Skip to main content
Fig. 1 | Malaria Journal

Fig. 1

From: Improvement of an in vitro drug selection method for generating transgenic Plasmodium berghei parasites

Fig. 1

Generation of mutants using pacPB driven by pbef- promoter. a Nucleotide sequences of original (pac) and codon-optimized pac (pacPB). In the pacPB sequence, modified nucleotides are highlighted. b Schematic diagram of the piggyBac transposon vector containing pacPB and egfp expression cassettes (pXL/pbef-1α-pacPB-egfp). ITR: inverted terminal repeat. c The eGFP-positive parasite ratio after each puromycin selection. The ratio was analysed using fluorescence microscopy. Each bar represents the mean ± SD of four independent experiments. **p < 0.01, n.s.: not significant (paired t-tests). d Fluorescence images of parasites after each selection. Parasites were stained with Hoechst 33342. The scale bar represents 10 µm. e Flow cytometry analysis of the typical transfection line of eGFP positive mutant ratio after each puromycin selection. Numbers above the bracketed lines indicate the percentage of parasites with eGFP expression. f Southern blot analysis of three pacPB integrated mutant clones. Genomic DNA was digested using NdeI and hybridized with a pacPB probe. Wild wild type, cl. clone. Insertion sites of piggyBac transposon were shown blow. U upper band of cl.1, L lower band of cl.1, Chr. chromosome. g Growth assay of two pacPB integrated clones. Female ICR mice were infected intravenously with 1000 infected P. berghei erythrocytes. Parasitaemia of infected mice (Wild: n = 5, cl.1: n = 4, cl.2: n = 3) was monitored daily by examination of blood smears. cl. clone, Wild wild type. h Growth inhibition of pXL/hdhfr-pac-egfp [8]-transfected parasites (pbhsp70-pac), pXL/hdhfr-pbef-1α-pac-egfp-transfected parasites (pbef--pac) obtained by pyrimethamine selection using hdhfr marker and pXL/pbef-1α-pacPB-egfp-transfected parasites (pbef--pacPB) was evaluated by determining schizont development within a range of puromycin concentrations. i Real-time quantitative RT-PCR analysis of marker gene expression in pbhsp70-pac, pbef--pac, and pbef--pacPB. The value was normalized to the level of pbhsp70 transcripts in each sample. **p < 0.01 (Dunnett’s multiple comparisons tests)

Back to article page