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

Braks, Joanna A. M.; Franke-Fayard, Blandine; Kroeze, Hans; Janse, Chris J.; Waters, Andrew P.

doi:10.1093/nar/gnj033

Cited by 74 publications

(102 citation statements)

References 29 publications

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“…FCU1 has been recently used as a negative selectable marker in Plasmodium, another protozoan parasite. Plasmodium berghei cells expressing FCU1 were found to be almost 1000 fold more sensitive to the prodrug in vitro and in vivo treatment with 5-FC killed over 99.9% of the infecting recombinant parasites in the erythrocytic-stage mouse model of malaria 11 . E. histolytica cells expressing FCU1 showed only a 30-fold increased sensitivity to the prodrug 5-FC in contrast to the sensitivity observed in P. berghei.…”

Section: Discussionmentioning

confidence: 99%

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Development of a Negative Selectable Marker for <em>Entamoeba histolytica</em>

Abhyankar

Haviland

Gilchrist

et al. 2010

JoVE

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Entamoeba histolytica is the causative agent of amebiasis and infects up to 10% of the world's population. The molecular techniques that have enabled the up-and down-regulation of gene expression rely on the transfection of stably maintained plasmids. While these have increased our understanding of Entamoeba virulence factors, the capacity to integrate exogenous DNA into genome, which would allow reverse genetics experiments, would be a significant advantage in the study of this parasite. The challenges presented by this organism include inability to select for homologous recombination events and difficulty to cure episomal plasmid DNA from transfected trophozoites. The later results in a high background of exogenous DNA, a major problem in the identification of trophozoites in which a bona fide genomic integration event has occurred. We report the development of a negative selection system based upon transgenic expression of a yeast cytosine deaminase and uracil phosphoribosyl transferase chimera (FCU1) and selection with prodrug 5-fluorocytosine (5-FC). The FCU1 enzyme converts non-toxic 5-FC into toxic 5-fluorouracil and 5-fluorouridine-5'-monophosphate. E. histolytica lines expressing FCU1 were found to be 30 fold more sensitive to the prodrug compared to the control strain. Protocol FCU Negative Selection System in Entamoeba histolytica Determination of Selection Efficiency Representative ResultsThe E. histolytica transfectants showed robust expression of codon optimized recombinant FCU1 (Figure 1).When this protocol is followed correctly it results in selective elimination of E. histolytica cells expressing FCU1 in presence of 0.5 mM 5-fluorocytosine. Control cells, in contrast continue to grow normally at up to 5 mM concentration of 5-FC and reach confluence between 72 and 96 hrs (Figure 2-a). The FCU1 carrying cells at 48 hrs are completely lysed when the treated wells are viewed under a microscope. These also show decreased fluorescence when stained with the vital dye CMFDA, which is used in quantitative studies involving large number of samples ( figure 2-b). The FCU1/5-FC system is an effective and powerful negative selection tool for E. histolytica trophozoites.

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Section: Discussionmentioning

confidence: 99%

“…In both P. berghei and P. falciparum the FCU1 marker has been used in targeted gene disruption. studies 11,12 . We aim to manipulate the Entamoeba genome using homologous recombination.…”

Section: Discussionmentioning

confidence: 99%

Development of a Negative Selectable Marker for <em>Entamoeba histolytica</em>

Abhyankar

Haviland

Gilchrist

et al. 2010

JoVE

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“…hDHFR has become more commonly used is its relative small size. This facilitates the generation of more complex 44,52,53 or PCR-based 54 transfection vectors.…”

Section: Selectionmentioning

confidence: 99%

“…yFcu metabolises 5-fluorocytosine (5-FC) into a toxic metabolite. 53 Thus, this selection cassette first allows the positive selection of successfully transfected parasites by pyrimethamine or WR99120 selection and may next be completely removed again following selection with 5-FC. This loss occurs by means of homologous recombination, using duplicated sequences flanking the selectable marker (Fig.…”

Section: Matz and Kooij Experimental Genetics In Plasmodium Bergheimentioning

confidence: 99%

Towards genome-wide experimental genetics in thein vivomalaria model parasitePlasmodium berghei

Matz

Kooij

2015

Pathogens and Global Health

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Plasmodium berghei was identified as a parasite of thicket rats (Grammomys dolichurus) and Anopheles dureni mosquitoes in African highland forests. Successful adaptation to a range of rodent and mosquito species established P. berghei as a malaria model parasite. The introduction of stable transfection technology, permitted classical reverse genetics strategies and thus systematic functional profiling of the gene repertoire. In the past 10 years following the publication of the P. berghei genome sequence, many new tools for experimental genetics approaches have been developed and existing ones have been improved. The infection of mice is the principal limitation towards a genome-wide repository of mutant parasite lines. In the past few years, there have been some promising and most welcome developments that allow rapid selection and isolation of recombinant parasites while simultaneously minimising animal usage. Here, we provide an overview of all the currently available tools and methods.

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“…This enzyme also converts 5-fluorocytosine (5FC) into 5-fluorouracil (5FU), a highly toxic compound. This suicide effect has been exploited in negative selection procedures for eucaryotic organisms (2,12,20,26).…”

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confidence: 99%