Lucas, Stuart James; (2003) Genetic manipulation and gene expression in the human malaria parasite Plasmodium falciparum. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Molecular genetic analysis of the most important malaria pathogen, Plasmodium falciparum, presents numerous challenges. The parasite is relatively refractory towards transfection, and the nature of its transcriptional regulation mechanisms remain to be elucidated. Both existing techniques and new approaches for the transfection of parasites within red blood cells were assessed, including electroporation, lipofection, pre-loading erythrocytes and applying morpholino antisense oligonucleotides. Of these only electroporation proved reliable, and although the efficiency of transfection was improved by optimization of this method it remained around 0.1%. In an attempt to identify transfected cells from the untransfected background, a plasmid was designed to express a c-myc epitope-tagged protein of the Rifin family on the surface of erythrocytes infected with transfected parasites. However, in transient transfection studies, expression of the Rifin was too low to detect at either the mRNA or the protein level. This Rifin expression problem raised the issue of transcriptional control in P. falciparum. Before promoter activity could be accurately measured, a quantitative assay for the chloramphenicol acetyltransferase (CAT) reporter gene product was established. A range of P. falciparum 5' untranslated regions from genes of biological interest were then cloned and used to drive CAT expression in a transient transfection system. Clear differences in promoter strength and stage specificity were observed, and regions containing important control sequences for the enolase, kahrp and mspl genes were identified. The expression patterns of several promoters differed from their predicted stage specificity; however, patterns much closer to their biological activity were observed after plasmid replication by the parasite, indicating a role for epigenetic factors in regulating gene expression. Stable transfection was established using selection for the human dihydrofolate reductase (hDHFR) drug resistance gene, and this strategy was used to try and express the Rifin gene either with c-myc tag or fused to Green Fluorescent Protein.

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