Figure 4

Identification of piggyBac -trapped promoters. A. FACS sorting of GFP-expressing parasites from a population with piggyBac inserts. Dot plots show GFP fluorescence and forward light scatter of (infected) erythrocytes of a control population without inserts (left plot) and of parent population P2 (see Figure 2A) with piggyBac inserts (right plot). Three populations (F1-F3) of GFP-expressing parasites were collected by sorting from gates F1 - F3. B. GFP-expression in blood stages of FACS-sorted populations F1-F3 and P1.5e (see Figure 2A). Gates: g1: infected erythrocytes (Hoechst positive) that are GFP-negative; g2: infected erythrocytes (Hoechst positive) that are GFP-positive. Percentages (mean + st. dev.) of GFP-positive infected cells: Control) 0.1%; F1) 41.6% + 3; F2) 4.0% + 0.7; F3) 28.1% + 2.9; P1e) 1. 6% + 0.2). C. PiggyBac insertions in GFP-expressing parasites of the F1-F3 populations shown by Southern analysis of chromosomes hybridized with pbdhfr/ts and gfp probes. D. Schematic representations of two piggyBac insertion sites identified by TAIL-PCR in the GFP-expressing parasites of the F1-F3 populations. Insertion location is compatible with GFP expression (black arrows; see also Additional file 1: Table S1). Grey arrow: location of integration primer (4571). E. Northern analysis of gfp-expression in blood stages of the Control and F1-F3 populations and subpopulation P1.5e. Loading control: ethidium bromide stained RNA. F. Confirmation of gfp transcription from the bir gene promoter in population F1 by RT-PCR (upper panel; see also Methods section; gDNA and cDNA (+RT or -RT enzyme) were obtained from F1 or Control parasites). Lanes: 1) Marker; 2) gDNA-F1; 3) cDNA-F1; 4) gDNA-Control; 5) cDNA-Control; 6) No DNA. Control of cDNA quality (lower panel) was performed by PCR across the two introns of the gene PBANKA_133840 (GeneDB [34]]). Lanes: 1) Marker 2) gDNA-Control; 3) cDNA-Control; 4) No cDNA-Control; 5) cDNA -F1; 6) No cDNA-F1; 7) No DNA.