A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle

Green fluorescent protein (GFP) is a well-established reporter protein for the examination of biological processes. This report describes a recombinant Plasmodium berghei, PbGFP(CON), that constitutively expresses GFP in a growth responsive manner in its cytoplasm from a transgene that is integrated into the genome and controlled by the strong promoter from a P. berghei elongation factor-lot gene. All life cycle forms of PbGFP(CON) except for mate gametes can be easily visualized by fluorescent microscopy. PbGFP(CON) showed similar growth characteristics to wild type P. berghei parasites throughout the whole life cycle and can therefore be used as a reference line for future investigations of parasite-host cell interactions. The principle of automated fluorescence-based counting and sorting of live parasites from host cell backgrounds and different parasite forms from complex mixtures such as asynchronous blood stages is established. PbGFP(CON) allows the visualization and investigation of live parasite stages that are difficult and labor-intensive to observe, such as the liver and mosquito stages. PbGFP(CON) can be employed to establish the phenotype of independent mutant parasites. With the recent development of a second, independent selectable marker in R berghei, PbGFP(CON) is a useful tool to investigate the effect of further genetic modifications on host-parasite interactions. (C) 2004 Elsevier B.V. All rights reserved.