DiCre-Mediated Gene Excision in Malaria Parasite

DiCre recombinase mediated excision of targeted DNA sequences in vivo was achieved by a single oral administration of 200μg rapamycin (1mg/ml stock, Rapamune, Pfizer) to mice. Excision of the GFP cassette in blood stage parasites was monitored by flow cytometry using a Guava EasyCyte 6/2L bench cytometer equipped with 488 nm and 532 nm lasers (Millipore) to detect GFP and mCherry, respectively. To analyze parasite development in the mosquito, rapamycin was administered to infected mice 24 hours prior to transmission to mosquitoes, as described [19 (link)]. Midguts were dissected out at day 14 post infection. The haemolymph was collected by flushing the haemocoel with complete DMEM, day 14 to 16 post infection. Salivary gland sporozoites were collected between 21–28 days post feeding from infected mosquitoes, by hand dissection and homogenization of isolated salivary glands in complete DMEM. Live samples (infected mosquito midguts or salivary glands, sporozoites) were mounted in PBS and visualized live using a Zeiss Axio Observer.Z1 fluorescence microscope equipped with a LD Plan-Neofluar 40x/0.6 Corr Ph2 M27 objective. The exposure time was set according to the positive control and maintained for both untreated and rapamycin-treated parasites, in order to allow comparisons. All images were processed with ImageJ for adjustment of contrast.