emGFP) genes was targeted adjacent to a silent BES promoter in procyclic form T. brucei and selected with 1 µg/ml puromycin. The reporter cells are unable to grow at a higher concentration of puromycin (100 µg/ml), due to BES promoter silencing. After stably integrating a transposase at an array of tubulin genes, cells were mutagenized using a transposon insertion. The transposon plasmid carries a mariner-donor containing HYG marker flanked by inverted repeats (IRs). Expansion of neomycin-selected cells was distributed in 96-well plates with 100 µg/ml of puromycin. Only transposon-mutant cells that had lost the ability to repress this silent BES locus can grow at this concentration of puromycin but parental cells cannot. 19 PUR^R clones were obtained and increased luciferase activity by 2–6-fold (Table S1). Black circle is telomere.