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01.12.2012 | Methodology | Ausgabe 1/2012 Open Access

Malaria Journal 1/2012

Plate-based transfection and culturing technique for genetic manipulation of Plasmodium falciparum

Zeitschrift:
Malaria Journal > Ausgabe 1/2012
Autoren:
Florence Caro, Mathew G Miller, Joseph L DeRisi
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​1475-2875-11-22) contains supplementary material, which is available to authorized users.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

FC designed and performed experiments, analyzed data and wrote the paper. MM designed and performed experiments. JDR conceived of the study, and participated in its design, coordination, data analysis, co-wrote the manuscript, and co-designed the figures. All authors read and approved the final manuscript.

Abstract

Genetic manipulation of malaria parasites remains an inefficient, time-consuming and resource-intensive process. Presented here is a set of methods for 96-well plate-based transfection and culture that improve the efficiency of genetic manipulation of Plasmodium falciparum. Compared to standard protocols plate-based transfection requires 20-fold less DNA, transient transfection efficiency achieved is approximately seven-fold higher, whilst stable transfection success rate is above 90%. Furthermore the utility of this set of protocols to generate a knockout of the PfRH3 pseudogene, screened by whole-cell PCR, is demonstrated. The methods and tools presented here will facilitate genome-scale genetic manipulation of P. falciparum.
Zusatzmaterial
Additional file 1: RLUC assay noise measurements. Boxplots of normalized RLU values for each of the noise measurements. Transfection noise was measured on 44 independently transfected and cultured wells. Assay noise was measured by pooling and re-splitting 40 transfection wells eliminating transfection noise. Luminometer reading noise was measured by pooling and re-splitting the lysate of 40 transfection wells prior to addition of RLUC substrate, eliminating transfection noise and assay noise. In each case, four wells of negative control plasmid, pfGNr, were transfected in parallel (bckg = background). (PDF 145 KB)
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Additional file 2: Transient transfection with an extended set of pulses. 6 μl of packed RBCs were transfected with 5 μg RLUC reporter plasmid, 12 mM Li2ATP and Buffer SE using pulses in the CM series and eight additional ones. Four replicas of each condition were setup. HC was determined measuring absorbance at 410 nm and normalizing to a standard curve of non-pulsed RBCs at known HC. In bold is the chosen pulse, CM-162. ± s.d., standard deviation. (PDF 396 KB)
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Additional file 3: Optimizing buffer and pulse codes for transient transfection in plates. (A) Set of the 31 pulses tested and their corresponding position on the electroporation plate. (B) Intensity heatmaps of RLUC reporter activity for direct ring and schizont electroporation. Transfection procedure: 4 ul packed RBCs or 6% ring or schizont stage parasites, were mixed with 5 μg of the RLUC reporter plasmid in one of three buffers (SE, SF, or SG). Each of the 31 pulses was delivered to the corresponding wells. pfGNr was used as negative control. Luciferase activity was measured 48 h and 72 h after electroporation, for RBC or schizonts, and for rings, respectively. In parallel with the ring and schizont transfections, RBCs were electroporated as in B using pulse CM-150 and buffer SE. (PDF 382 KB)
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Additional file 4: Optimization transfection mixture components. (A) Transfection efficiency using different ATP salts. 6 μl packed RBCs were transfected (pulse CM-162) with 5 μg of the RLUC reporter plasmid, in Buffer SE and 6.25 mM final concentration of different ATP salts (Na2ATP, Li2ATP and MgATP). RLUC reporter signal was measured 48 h later for six replicas of each condition. Error bars, s.e.m. (B) Transfection efficiency using different volumes of packed RBCs. Three different volumes (2, 4 and 6 μl) of packed RBCs were transfected (pulse CM-162) with 2.5, 5 or 10 μg RLUC reporter plasmid, in Buffer SE with or without 10 mM Li2ATP. Table shows RLU values measured 48 h later for two replicas of each condition. (PDF 345 KB)
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Additional file 5: Optimization of hematocrit for culture in 96-well plates. 200 μl of 0.025, 0.013, 0.006, 0.003% parasitaemia cultures (ring stage) were plated at 1, 2, 3, 4, and 5% HC in a 96-well flat-bottom plate, in triplicate. Five days later parasitaemia was measured by mMSF assay for each condition. Error bars, standard deviation. (PDF 267 KB)
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Additional file 6: Modified MSF assay. (A) For each tested condition, 200 μl of 0.8% parasitaemia cultures at 2% HC, were set up in columns 1 to 10 of five 96-well plates, uninfected RBCs were plated in column 11 for background signal measurement, and column 12 was used to generate a standard curve for parasitaemia. Final parasitaemia was measured 72 h later by MSF assay using five different manipulations before the measurement; media change, spent media was replaced with fresh media; no media change, culture was resuspended in the spent media; media wash/PBS resuspend, spent media was replaced with fresh media and culture was washed with 1X PBS; PBS resuspend, spent media was replaced with 1X PBS; PBS washed, spent media was replaced with 1X PBS and further washed in PBS. (B) For each of the five manipulations the mean of 10 column measurements (n = 80), their corresponding mean background signals (n = 8) and signal to background ratios, are listed. (C) Standard curve obtained using the PBS resuspend manipulation. Note that parasitaemias as low as 0.5% can be detected. (PDF 380 KB)
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Authors’ original file for figure 1
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Authors’ original file for figure 2
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Authors’ original file for figure 3
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Authors’ original file for figure 4
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