Parasite culture
The P. falciparum clone AHEI was cultured using standard protocols in the presence of 5nM WR99210 [10]. Synchronization to prepare ring stage IE was carried out using 5% D-sorbitol [11]. Enrichment of late trophozoite/schizont IE was carried out using Plasmagel (Bellon, France) density gradient flotation [12]. Giemsa-stained thin blood smears were used to monitor parasite staging and parasitaemia. Immediately prior to transfection, AHEI were passaged into freshly isolated O+ erythrocytes (National Blood and Transfusion Service, UK), with this same single source of erythrocytes used in all experiments for the remainder of the study.
Transfection and time-course of sampling
Large-scale preparation of plasmids pΔ1 and pINT was carried out using a commercial maxipreparation kit (Qiagen, UK). Plasmid preparations were pooled and the same common source of plasmid used in all subsequent experiments. pΔ1 contains a luciferase reporter gene flanked by 1418 bp and 647 bp of 5′ and 3′ Pfpcna sequences, respectively, in a pDCatt P backbone to facilitate integration into the genome of the P. falciparum clone AHEI when cotransfected with pINT [10, 13]. Four different protocols were investigated here, each performed in triplicate. Transfections 1–3 represent the direct electroporation of 40 μg each of pΔ1/pINT into ring stage IE (Figure 1A, Protocol 1) with transfections 4–6 using 40 μg each of pΔ1/pINT preloaded into erythrocytes (Protocol 2). Transfections 7–12 utilised a “double-tap” combination of these protocols; transfections 7–9 using double the amount (40 μg each of pΔ1/pINT twice, Protocol 3) of DNA compared to transfections 10–12 (20 μg each of pΔ1/pINT twice, Protocol 4). Thus, transfections 10–12 use the same overall amount of DNA as do transfections 1–6. As transfections 4–6 start 1 day later than the remainder, which all employ direct electroportation of ring IE on day 0, all calculations assume a start from day 1, although data is plotted from day 0 for common illustrative purposes.
Transfections of ring stage IE (transfections 1–3, 7–12) were carried out on 1.25x108 parasites in a total volume of 400 μl in 1x cytomix [6]. Chilled 4 mm electroporation cuvettes were used with a GenePulser II electroporator (Biorad) set at 0.31KV and 950 μF. Immediately following electroporation, the IE were transferred to 10 ml of prewarmed complete medium, gassed (1% O2, 3% CO2, balance N2) and incubated at 37 °C. To preload erythrocytes, the appropriate amount of plasmid was added to 150 μl of packed erythrocytes in a total volume of 400 μl of 1x cytomix [7]. The same electroporation conditions as above were used. These preloaded erythrocytes were either mixed with the parasites transfected on day 0 (transfections 7–12) or with 2x108 trophozoite/schizont stage IE (transfections 4–6). All transfection experiments were then matched to a 3% haematocrit (HCT), which was maintained over the remainder of the experiment.
From Day 2, drug selection was applied to the transfected culture by supplementing complete medium with 5nM WR99210, 2.5 μg/ml Blasticidin S and 125 μg/ml Neomycin; the latter two drugs representing selection for the pΔ1 and pINT plasmids, respectively. Throughout the selection procedure a common source of complete culture medium was used for all experiments. From Day 3, 200 μl of each transfected culture was periodically harvested approximately every 2 days (Mon, Wed, Fri). At this time, complete culture medium was replaced, washed erythrocytes added to maintain a 3% haematocrit and all flasks gassed before placing back at 37 °C. The 200 μl sample provided 3x40μl samples for determination of luciferase activity using an improved single-step lysis protocol [9] and IE to prepare a thin-blood smear for microscopic examination. To each 40 μl sample, 10 μl of 5x Passive Lysis Buffer (Promega, UK) was mixed, aliquoted into a well containing 50 μl of luciferase assay buffer (Promega, UK) on a 96-well white multiplate (Greiner, UK) and bioluminescence (in relative light units, RLU) measured for 2 sec in a GloMax Multi Detection System (Promega, UK).
Analysis of growth and transfection efficiency
To correlate RLU with parasite number a standard curve was generated. Pfluc is an AHEI derivative in which the pΔ1 plasmid is integrated into the cg6 locus on chromosome 7 [9, 13]. A serial three-fold dilution of a 1% starting parasitaemia at a 3% HCT was performed, maintaining a 3% HCT in each sample due to the quenching effect of released haemoglobin on luciferase bioluminescence [9]. Five replicates were prepared and the parasite number per sample plotted against mean log10 transformed RLU. Using a 1/250th sampling volume (40 μl from 10 ml), an extrapolation to provide an estimate of the increase in parasite number over time in each transfected culture can be made. A logarithmic non-linear regression of parasite numbers over time (GraphPad Prism v5.01) allows the rate constant (k), i.e. the fold increase in parasite numbers per asexual cycle, to be determined. These values ranged from between 2.25-2.75 across the 10 experiments for which these could be plotted. Since there was no correlation between the rate constant and the protocol employed, a median of 2.5-fold increase in parasite numbers per cycle was used in all subsequent calculations. Using a generalised formula for logarithmic growth (N = Noekt, where N represents the numbers of parasite following t cycles of growth at the rate constant k from an initial population of No) the number of parasites that were successfully initially transfected could be estimated and are indicated here as a proportion of the input parasite numbers to represent transfection efficiency. For each experiment, No was determined from two timepoints on the curve, and the mean transfection efficiency reported.