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Background: Plasmodium knowlesi is now the major cause of human malaria in Malaysia, complicating malaria control efforts that must attend to the elimination of multiple Plasmodium species. Recent advances in the cultivation of P. knowlesi erythrocytic-stage parasites in vitro, transformation with exogenous DNA, and infection of mosquitoes with gametocytes from culture have opened up studies of this pathogen without the need for resource-intensive and costly non-human primate (NHP) models. For further understanding and development of methods for parasite transformation in malaria research, this study examined the activity of various trans-species transcriptional control sequences and the influence of Plasmodium vivax centromeric (pvcen) repeats in plasmid-transfected P. knowlesi parasites., Methods: In vitro cultivated P. knowlesi parasites were transfected with plasmid constructs that incorporated Plasmodium vivax or Plasmodium falciparum 5' UTRs driving the expression of bioluminescence markers (firefly luciferase or Nanoluc). Promoter activities were assessed by bioluminescence, and parasites transformed with human resistant allele dihydrofolate reductase-expressing plasmids were selected using antifolates. The stability of transformants carrying pvcen-stabilized episomes was assessed by bioluminescence over a complete parasite life cycle through a rhesus macaque monkey, mosquitoes, and a second rhesus monkey., Results: Luciferase expression assessments show that certain P. vivax promoter regions, not functional in the more evolutionarily-distant P. falciparum, can drive transgene expression in P. knowlesi. Further, pvcen repeats may improve the stability of episomal plasmids in P. knowlesi and support detection of NanoLuc-expressing elements over the full parasite life cycle from rhesus macaque monkeys to Anopheles dirus mosquitoes and back again to monkeys. In assays of drug responses to chloroquine, G418 and WR9910, anti-malarial half-inhibitory concentration (IC 50 ) values of blood stages measured by NanoLuc activity proved comparable to IC 50 values measured by the standard SYBR Green method., Conclusion: All three P. vivax promoters tested in this study functioned in P. knowlesi, whereas two of the three were inactive in P. falciparum. NanoLuc-expressing, centromere-stabilized plasmids may support high-throughput screenings of P. knowlesi for new anti-malarial agents, including compounds that can block the development of mosquito- and/or liver-stage parasites.

Sonoporation has not been widely explored as a strategy for the transfection of heterologous genes into notoriously difficult-to-transfect mammalian cell lines such as B cells. This technology utilizes ultrasound to create transient pores in the cell membrane, thus allowing the uptake of extraneous DNA into eukaryotic and prokaryotic cells, which is further enhanced by cationic microbubbles. This study investigates the use of sonoporation to deliver a plasmid encoding green fluorescent protein (GFP) into three human B-cell lines (Ramos, Raji, Daudi). A higher transfection efficiency (TE) of >42% was achieved using sonoporation compared with <3% TE using the conventional lipofectamine method for Ramos cells. Upon further antibiotic selection of the transfected population for two weeks, we successfully enriched a stable population of GFP-positive Ramos cells (>70%). Using the same strategy, Raji and Daudi B cells were also successfully transfected and enriched to 67 and 99% GFP-positive cells, respectively. Here, we present sonoporation as a feasible non-viral strategy for stable and highly efficient heterologous transfection of recalcitrant B-cell lines. This is the first demonstration of a non-viral method yielding transfection efficiencies significantly higher (42%) than the best reported values of electroporation (30%) for Ramos B-cell lines., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)