Negative selection using yeast cytosine deaminase/uracil phosphoribosyl transferase in Plasmodium falciparum for targeted gene deletion by double crossover recombination.

The ability to genetically manipulatePlasmodium falciparum as been established for a number of years and it is an important ool for functional analysis of parasite genes using gene disuption [1], allelic exchange [2] and transgene expression [3]. his was established using positive selection for maintenance of ircular plasmids carrying a drug selectable marker such as Toxplasma gondii dihydrofolate reductase (TgDHFR) [4,5], or the uman dhfr gene [6] conferring resistance to pyrimethamine or R99210 respectively although more recently other selection ystems have been used [7]. Transfected P. falciparum parasites arrying copies of these plasmids integrated into the genome by omologous recombination were generally selected over time y cycles of growth on and off drug selection. The episomal lasmids are lost rapidly in the absence of drug selection as they re partitioned unevenly amongst the daughter progeny whereas he plasmids integrated into the genome are segregated normally ith each chromosome [8]. This method ensured that after subequent on/off drug cycles all transfectedP. falciparum parasites btained had integrated the plasmid by single crossover homoloous recombination. Whilst this proved to be an extremely useful ethod for gene disruption it was limited because of the long eriods of culture required to derive parasites with integrated opies of the plasmid due to persistence of the circular episomal orms. Another drawback is that with a single cross over the