Expansion of experimental genetics approaches for Plasmodium berghei with versatile transfection vectors

Kooij, Taco W. A.; Rauch, Manuel; Matuschewski, Kai

doi:10.1016/j.molbiopara.2012.06.001

Cited by 74 publications

(99 citation statements)

References 18 publications

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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%

“…A novel approach uses the significantly stronger HSP70 promoter, resulting in cytosolic fluorescence levels that are an order of magnitude higher, thus resulting in an almost absolute separation of wild-type and fluorescent mutant parasites. 44,63 This method has been shown to be efficient even in the presence of a 100-fold excess of wild-type parasites, provided that the parasitaemia of the donor mice does not exceed 1%. At higher parasitaemias, sorting efficiencies decline because of the presence of double-infected erythrocytes harbouring a transfected and a wild-type parasite.…”

Section: Isolationmentioning

confidence: 99%

“…The high levels of fluorescence also facilitate imaging of live and fixed parasites in all life cycle stages, even of the extremely thin and highly motile male microgametes. 44 Most importantly though, the flow-cytometric isolation of mutant parasite lines leads to an 80-90% reduction in the use of experimental animals.…”

Section: Isolationmentioning

confidence: 99%

“…32,62 Despite being relatively inefficient compared to traditional methods using drug selection, this method was employed to generate the widely used reference strain GFP CON . Although largely untested, one might speculate that the improved isolation tools and methods 44,63 might facilitate a more reliable use of flow cytometrybased isolation in the absence of drug pressure. Such an approach could even be expanded to multiple manipulation rounds by using multiple fluorescent markers with different colours.…”

Section: Sequential Genetic Modificationmentioning

confidence: 99%

“…Recently, we have introduced the Berghei Adaptable Transfection (pBAT) plasmid system that allows the generation of deletion and tagging constructs from the same intermediate construct. 44 These plasmids have been optimised for size and ease of cloning and combine (i) a recyclable, drugselectable cassette and (ii) a bright fluorescence cassette flanked by multiple cloning sites and transgene targetting sequences. A variety of plasmids are available that include amino-or carboxy-terminal tagging sequences.…”

Section: Visualizationmentioning

confidence: 99%

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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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“…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%

Section: Isolationmentioning

confidence: 99%

Section: Isolationmentioning

confidence: 99%

Section: Sequential Genetic Modificationmentioning

confidence: 99%

Section: Visualizationmentioning

confidence: 99%

See 3 more Smart Citations

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

Matz

Kooij

2015

Pathogens and Global Health

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Plasmodium berghei Gamete Egress Protein is required for fertility of both genders

et al. 2020

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Male and female Plasmodium gametocytes ingested by the Anopheles mosquitoes during a blood meal egress from the red blood cells by rupturing the two surrounding membranes, the parasitophorous vacuole and the red blood cell membranes. Proteins of the so‐called osmiophilic bodies, (OBs), secretory organelles resident in the cytoplasm, are important players in this process. Once gametes emerge, the female is ready to be fertilized while the male develops into motile flagellar gametes. Here, we describe the function(s) of PBANKA_1115200, which we named Gamete Egress Protein (GEP), a protein specific to malaria parasites. GEP is restricted to gametocytes, expressed in gametocytes of both genders and partly localizes to the OBs. A mutant lacking the protein shows aberrant rupture of the two surrounding membranes, while OBs discharge is delayed but not aborted. Moreover, we identified a second function of GEP during exflagellation since the axonemes of the male flagellar gametes were not motile. Genetic crossing experiments reveal that both genders are unable to establish infections in mosquitoes and thus the lack of GEP leads to a complete block in Plasmodium transmission from mice to mosquitoes. The combination of our results reveals essential and pleiotropic functions of GEP in Plasmodium gametogenesis.

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Generation of Transgenic Rodent Malaria Parasites Expressing Human Malaria Parasite Proteins

Salman

Mogollon

Lin

et al. 2015

Methods in Molecular Biology

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We describe methods for the rapid generation of transgenic rodent Plasmodium berghei (Pb) parasites that express human malaria parasite (HMP) proteins, using the recently developed GIMO-based transfection methodology. Three different genetic modifications are described resulting in three types of transgenic parasites. (1) Additional Gene (AG) mutants. In these mutants the HMP gene is introduced as an "additional gene" into a silent/neutral locus of the Pb genome under the control of either a constitutive or stage-specific Pb promoter. This method uses the GIMO-transfection protocol and AG mutants are generated by replacing the positive-negative selection marker (SM) hdhfr::yfcu cassette in a neutral locus of a standard GIMO mother line with the HMP gene expression cassette, resulting in SM free transgenic parasites. (2) Double-step Replacement (DsR) mutants. In these mutants the coding sequence (CDS) of the Pb gene is replaced with the CDS of the HMP ortholog in a two-step GIMO-transfection procedure. This process involves first the replacement of the Pb CDS with the hdhfr::yfcu SM, followed by insertion of the HMP ortholog at the same locus thereby replacing hdhfr::yfcu with the HMP CDS. These steps use the GIMO-transfection protocol, which exploits both positive selection for Pb orthologous gene-deletion and negative selection for HMP gene-insertion, resulting in SM free transgenic parasites. (3) Double-step Insertion (DsI) mutants. When a Pb gene is essential for blood stage development the DsR strategy is not possible. In these mutants the HMP expression cassette is first introduced into the neutral locus in a standard GIMO mother line as described for AG mutants but under the control elements of the Pb orthologous gene; subsequently, the Pb ortholog CDS is targeted for deletion through replacement of the Pb CDS with the hdhfr::yfcu SM, resulting in transgenic parasites with a new GIMO locus permissive for additional gene-insertion modifications.The different types of transgenic parasites can be exploited to examine interactions of drugs/inhibitors or immune factors with HMP molecules in vivo. Mice either immunized with HMP-vaccines or treated with specific drugs can be infected/challenged with these transgenic mutants to evaluate drug or vaccine efficacy in vivo.

show abstract

Expansion of experimental genetics approaches for Plasmodium berghei with versatile transfection vectors

Cited by 74 publications

References 18 publications

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

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

Plasmodium berghei Gamete Egress Protein is required for fertility of both genders

Generation of Transgenic Rodent Malaria Parasites Expressing Human Malaria Parasite Proteins

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