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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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Diagrammatic representation of strategy to generate transgenic parasites by homologous recombination of Pvdhfr-ts gene replacing endogenous dhfr-ts locus. (A) Generation of transgenic P. falciparum stably expressing wild-type PvDHFR-TS. The endogenous Pfdhfr-ts locus was targeted via double homologous recombination with a plasmid containing expressing cassettes of wild-type Pvdhfr-ts, and blasticidin-S deaminase (bsd) marker. Yeast cytosine deaminase-uracil phosphoribosyl transferase (yfcu) was used as negative selectable marker. (B) Generation of transgenic P. berghei stably expressing wild-type PvDHFR-TS. The endogenous Pbdhfr-ts locus was targeted with a linearized plasmid containing expressing cassettes of wild-type Pvdhfr-ts, and fusion gene of human dhfr (hdhfr) and yeast yfcu as positive and negative selectable markers respectively. After double homologous recombination, the drug selectable markers are excised by single homologous recombination via the Pbdhfr-ts 3'UTR repeated sequence elements (blue boxes), whilst retaining the wild-type Pvdhfr-ts gene. (C), Generation of transgenic P. berghei stably expressing mutant PvDHFR-TS SP21. The endogenous Pbdhfr-ts locus was targeted with a linearized plasmid containing expressing cassettes of mutant Pvdhfr-ts sp21 (Pvsp21) flanked with 5' and 3' UTR of Pbdhfr-ts which also serve as the sites for double homologous recombination. Specific probes for Southern analysis are located by bold horizontal line. E: EcoRI, H: HindIII, K: KpnI.
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Figure 1: Diagrammatic representation of strategy to generate transgenic parasites by homologous recombination of Pvdhfr-ts gene replacing endogenous dhfr-ts locus. (A) Generation of transgenic P. falciparum stably expressing wild-type PvDHFR-TS. The endogenous Pfdhfr-ts locus was targeted via double homologous recombination with a plasmid containing expressing cassettes of wild-type Pvdhfr-ts, and blasticidin-S deaminase (bsd) marker. Yeast cytosine deaminase-uracil phosphoribosyl transferase (yfcu) was used as negative selectable marker. (B) Generation of transgenic P. berghei stably expressing wild-type PvDHFR-TS. The endogenous Pbdhfr-ts locus was targeted with a linearized plasmid containing expressing cassettes of wild-type Pvdhfr-ts, and fusion gene of human dhfr (hdhfr) and yeast yfcu as positive and negative selectable markers respectively. After double homologous recombination, the drug selectable markers are excised by single homologous recombination via the Pbdhfr-ts 3'UTR repeated sequence elements (blue boxes), whilst retaining the wild-type Pvdhfr-ts gene. (C), Generation of transgenic P. berghei stably expressing mutant PvDHFR-TS SP21. The endogenous Pbdhfr-ts locus was targeted with a linearized plasmid containing expressing cassettes of mutant Pvdhfr-ts sp21 (Pvsp21) flanked with 5' and 3' UTR of Pbdhfr-ts which also serve as the sites for double homologous recombination. Specific probes for Southern analysis are located by bold horizontal line. E: EcoRI, H: HindIII, K: KpnI.

Mentions: Wild-type and mutant P. vivax dhfr-ts genes were kindly given by Ubolsree Leartsakulpanich, BIOTEC, Thailand [11]. For generation of transgenic P. falciparum parasite stably expressing PvDHFR-TS, a transfection plasmid was constructed containing three expression cassettes: 1) blasticidin-S deaminase (bsd) as a positive selection marker under the control of 5' flanking region of P. falciparum camodulin gene (Pfcam) and 3' UTR of P. falciparum histidine rich protein 2 (Pfhrp2) [26], 2) A fusion gene of cytosine deaminase and uracil phosphoribosyl transferase (yfcu) from Saccharomyces cerevisiae as a negative selection marker under the control of 5' flanking region of P. falciparum heat shock protein 86 gene (Pfhsp86) and 3' UTR of P. berghei dihydrofolate reductase (Pbdhfr) [27], and 3) The wild-type dhfr-ts gene of P. vivax under the control of 5' flanking region of Pfdhfr and 3' UTR of Pfhrp2 (Figure 1A). The 5' flanking region and truncated 5' coding sequence of Pfdhfr-ts are the sites for homologous recombination and replacement of Pfdhfr-ts with Pvdhfr-ts gene in the P. falciparum parasite chromosome 4.


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)

Diagrammatic representation of strategy to generate transgenic parasites by homologous recombination of Pvdhfr-ts gene replacing endogenous dhfr-ts locus. (A) Generation of transgenic P. falciparum stably expressing wild-type PvDHFR-TS. The endogenous Pfdhfr-ts locus was targeted via double homologous recombination with a plasmid containing expressing cassettes of wild-type Pvdhfr-ts, and blasticidin-S deaminase (bsd) marker. Yeast cytosine deaminase-uracil phosphoribosyl transferase (yfcu) was used as negative selectable marker. (B) Generation of transgenic P. berghei stably expressing wild-type PvDHFR-TS. The endogenous Pbdhfr-ts locus was targeted with a linearized plasmid containing expressing cassettes of wild-type Pvdhfr-ts, and fusion gene of human dhfr (hdhfr) and yeast yfcu as positive and negative selectable markers respectively. After double homologous recombination, the drug selectable markers are excised by single homologous recombination via the Pbdhfr-ts 3'UTR repeated sequence elements (blue boxes), whilst retaining the wild-type Pvdhfr-ts gene. (C), Generation of transgenic P. berghei stably expressing mutant PvDHFR-TS SP21. The endogenous Pbdhfr-ts locus was targeted with a linearized plasmid containing expressing cassettes of mutant Pvdhfr-ts sp21 (Pvsp21) flanked with 5' and 3' UTR of Pbdhfr-ts which also serve as the sites for double homologous recombination. Specific probes for Southern analysis are located by bold horizontal line. E: EcoRI, H: HindIII, K: KpnI.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Diagrammatic representation of strategy to generate transgenic parasites by homologous recombination of Pvdhfr-ts gene replacing endogenous dhfr-ts locus. (A) Generation of transgenic P. falciparum stably expressing wild-type PvDHFR-TS. The endogenous Pfdhfr-ts locus was targeted via double homologous recombination with a plasmid containing expressing cassettes of wild-type Pvdhfr-ts, and blasticidin-S deaminase (bsd) marker. Yeast cytosine deaminase-uracil phosphoribosyl transferase (yfcu) was used as negative selectable marker. (B) Generation of transgenic P. berghei stably expressing wild-type PvDHFR-TS. The endogenous Pbdhfr-ts locus was targeted with a linearized plasmid containing expressing cassettes of wild-type Pvdhfr-ts, and fusion gene of human dhfr (hdhfr) and yeast yfcu as positive and negative selectable markers respectively. After double homologous recombination, the drug selectable markers are excised by single homologous recombination via the Pbdhfr-ts 3'UTR repeated sequence elements (blue boxes), whilst retaining the wild-type Pvdhfr-ts gene. (C), Generation of transgenic P. berghei stably expressing mutant PvDHFR-TS SP21. The endogenous Pbdhfr-ts locus was targeted with a linearized plasmid containing expressing cassettes of mutant Pvdhfr-ts sp21 (Pvsp21) flanked with 5' and 3' UTR of Pbdhfr-ts which also serve as the sites for double homologous recombination. Specific probes for Southern analysis are located by bold horizontal line. E: EcoRI, H: HindIII, K: KpnI.
Mentions: Wild-type and mutant P. vivax dhfr-ts genes were kindly given by Ubolsree Leartsakulpanich, BIOTEC, Thailand [11]. For generation of transgenic P. falciparum parasite stably expressing PvDHFR-TS, a transfection plasmid was constructed containing three expression cassettes: 1) blasticidin-S deaminase (bsd) as a positive selection marker under the control of 5' flanking region of P. falciparum camodulin gene (Pfcam) and 3' UTR of P. falciparum histidine rich protein 2 (Pfhrp2) [26], 2) A fusion gene of cytosine deaminase and uracil phosphoribosyl transferase (yfcu) from Saccharomyces cerevisiae as a negative selection marker under the control of 5' flanking region of P. falciparum heat shock protein 86 gene (Pfhsp86) and 3' UTR of P. berghei dihydrofolate reductase (Pbdhfr) [27], and 3) The wild-type dhfr-ts gene of P. vivax under the control of 5' flanking region of Pfdhfr and 3' UTR of Pfhrp2 (Figure 1A). The 5' flanking region and truncated 5' coding sequence of Pfdhfr-ts are the sites for homologous recombination and replacement of Pfdhfr-ts with Pvdhfr-ts gene in the P. falciparum parasite chromosome 4.

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