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        Episomal Transformation of Plasmodium berghei

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        Genetic transformation of an organism is a fundamental investigational tool that allows the researcher to gain an insight into the basic cellular biology. Numerous areas of parasite biology can be addressed simply by introducing plasmids that are maintained as episomes into the cell of choice and modifying phenotype through the expression of plasmid-borne transgenes. The methodologies to achieve this in a stable drug selectable fashion were developed for malaria parasites 7 yr ago (1 -4 ). These have already yielded insights into gene transcription promoter structure (3 ,5 ), reporter gene expression (2 ,3 ,6 ), the requirements for stage-specific localization of malaria proteins (7 ,8 ), and organelle targeting (9 ). Attempts have been made to complement genetic mutants through episomal transgene expression of a normal copy of the defective gene making use of a second selectable maker that is now available (10 ,11 ). Little experimentation has yet been performed investigating what is the most obvious application that is the over expression of transgene-encoded proteins creating novel phenotypes such as drug resistance and dominant negative phenotypes. All transfection procedures take 2-3 wk in the P. berghei model and the integration of intact episomes under these conditions into the genome of P. berghei has not yet been demonstrated. In the human parasite, P. falciparum episome integration is a commonly observed mechanism, although a protracted selection procedure after transfection is necessary to obtain integrants (12 -14 ). In P. berghei , site-directed integration of linear pieces of DNA is relatively efficient (see Chapter 29), whereas integration of intact plasmids might not occur at an observable frequency under normal transfection procedures.
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