Rapid activation of distinct member of multigene families in Plasmodium spp.

The objective of this study is to provide a novel method to study multigene proteins in Plasmodium spp. The method is based on selection linked integration (SLI), which allows positive selection of genomic integration events. By targeting specific members of multigene families, parasites are being selected for not only genomic integration but also expression of the targeted gene under its endogenous promoter P. falciparum parasites strain 3D7 transfected with pSLI plasmids targeting either a specific rifin, stevor or var gene. Transfectant were selected for genomic integration and class switch. Integration was confirmed by PCR and sequencing prior to RNA isolation and microarray hybridization. In brief, 20 ml parasite cultures were synchronized twice per day with 5% (v/v) sorbitol in water to obtain tightly synchronized ring-stage parasites. A total of 12 RNA samples were collected, including 3 parental 3D7 controls, at 18, 24 and 30 hours post invasion. RNA were extracted and synthesis and amplification of cDNA was carried out as previosuly described in Bozdech, Z., S. Mok & A. P. Gupta, (2013). cDNA generated was used for microarray hybridizations against a common RNA reference pool of P. falciparum 3D7 strain.