New selectable markers and single crossover integration for the highly versatile Plasmodium knowlesi transfection system

Wel, Annemarie van der; Kocken, Clemens H. M.; Pronk, Tamarah C; Franke-Fayard, Blandine; Thomas, Alan W.

doi:10.1016/j.molbiopara.2003.10.019

Cited by 11 publications

(12 citation statements)

References 41 publications

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“…The procedure resulted in the isolation of mutants that contain disrupted genes through double crossover recombination events. For both P.falciparum and P.knowlesi it has been demonstrated that mutants expressing hsvTK are effectively killed in vitro by the drug GCV ( 5 , 9 ). The use of b cd in P.falciparum to select for double crossover integration events was assessed and discarded because for unclear reasons selection resulted in parasites that were resistant to 5-FC and had not undergone double crossover integration ( 9 ).…”

Section: Discussionmentioning

confidence: 99%

“…The two rodent malaria parasites cannot be grown in long term in vitro cultures and selection of mutants is therefore carried out in vivo in laboratory rodents, which places constraints on the range of selectable markers that can be used. For example, the combination of puromycin and puromycin acetyl transferase that has been used for in vitro selection of mutants of P.falciparum and P.knowlesi ( 4 , 5 ) cannot be applied in the rodent parasites as the drug is lethal to laboratory animals. Currently only three drug-selectable markers are available for use with rodent malaria parasites.…”

Section: Introductionmentioning

confidence: 99%

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Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium

Braks

Franke-Fayard

Kroeze

et al. 2006

Nucleic Acids Research

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A limitation of transfection of malaria parasites is the availability of only a low number of positive selectable markers for selection of transformed mutants. This is exacerbated for the rodent parasite Plasmodium berghei as selection of mutants is performed in vivo in laboratory rodents. We here report the development and application of a negative selection system based upon transgenic expression of a bifunctional protein (yFCU) combining yeast cytosine deaminase and uridyl phosphoribosyl transferase (UPRT) activity in P.berghei followed by in vivo selection with the prodrug 5-fluorocytosine (5-FC). The combination of yfcu and a positive selectable marker was used to first achieve positive selection of mutant parasites with a disrupted gene in a conventional manner. Thereafter through negative selection using 5-FC, mutants were selected where the disrupted gene had been restored to its original configuration as a result of the excision of the selectable markers from the genome through homologous recombination. This procedure was carried out for a Plasmodium gene (p48/45) encoding a protein involved in fertilization, the function of which had been previously implied through gene disruption alone. Such reversible recombination can therefore be employed for both the rapid analysis of the phenotype by targeted disruption of a gene and further associate phenotype and function by genotype restoration through the use of a single plasmid and a single positive selectable marker. Furthermore the negative selection system may also be adapted to facilitate other procedures such as ‘Hit and Run’ and ‘vector recycling’ which in principle will allow unlimited manipulation of a single parasite clone. This is the first demonstration of the general use of yFCU in combination with a positive selectable marker in reverse genetics approaches and it should be possible to adapt its use to many other biological systems.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium

Braks

Franke-Fayard

Kroeze

et al. 2006

Nucleic Acids Research

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“…Pk H SSUD attB /BSD-GFP (SSUD: small subunit of the D-type ribosomal RNA gene) was generated in our lab as follows. The targeting vector, pDEF-DSSU-hDHFR 63 , was integrated into Pk H (provided by A.W. Thomas; Biomedical Primate Research Center, Rijswijk) to form Pk H SSUD.…”

Section: Methodsmentioning

confidence: 99%

Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite

Dankwa¹,

Lim²,

Bei³

et al. 2016

Nat Commun

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Plasmodium knowlesi is a zoonotic parasite transmitted from macaques causing malaria in humans in Southeast Asia. Plasmodium parasites bind to red blood cell (RBC) surface receptors, many of which are sialylated. While macaques synthesize the sialic acid variant N-glycolylneuraminic acid (Neu5Gc), humans cannot because of a mutation in the enzyme CMAH that converts N-acetylneuraminic acid (Neu5Ac) to Neu5Gc. Here we reconstitute CMAH in human RBCs for the reintroduction of Neu5Gc, which results in enhancement of P. knowlesi invasion. We show that two P. knowlesi invasion ligands, PkDBPβ and PkDBPγ, bind specifically to Neu5Gc-containing receptors. A human-adapted P. knowlesi line invades human RBCs independently of Neu5Gc, with duplication of the sialic acid-independent invasion ligand, PkDBPα and loss of PkDBPγ. Our results suggest that absence of Neu5Gc on human RBCs limits P. knowlesi invasion, but that parasites may evolve to invade human RBCs through the use of sialic acid-independent pathways.

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“…falciparum or P . knowlesi ring-stage cultures as previously described [13,26,27]. Briefly, the concentration range in each of the JC-1 assays was based on the IC 50 values, previously obtained using pLDH and hypoxanthine radioactive testing for each drug on the specific parasite strain.…”

Section: Methodsmentioning

confidence: 99%

A novel live-dead staining methodology to study malaria parasite viability

Pasini

Ierssel

Vial

et al. 2013

Malar J

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BackgroundMalaria is a major health and socio-economical problem in tropical and sub-tropical areas of the world. Several methodologies have been used to assess parasite viability during the adaption of field strains to culture or the assessment of drug potential, but these are in general not able to provide an accurate real-time assessment of whether parasites are alive or dead.MethodsDifferent commercial dyes and kits were assessed for their potential to allow for the real-time detection of whether a blood stage malaria parasite is dead or alive.ResultsHere, a methodology is presented based on the potential-sensitive mitochondrial probe JC-1, which allows for the real-time visualization of live (red staining) and/or dead (absence of red staining) blood stage parasites in vitro and ex vivo. This method is applicable across malaria parasite species and strains and allows to visualize all parasite blood stages including gametocytes. Further, this methodology has been assessed also for use in drug sensitivity testing.ConclusionsThe JC-1 staining approach is a versatile methodology that can be used to assess parasite viability during the adaptation of field samples to culture and during drug treatment. It was found to hold promise in the assessment of drugs expected to lead to delayed death phenotypes and it currently being evaluated as a method for the assessment of parasite viability during the adaptation of patient-derived Plasmodium vivax to long-term in vitro culture.

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New selectable markers and single crossover integration for the highly versatile Plasmodium knowlesi transfection system

Cited by 11 publications

References 41 publications

Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium

Development and application of a positive-negative selectable marker system for use in reverse genetics in Plasmodium

Ancient human sialic acid variant restricts an emerging zoonotic malaria parasite

A novel live-dead staining methodology to study malaria parasite viability

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