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Transgenic Plasmodium parasites stably expressing Plasmodium vivax dihydrofolate reductase-thymidylate synthase as in vitro and in vivo models for antifolate screening.

Somsak V, Uthaipibull C, Prommana P, Srichairatanakool S, Yuthavong Y, Kamchonwongpaisan S - Malar. J. (2011)

Bottom Line: To facilitate the development of anti-P. vivax drugs, bacterial and yeast surrogate models expressing the validated P. vivax target dihydrofolate reductase-thymidylate synthase (DHFR-TS) have been generated; however, they can only be used as primary screening models because of significant differences in enzyme expression level and in vivo drug metabolism between the surrogate models and P. vivax parasites.The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites.A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-P. vivax drugs in general.

View Article: PubMed Central - HTML - PubMed

Affiliation: Protein-Ligand Engineering and Molecular Biology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Thailand Science Park, Pathumthani 12120, Thailand.

ABSTRACT

Background: Plasmodium vivax is the most prevalent cause of human malaria in tropical regions outside the African continent. The lack of a routine continuous in vitro culture of this parasite makes it difficult to develop specific drugs for this disease. To facilitate the development of anti-P. vivax drugs, bacterial and yeast surrogate models expressing the validated P. vivax target dihydrofolate reductase-thymidylate synthase (DHFR-TS) have been generated; however, they can only be used as primary screening models because of significant differences in enzyme expression level and in vivo drug metabolism between the surrogate models and P. vivax parasites.

Methods: Plasmodium falciparum and Plasmodium berghei parasites were transfected with DNA constructs bearing P. vivax dhfr-ts pyrimethamine sensitive (wild-type) and pyrimethamine resistant (mutant) alleles. Double crossover homologous recombination was used to replace the endogenous dhfr-ts of P. falciparum and P. berghei parasites with P. vivax homologous genes. The integration of Pvdhfr-ts genes via allelic replacement was verified by Southern analysis and the transgenic parasites lines validated as models by standard drug screening assays.

Results: Transgenic P. falciparum and P. berghei lines stably expressing PvDHFR-TS replacing the endogenous parasite DHFR-TS were obtained. Anti-malarial drug screening assays showed that transgenic parasites expressing wild-type PvDHFR-TS were pyrimethamine-sensitive, whereas transgenic parasites expressing mutant PvDHFR-TS were pyrimethamine-resistant. The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites.

Conclusion: With the permanent integration of Pvdhfr-ts gene in the genome, the transgenic Plasmodium lines expressing PvDHFR-TS are genetically stable and will be useful for screening anti-P. vivax compounds targeting PvDHFR-TS. A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-P. vivax drugs in general.

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Southern analysis of transgenic parasites to confirm allelic replacement of dhfr-ts of Plasmodium falciparum (A) and Plasmodium berghei (B) with dhfr-ts gene from P. vivax. DNA probes specific to Pvdhfr-ts (PvDT), Pfdhfr (PfD) and 3'UTR of Pbdhfr-ts (3'UTR PbDT) were used to detect restriction-digested fragments from genomic DNA of transgenic parasites. Pl: pCBVD plasmid control; K1: P. falciparum K1CB1; B2: Transgenic PfPvDTclB2; PbGFP: P. berghei GFP; WT: transgenic PbPvDTcl4; SP: transgenic PbPvSP21cl2.
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Figure 2: Southern analysis of transgenic parasites to confirm allelic replacement of dhfr-ts of Plasmodium falciparum (A) and Plasmodium berghei (B) with dhfr-ts gene from P. vivax. DNA probes specific to Pvdhfr-ts (PvDT), Pfdhfr (PfD) and 3'UTR of Pbdhfr-ts (3'UTR PbDT) were used to detect restriction-digested fragments from genomic DNA of transgenic parasites. Pl: pCBVD plasmid control; K1: P. falciparum K1CB1; B2: Transgenic PfPvDTclB2; PbGFP: P. berghei GFP; WT: transgenic PbPvDTcl4; SP: transgenic PbPvSP21cl2.

Mentions: Pyrimethamine-resistant P. falciparum strain K1CB1 was transfected with plasmid DNA for replacement of the endogenous Pfdhfr-ts with wild-type Pvdhfr-ts. Following rounds of positive and negative selections, transgenic P. falciparum were obtained. A clonal parasite line designated PfPvDTclB2 was obtained and gene replacement verified by Southern blotting analysis. As shown in Figure 2A, hybridization with a Pvdhfr-ts DNA probe showed the expected 3.7 kb and 5.9 kb EcoRI-digested fragments from transgenic PfPvDTclB2 genomic DNA and control plasmid DNA, respectively, confirming the integration of Pvdhfr-ts at the correct site, while no signal was detected for parental P. falciparum K1CB1 line. Another DNA probe specific to Pfdhfr could detect the expected 8.8 kb, 8.0 kb and 5.4 kb EcoRI-digested fragments from genomic DNA of transgenic PfPvDTclB2, parental P. falciparum K1CB1 line and control plasmid DNA, respectively. From these results, it can be concluded that transgenic PfPvDTclB2 parasites stably express wild-type Pvdhfr-ts instead of endogenous Pfdhfr-ts; furthermore, these parasites are clonal, lack episomal plasmid DNA and can be stably maintained without drug selection.


Transgenic Plasmodium parasites stably expressing Plasmodium vivax dihydrofolate reductase-thymidylate synthase as in vitro and in vivo models for antifolate screening.

Somsak V, Uthaipibull C, Prommana P, Srichairatanakool S, Yuthavong Y, Kamchonwongpaisan S - Malar. J. (2011)

Southern analysis of transgenic parasites to confirm allelic replacement of dhfr-ts of Plasmodium falciparum (A) and Plasmodium berghei (B) with dhfr-ts gene from P. vivax. DNA probes specific to Pvdhfr-ts (PvDT), Pfdhfr (PfD) and 3'UTR of Pbdhfr-ts (3'UTR PbDT) were used to detect restriction-digested fragments from genomic DNA of transgenic parasites. Pl: pCBVD plasmid control; K1: P. falciparum K1CB1; B2: Transgenic PfPvDTclB2; PbGFP: P. berghei GFP; WT: transgenic PbPvDTcl4; SP: transgenic PbPvSP21cl2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3198988&req=5

Figure 2: Southern analysis of transgenic parasites to confirm allelic replacement of dhfr-ts of Plasmodium falciparum (A) and Plasmodium berghei (B) with dhfr-ts gene from P. vivax. DNA probes specific to Pvdhfr-ts (PvDT), Pfdhfr (PfD) and 3'UTR of Pbdhfr-ts (3'UTR PbDT) were used to detect restriction-digested fragments from genomic DNA of transgenic parasites. Pl: pCBVD plasmid control; K1: P. falciparum K1CB1; B2: Transgenic PfPvDTclB2; PbGFP: P. berghei GFP; WT: transgenic PbPvDTcl4; SP: transgenic PbPvSP21cl2.
Mentions: Pyrimethamine-resistant P. falciparum strain K1CB1 was transfected with plasmid DNA for replacement of the endogenous Pfdhfr-ts with wild-type Pvdhfr-ts. Following rounds of positive and negative selections, transgenic P. falciparum were obtained. A clonal parasite line designated PfPvDTclB2 was obtained and gene replacement verified by Southern blotting analysis. As shown in Figure 2A, hybridization with a Pvdhfr-ts DNA probe showed the expected 3.7 kb and 5.9 kb EcoRI-digested fragments from transgenic PfPvDTclB2 genomic DNA and control plasmid DNA, respectively, confirming the integration of Pvdhfr-ts at the correct site, while no signal was detected for parental P. falciparum K1CB1 line. Another DNA probe specific to Pfdhfr could detect the expected 8.8 kb, 8.0 kb and 5.4 kb EcoRI-digested fragments from genomic DNA of transgenic PfPvDTclB2, parental P. falciparum K1CB1 line and control plasmid DNA, respectively. From these results, it can be concluded that transgenic PfPvDTclB2 parasites stably express wild-type Pvdhfr-ts instead of endogenous Pfdhfr-ts; furthermore, these parasites are clonal, lack episomal plasmid DNA and can be stably maintained without drug selection.

Bottom Line: To facilitate the development of anti-P. vivax drugs, bacterial and yeast surrogate models expressing the validated P. vivax target dihydrofolate reductase-thymidylate synthase (DHFR-TS) have been generated; however, they can only be used as primary screening models because of significant differences in enzyme expression level and in vivo drug metabolism between the surrogate models and P. vivax parasites.The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites.A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-P. vivax drugs in general.

View Article: PubMed Central - HTML - PubMed

Affiliation: Protein-Ligand Engineering and Molecular Biology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Thailand Science Park, Pathumthani 12120, Thailand.

ABSTRACT

Background: Plasmodium vivax is the most prevalent cause of human malaria in tropical regions outside the African continent. The lack of a routine continuous in vitro culture of this parasite makes it difficult to develop specific drugs for this disease. To facilitate the development of anti-P. vivax drugs, bacterial and yeast surrogate models expressing the validated P. vivax target dihydrofolate reductase-thymidylate synthase (DHFR-TS) have been generated; however, they can only be used as primary screening models because of significant differences in enzyme expression level and in vivo drug metabolism between the surrogate models and P. vivax parasites.

Methods: Plasmodium falciparum and Plasmodium berghei parasites were transfected with DNA constructs bearing P. vivax dhfr-ts pyrimethamine sensitive (wild-type) and pyrimethamine resistant (mutant) alleles. Double crossover homologous recombination was used to replace the endogenous dhfr-ts of P. falciparum and P. berghei parasites with P. vivax homologous genes. The integration of Pvdhfr-ts genes via allelic replacement was verified by Southern analysis and the transgenic parasites lines validated as models by standard drug screening assays.

Results: Transgenic P. falciparum and P. berghei lines stably expressing PvDHFR-TS replacing the endogenous parasite DHFR-TS were obtained. Anti-malarial drug screening assays showed that transgenic parasites expressing wild-type PvDHFR-TS were pyrimethamine-sensitive, whereas transgenic parasites expressing mutant PvDHFR-TS were pyrimethamine-resistant. The growth and sensitivity to other types of anti-malarial drugs in the transgenic parasites were otherwise indistinguishable from the parental parasites.

Conclusion: With the permanent integration of Pvdhfr-ts gene in the genome, the transgenic Plasmodium lines expressing PvDHFR-TS are genetically stable and will be useful for screening anti-P. vivax compounds targeting PvDHFR-TS. A similar approach could be used to generate transgenic models specific for other targets of interest, thus facilitating the development of anti-P. vivax drugs in general.

Show MeSH
Related in: MedlinePlus