Figure 1.

Optimisation of rapamycin-induced recombination. (a) Parasites containing a modified PFA0210c ²² were used to test DiCre-mediated recombination efficiency. For this a recodonised version of part of the ORF of PFA0210c was flanked by a loxPint sequence upstream and a loxP sequence downstream located within the endogenous locus on chromosome 1 in the 1G5DC parasite line. (b) The efficiency of excision was tested whilst altering the concentration and length of exposure to rapamycin (rapa). Either DMSO alone (0 nM) or increasing concentrations (in nM) of rapamycin were added to transgenic PFA0210c-loxP parasites as indicated for 4, 2, 1 h or 30 min. The degree of recombination was determined by PCR using primers 13 and 14 depicted as arrows in panel a). A lack of recombination and no excision resulted in a PCR product of 1580 bp (u) whereas successful excision resulted in a 562 bp product (e). The sizes of DNA markers (in kb) are depicted on the left. (c) The timing of excision was determined following addition of either 100 nM rapamycin for 4 h (100) or 10 nM rapamycin for 30 min (10) at very early ring stages. Parasites were harvested 2, 4, 8, 20, 30 or 44 h after the onset of treatment. Genomic DNA was extracted from saponin-treated parasite pellets and excision was determined by PCR as described in panel b. (d) Graph depicting the degree of excision (in %) over time elapsed after start of rapamycin treatment. For this graph, the data in panel c) was semi-quantitatively analysed. Pixel intensities of stained DNA bands were measured using ImageJ and the ratio of excised vs. unexcised was plotted against time after rapamycin addition, comparing two treatment methods (100 nM rapamycin for 4 h and 10 nM rapamycin for 30 min).