A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle

Franke-Fayard, Blandine; Trueman, Holly E.; Ramesar, Jai; Mendoza, Jacqui; Keur, Maarten van der; Linden, Reinier van der; Sinden, Robert E.; Waters, Andrew P.; Janse, Chris J.

doi:10.1016/j.molbiopara.2004.04.007

Cited by 443 publications

(429 citation statements)

References 29 publications

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“…P. berghei NK65 clone, expressing GFP under the control of the csp promoter (stage-specific fluorescence at the oocyst, sporozoite and early liver stages) 6 . P. berghei ANKA clone, PbGFP CON , expressing GFP under the control of the ef-1a promoter (the parasite is fluorescent at all stages) 7 . Mice: 4-7-week-old hairless SKH1 females (Charles River Laboratories) !…”

Section: Reagentsmentioning

confidence: 99%

“…The rodent systems offer an additional key advantage, the possibility of studying parasites in as near as possible to their natural environment. A number of wild-type fluorescent sporozoites expressing a fluorescent marker (GFP or RFP) through a variety of stage-specific or constitutive promoters are now available in both P. berghei 6,7 and P. yoelii 8 , which can now be imaged in the dermis or liver of rodent hosts [9][10][11][12] .…”

Section: Introductionmentioning

confidence: 99%

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Imaging malaria sporozoites in the dermis of the mammalian host

et al. 2007

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The initial phase of malaria infection is the pre-erythrocytic phase, which begins when parasites are injected by the mosquito into the dermis and ends when parasites are released from hepatocytes into the blood. We present here a protocol for the in vivo imaging of GFP-expressing sporozoites in the dermis of rodents, using the combination of a high-speed spinning-disk confocal microscope and a high-speed charge-coupled device (CCD) camera permitting rapid in vivo acquisitions. The steps of this protocol indicate how to infect mice through the bite of infected Anopheles stephensi mosquitoes, record the sporozoites' fate in the mouse ear and to present the data as maximum-fluorescence-intensity projections, time-lapse representations and movie clips. This protocol permits investigating the various aspects of sporozoite behavior in a quantitative manner, such as motility in the matrix, cell traversal, crossing the endothelial barrier of both blood and lymphatic vessels and intravascular gliding. Applied to genetically modified parasites and/or mice, these imaging techniques should be useful for studying the cellular and molecular bases of Plasmodium sporozoite infection in vivo. INTRODUCTIONThe notion that Anopheles mosquitoes inject Plasmodium sporozoites into the dermis of the host, rather than directly into the blood circulation, was first suggested in the 1930s 1 and has since received experimental confirmation 2-4 . Sporozoites are ejected when the mosquito salivates 5 , especially when it probes the dermis, searching for a blood source. However, given the small number of sporozoites injected through its bite and the highly motile behavior of these sporozoites, the dermal phase of sporozoite infection is still poorly characterized. Only now, with the development of tools for studying the fast dynamics of sporozoites in real time, can their exact in vivo fate be analyzed.A fundamental feature of the malarial sporozoite is its motility and migratory behavior. Like other apicomplexan parasites, this needle-shaped cell (10 mm in length, 1 mm in width) moves by gliding over a substrate at very high speeds (up to 4 mm s À1 ). The sporozoite and its gliding properties have mainly been studied in species of Plasmodium that infect rodents (P. berghei and P. yoelii), which offer, over the human-infecting parasite species, the advantages of safety in handling the parasites and ease in manipulating their genome. The rodent systems offer an additional key advantage, the possibility of studying parasites in as near as possible to their natural environment. A number of wild-type fluorescent sporozoites expressing a fluorescent marker (GFP or RFP) through a variety of stage-specific or constitutive promoters are now available in both P. berghei 6,7 and P. yoelii 8 , which can now be imaged in the dermis or liver of rodent hosts 9-12 .Imaging techniques have been revolutionized by advancements in both microscope instrumentation and data collection/processing software. Although one-photon methods are more limited than two-phot...

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Section: Reagentsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Imaging malaria sporozoites in the dermis of the mammalian host

et al. 2007

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“…To better understand the molecular events involved in this complex journey within the invertebrate and vertebrate hosts transgenic parasites that express GFP or a red fluorescent protein, RedStar, under the control of the sporozoite-specific cir-cumsporozoite protein (CSP) or the constitutive elongation factor 1α (EF1α) promoter have been generated [14][15][16]. These transgenic parasites were a prerequisite for the application of intravital microscopy to describe the Plasmodium life cycle in whole animals [9,11,12,16].…”

Section: Introductionmentioning

confidence: 99%

Transgenic Plasmodium berghei sporozoites expressing β-galactosidase for quantification of sporozoite transmission

Engelmann¹,

Sinnis²,

Matuschewski³

2006

Molecular and Biochemical Parasitology

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Malaria transmission occurs during a blood-meal of an infected Anopheles mosquito. Visualization and quantification of sporozoites along the journey from the mosquito midgut, where they develop, to the vertebrate liver, their final target organ, is important for understanding many aspects of sporozoite biology. Here we describe the generation of Plasmodium berghei parasites that express the reporter gene lacZ as a stable transgene, under the control of the sporozoite-specific CSP promoter. Transgenic sporozoites expressing β-galactosidase can be simply visualized and quantified in an enzymatic assay. In addition, these sporozoites can be used to quantify sporozoites deposited in subcutaneous tissue during natural infection.

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“…P. berghei NK65 clone, PbFluspo, expressing GFP under the control of the cs promoter (stage-specific fluorescence at the oocyst, sporozoite and early liver stages) 14 . P. berghei ANKA clone, WT: GFP, expressing GFP under the control of the hsp70 promoter 19 , or P. berghei ANKA clone, PbGFP CON , expressing GFP under the control of the ef-1 a promoter 15 ; the parasites are fluorescent at all stages . Mice: 4-to 7-week-old C57BL/6J@Rj (Elevage Janvier).…”

Section: Reagentsmentioning

confidence: 99%

“…Among these techniques are (i) laser scanning confocal microscopy, which may cause high cell damage in living cells owing to the generation of oxygen radicals and is limited by the slow speed of data acquisition, (ii) multi-photon microscopy 12 , which offers the advantage of deep tissue penetration with minimal cell damage but is also limited by the slow speed of data acquisition, and (iii) highspeed spinning disk confocal microscopy 13 , which causes little cell damage and allows high-speed data acquisition. Combined with the construction of recombinant rodent malaria parasites [14][15][16] that express the GFP at the pre-erythrocytic stages, these developments now permit in vivo analysis of Plasmodium behavior in rodent systems.…”

Section: Introductionmentioning

confidence: 99%

In vivo imaging of malaria parasites in the murine liver

et al. 2007

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The form of the malaria parasite inoculated by the mosquito, called the sporozoite, transforms inside the host liver into thousands of a new form of the parasite, called the merozoite, which infects erythrocytes. We present here a protocol to visualize in vivo the behavior of Plasmodium berghei parasites in the hepatic tissue of the murine host. The use of GFP-expressing parasites and a high-speed spinning disk confocal microscope allows for the acquisition of four-dimensional images, which provide a time lapse view of parasite displacement and development in tissue volumes. These data can be analyzed to give information on the early events of sporozoite penetration of the hepatic tissue, that is, sporozoite gliding in the liver sinusoids, crossing the sinusoidal barrier, gliding in the parenchyma and traversal of hepatocytes, and invasion of a final hepatocyte, as well as the terminal events of merosome and merozoite release from infected hepatocytes. Combined with the use of mice expressing fluorescent cell types or cell markers, the system will provide useful information not only on the primary infection process, but also on parasite interactions with the host immune cells in the liver.

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A Plasmodium berghei reference line that constitutively expresses GFP at a high level throughout the complete life cycle

Cited by 443 publications

References 29 publications

Imaging malaria sporozoites in the dermis of the mammalian host

Imaging malaria sporozoites in the dermis of the mammalian host

Transgenic Plasmodium berghei sporozoites expressing β-galactosidase for quantification of sporozoite transmission

In vivo imaging of malaria parasites in the murine liver

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