High efficiency transfection of Plasmodium berghei facilitates novel selection procedures

Janse, Chris J.; Franke-Fayard, Blandine; Mair, Gunnar R.; Ramesar, Jai; Thiel, Corinna; Engelmann, Sabine; Matuschewski, Kai; Gemert, Geert Jan van; Sauerwein, Robert W.; Waters, Andrew P.

doi:10.1016/j.molbiopara.2005.09.007

Cited by 420 publications

(522 citation statements)

References 24 publications

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“…Using established transfection methodologies [24] we introduced a GFP tag into each locus, bodies, similar to that observed for the LCCL protein family in ookinete stages [45]. The LCCL and COWP protein families, along with the CPW-WPC family are conserved among pre-apicomplexan alveolates, and are likely to be markers for an archetypal preapicomplexan/ alveolate structure.…”

Section: Cpw-wpc Proteins Are Composed Of Arrays Of a Domain Conservementioning

confidence: 99%

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Translational repression of the cpw-wpc gene family in the malaria parasite Plasmodium

Rao

Santos

Pain³

et al. 2016

Parasitology International

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Please cite this article as: Rao Pavitra N., Santos Jorge M., Pain Arnab, Templeton Thomas J., Mair Gunnar R., Translational repression of the cpw-wpc gene family in the malaria parasite Plasmodium, Parasitology International (2016), doi: 10.1016/j.parint.2016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Section: Cpw-wpc Proteins Are Composed Of Arrays Of a Domain Conservementioning

confidence: 99%

“…respectively. Prior to transfection pLIS0453 was linearized with EcoRI, and pLIS0454 with BsmI, and transfected into P. berghei ANKA following established methodologies [24] resulting in the following mutant lines: T0453 (pbanka_1352500::gfp) and T0454…”

Section: Transfection Of P Falciparummentioning

confidence: 99%

Translational repression of the cpw-wpc gene family in the malaria parasite Plasmodium

Rao

Santos

Pain³

et al. 2016

Parasitology International

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“…Successful transfection in P. berghei relies on the electroporation of purified mature schizonts, so to mimic similar conditions, highly synchronous P. knowlesi A1-H.1 schizonts were produced by centrifugation on Nycodenz cushions. The Amaxa electroporation system has provided significant improvements in transfection efficiency in P. berghei (24), so PkconGFP ep was introduced into the P. knowlesi schizonts by electroporation with the Amaxa 4D electroporator. Drug selection (2.5 nM WR99210) was applied ∼18 h postelectroporation.…”

Section: High-efficiency Transfection With Episomal Constructs Allowsmentioning

confidence: 99%

“…This integration can currently be achieved with P. falciparum but requires transfection with circular plasmid, which is maintained episomally and can only be removed by either repeated cycles of drug selection or the use of negative selection markers (25). To determine whether the observed high-transfection efficiency facilitated genomic modification of our human-adapted P. knowlesi parasites, we modified PkconGFP ep to incorporate an ∼1.2-kb region of the Pkp230p gene (PlasmoDB ID PKH_041110), which is dispensable in P. berghei and was therefore considered a suitable targeting sequence for homologous integration (24). The resulting construct, called PkconGFP p230p , was linearized using a KpnI site situated within the targeting region to prevent its maintenance as a stable episome and to promote integration through single crossover homologous recombination into the P. knowlesi p230p locus (Fig.…”

Section: Rapid Genomic Integration By Homologous Recombination Of Linearmentioning

confidence: 99%

Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

Moon

Hall

Rangkuti

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

264

393

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Research into the aetiological agent of the most widespread form of severe malaria, Plasmodium falciparum, has benefitted enormously from the ability to culture and genetically manipulate blood-stage forms of the parasite in vitro. However, most malaria outside Africa is caused by a distinct Plasmodium species, Plasmodium vivax, and it has become increasingly apparent that zoonotic infection by the closely related simian parasite Plasmodium knowlesi is a frequent cause of life-threatening malaria in regions of southeast Asia. Neither of these important malarial species can be cultured in human cells in vitro, requiring access to primates with the associated ethical and practical constraints. We report the successful adaptation of P. knowlesi to continuous culture in human erythrocytes. Humanadapted P. knowlesi clones maintain their capacity to replicate in monkey erythrocytes and can be genetically modified with unprecedented efficiency, providing an important and unique model for studying conserved aspects of malarial biology as well as speciesspecific features of an emerging pathogen.invasion | transfection T he development of a continuous culture system for asexual blood stages of the most deadly human malaria parasite, Plasmodium falciparum (1, 2), proved a milestone in malaria research, enabling genetic modification of the parasite (3), high-throughput drug screening (4), and other fundamental advances in parasite biology. Adaptation of other human malaria parasite species to in vitro culture has proved more challenging, and none of the additional four parasite species that cause human malaria can be continuously maintained in human RBC. This difficulty is a significant obstacle to studying these pathogens, which differ from P. falciparum in important aspects of biology and the pathology they cause. Furthermore, although considerable progress has been made in the development of transgenic technologies for Plasmodium, P. falciparum remains poorly amenable to genetic manipulation, with a typical transfection efficiency of only ∼10 −6 (5). Additional in vitro human malaria parasite models that are genetically tractable and that complement the P. falciparum system have tremendous potential.Much of the early work on the mechanics of RBC invasion by the malaria parasite used the simian parasite Plasmodium knowlesi. This species has a 24-h erythrocytic life cycle and large, long-lived invasive merozoites, facilitating the use of electron and video microscopy to dissect the dynamics of erythrocyte invasion (6-8). P. knowlesi can be cultured in vitro in rhesus monkey (Macaca mulata) RBC with rhesus or human serum (9, 10). Importantly, P. knowlesi is amenable to genetic manipulation, with reported transfection efficiencies similar to those achieved with the rodent malaria model Plasmodium berghei and far surpassing those attained in P. falciparum (10, 11). P. knowlesi is phylogenetically closely related to Plasmodium vivax, the most important cause of malaria outside of Africa (12), so its study can provide insights ...

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“…Recently, it has been shown that AMAXA nucleofection technology can be used to successfully transform P. berghei schizonts when using the U33 or V13 programs with human T cell solution [11]. The main advantages of the AMAXA system include the need for less DNA (only 5 g), and higher transfection efficiency (10 −2 to 10 −3 for linear DNA; 10 −3 to 10 −4 for circular DNA) [11].…”

Section: Amaxa Transfection System Can Be Used To Transform P Yoeliimentioning

confidence: 99%

Evaluation of Purine Salvage as a Chemotherapeutic Target in the Plasmodium yoelli Rodent Model

Kim¹

2007

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. (From -To) Because resistance to current antimalarials is widespread, new targets for malaria chemotherapy are needed to protect military personnel stationed in developing countries. Malaria parasites cannot make purines needed for RNA and DNA and must salvage purines from their host. Our preliminary studies reveal purine salvage is unique in malaria parasites. We would like to determine whether the unique activities of the malaria enzymes can be exploited to develop specific treatments for malaria that will be effective but not toxic. While study of drug targets in vivo is critical for all infectious diseases, evaluation in an animal model is especially critical for evaluation of purine salvage as a drug target. We perform our studies in Plasmodium yoelii, a rodent malaria whose genome has been sequenced and for which there are techniques for genetic manipulation. We have refined techniques for transformation of this rodent malaria species and have developed GFP reporter parasite lines. Using this system we have genetically disrupted purine salvage enzyme purine nucleoside phosphorylase (PNP) and are attempting to disrupt adenosine deaminase (ADA). We are testing the effects of malaria-specific PNP inhibitors on malaria infection in mice. These novel drugs will be tested in combination with other antimalarials and will also be evaluated for efficacy against exoerythrocytic malaria forms. We hope these experiments will lead to the development of new effective and nontoxic agents that can protect our military personnel from the lethal effects of malaria infection. REPORT DATE (DD-MM-YYYY) 01-03-2006 REPORT TYPE Annual DATES COVERED SUBJECT TERMS Introduction:Because resistance to current antimalarials is widespread, new targets for malaria chemotherapy are needed to protect military personnel stationed in developing countries. Malaria parasites cannot make purines needed for RNA and DNA and must salvage purines from their host. Our preliminary studies reveal purine salvage is unique in malaria parasites. We would like to determine whether the unique activities of the malaria enzymes can be exploited to develop...

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High efficiency transfection of Plasmodium berghei facilitates novel selection procedures

Cited by 420 publications

References 24 publications

Translational repression of the cpw-wpc gene family in the malaria parasite Plasmodium

Translational repression of the cpw-wpc gene family in the malaria parasite Plasmodium

Adaptation of the genetically tractable malaria pathogen Plasmodium knowlesi to continuous culture in human erythrocytes

Evaluation of Purine Salvage as a Chemotherapeutic Target in the Plasmodium yoelli Rodent Model

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