Figure 3.

Regulated expression of Renilla luciferase using variable blasticidin selection. P. falciparum NF54 parasites stably transformed with pHBIRH containing var intron PFC0005w were grown in presence of different blasticidin concentrations; 2 μg/ml (black), 5 μg/ml (dark grey), 10 μg/ml (light grey) and 20 μg/ml (white). A: Renilla luciferase activity measured 16–18 h (ring) and 40–42 h (late troph) after invasion. B: DNA was extracted from parasite cultures and the copy numbers of blasticidin (bsd), Renilla luciferase (ren) and the Plasmodium genes kahrp, sbp1 and msp1 were determined by quantitative realtime PCR. Values were standardized to the control gene seryl-tRNA synthetase. Samples were measured in triplicate and error bars indicate the standard deviation. C: mRNA levels were determined by quantitative realtime PCR analysis of cDNA. RNA was extracted from synchronized parasite cultures in the late trophozoite stage. ct values were standardized to control gene seryl-tRNA synthetase and relative mRNA copy numbers are displayed as percentile of msp1 (100%).