Limits...
Centromere plasmid: a new genetic tool for the study of Plasmodium falciparum.

Iwanaga S, Kato T, Kaneko I, Yuda M - PLoS ONE (2012)

Bottom Line: This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum.In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence.Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions.

View Article: PubMed Central - PubMed

Affiliation: Mie University, School of Medicine, Tsu, Japan. iwanaga@doc.medic.mie-u.ac.jp

ABSTRACT
The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite.

Show MeSH

Related in: MedlinePlus

The Growth of the Transgenic Parasite after Transfection with pFCEN.The transfection of the parasite with pFCEN (red line) and pCon (blue line) were performed as described in the Materials and Methods. After 48 hours, the drug screening of the transgenic parasites was initiated by supplementing the culture medium with pyrimethamine. The parasitemia of the transgenic parasites was monitored every 48 hours after transfection. The transfection experiments for each plasmid were performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3316556&req=5

pone-0033326-g003: The Growth of the Transgenic Parasite after Transfection with pFCEN.The transfection of the parasite with pFCEN (red line) and pCon (blue line) were performed as described in the Materials and Methods. After 48 hours, the drug screening of the transgenic parasites was initiated by supplementing the culture medium with pyrimethamine. The parasitemia of the transgenic parasites was monitored every 48 hours after transfection. The transfection experiments for each plasmid were performed in triplicate.

Mentions: The constructed pFCEN plasmid was introduced into the wild-type P. falciparum 3D7 strain using DNA-preloaded red blood cells (RBCs), and the parasitemia of the transfected parasites was then monitored every 48 hours. Treatment with pyrimethamine for screening the parasites that contained pFCEN was initiated 4 days after the transfection. The transfected parasite culture was diluted 2-folds with complete medium containing fresh RBCs every 10 days to compensate the loss of old RBCs. In this study, we used pCon as the negative control plasmid; this plasmid includes both the hdhfr and gfp genes but not the centromere (the pfcen5-1.5 sequence). As shown in Figure 3, the parasites transfected with pFCEN emerged more rapidly than the control parasites carrying pCon. The parasitemia of the parasites transfected with pFCEN was approximately 2.8% at 20 days after transfection, whereas that of the control parasites was only 0.08% at the same time interval. It ultimately took 28 days for the parasitemia of the control parasites to reach 2.9%. Because the multiplication rate (every 48 hours) of the parasites carrying pCon was estimated to be 2.9 based on the parasitemia, we estimated that the transfection efficiency of pFCEN was approximately 70-fold higher than that of pCon.


Centromere plasmid: a new genetic tool for the study of Plasmodium falciparum.

Iwanaga S, Kato T, Kaneko I, Yuda M - PLoS ONE (2012)

The Growth of the Transgenic Parasite after Transfection with pFCEN.The transfection of the parasite with pFCEN (red line) and pCon (blue line) were performed as described in the Materials and Methods. After 48 hours, the drug screening of the transgenic parasites was initiated by supplementing the culture medium with pyrimethamine. The parasitemia of the transgenic parasites was monitored every 48 hours after transfection. The transfection experiments for each plasmid were performed in triplicate.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3316556&req=5

pone-0033326-g003: The Growth of the Transgenic Parasite after Transfection with pFCEN.The transfection of the parasite with pFCEN (red line) and pCon (blue line) were performed as described in the Materials and Methods. After 48 hours, the drug screening of the transgenic parasites was initiated by supplementing the culture medium with pyrimethamine. The parasitemia of the transgenic parasites was monitored every 48 hours after transfection. The transfection experiments for each plasmid were performed in triplicate.
Mentions: The constructed pFCEN plasmid was introduced into the wild-type P. falciparum 3D7 strain using DNA-preloaded red blood cells (RBCs), and the parasitemia of the transfected parasites was then monitored every 48 hours. Treatment with pyrimethamine for screening the parasites that contained pFCEN was initiated 4 days after the transfection. The transfected parasite culture was diluted 2-folds with complete medium containing fresh RBCs every 10 days to compensate the loss of old RBCs. In this study, we used pCon as the negative control plasmid; this plasmid includes both the hdhfr and gfp genes but not the centromere (the pfcen5-1.5 sequence). As shown in Figure 3, the parasites transfected with pFCEN emerged more rapidly than the control parasites carrying pCon. The parasitemia of the parasites transfected with pFCEN was approximately 2.8% at 20 days after transfection, whereas that of the control parasites was only 0.08% at the same time interval. It ultimately took 28 days for the parasitemia of the control parasites to reach 2.9%. Because the multiplication rate (every 48 hours) of the parasites carrying pCon was estimated to be 2.9 based on the parasitemia, we estimated that the transfection efficiency of pFCEN was approximately 70-fold higher than that of pCon.

Bottom Line: This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum.In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence.Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions.

View Article: PubMed Central - PubMed

Affiliation: Mie University, School of Medicine, Tsu, Japan. iwanaga@doc.medic.mie-u.ac.jp

ABSTRACT
The introduction of transgenes into Plasmodium falciparum, a highly virulent human malaria parasite, has been conducted either by single crossover recombination or by using episomal plasmids. However, these techniques remain insufficient because of the low transfection efficiency and the low frequency of recombination. To improve the genetic manipulation of P. falciparum, we developed the centromere plasmid as a new genetic tool. First, we attempted to clone all of the predicted centromeres from P. falciparum into E. coli cells but failed because of the high A/T contents of these sequences. To overcome this difficulty, we identified the common sequence features of the centromere of Plasmodium spp. and designed a small centromere that retained those features. The centromere plasmid constructed with the small centromere sequence, pFCEN, segregated into daughter parasites with approximately 99% efficiency, resulting in the stable maintenance of this plasmid in P. falciparum even in the absence of drug selection. This result demonstrated that the small centromere sequence harboured in pFCEN could function as an actual centromere in P. falciparum. In addition, transgenic parasites were more rapidly generated when using pFCEN than when using the control plasmid, which did not contain the centromere sequence. Furthermore, in contrast to the control plasmid, pFCEN did not form concatemers and, thus, was maintained as a single copy over multiple cell divisions. These unique properties of the pFCEN plasmid will solve the current technical limitations of the genetic manipulation of P. falciparum, and thus, this plasmid will become a standard genetic tool for the study of this parasite.

Show MeSH
Related in: MedlinePlus