Fig. 7.

(a) Plasmids transfected into 3D7-infected erythrocytes carried the P. falciparum heat shock protein 86 promoter (5′ hsp86) driving transcription of the blasticidin deaminase gene (bsd) followed by the Plasmodium berghei dihydrofolate reductase transcriptional terminator (Pb dhfr 3′) followed by the ItG upsE driving transcription of human dihydrofolate reductase (hdhfr) followed by the P. falciparum histidine-rich protein 2 transcriptional terminator (hrp2 3′) followed by either the E8B or CS2 var2csa intron in plasmids pHBEiE and pHBEiC, respectively. The plasmid pHBEiER is the same as pHBEiE except for the inclusion of the TARE 6 (rep20) upstream of the upsE. (b) Plasmids were maintained in 3D7 parasites by growth in the presence of blasticidin-S, and the levels of hdhfr transcription were determined at different time points following transfection by Q-RT-PCR. Absolute quantitation was performed using standard curves of serially diluted, cloned hdhfr DNA of known concentration. The results were normalised using skeleton binding protein 1 cDNA levels to allow comparison of equivalent quantities of parasite material and then expressed as relative units of hdhfr cDNA per plasmid by dividing by the number of plasmids per genome. Plasmid number was determined by using Q-RT-PCR of transfected parasite gDNA to determine hdhfr normalised with sbp. (c) Northern blots of RNA from transfected parasites and nontransfected 3D7 parasites serially probed with sequence from bsd, hdhfr, and a conserved var exon 2.