RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-215
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*RMgm-215| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Gene tagging |
| Reference (PubMed-PMID number) | |
| MR4 number | |
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| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei ANKA |
| Name parent line/clone | P. berghei ANKA 2.34 |
| Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). |
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| The mutant parasite was generated by | |
| Name PI/Researcher | M. Tufet-Bayona, C.J. Janse, R.E. Sinden, B. Franke-Fayard |
| Name Group/Department | Leiden Malaria Research Group |
| Name Institute | Leiden University Medical Center (LUMC) |
| City | Leiden |
| Country | The Netherlands |
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| Name of the mutant parasite | |
| RMgm number | RMgm-215 |
| Principal name | PRP2-GFP |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype | |
| Asexual blood stage | PRP2-GFP is expressed in trophozoites as early as 8 hours after invasion. In these stages a diffuse, weak fluorescence signal is observed in cytoplasmic areas close to the nucleus. In mature trophozoites and immature schizonts, the fluorescence intensity increases progressively, but its distribution in the cytoplasm is unchanged. PRP2-GFP expression is observed in mature schizonts and merozoites. Merozoites showed a 'two-dot' staining pattern which is characteristic for a location in the two rhoptry-organelles. |
| Gametocyte/Gamete | Not different from wild type |
| Fertilization and ookinete | Not different from wild type |
| Oocyst | No PRP2-GFP expression is observed in gametocytes and ookinetes. PRP2-GFP is expressed during early oocyst development (days 3–5), a weak fluorescence pervaded the entire cytoplasm. In maturing oocysts (days 10–12), the intensity of fluorescence increases and is restricted to the forming sporoblasts. In the mature oocysts containing sporozoites, fluorescence is present in all sporozoites as a single intense fluorescent dot posterior to the nuclei. Free sporozoites released from ruptured oocysts revealed a similar pattern of fluorescence. |
| Sporozoite | In the mature oocysts containing sporozoites, fluorescence is present in all sporozoites as a single intense fluorescent dot posterior to the nuclei. Free sporozoites released from ruptured oocysts revealed a similar pattern of fluorescence. Compared to oocyst-derived sporozoites, salivary gland sporozoites show a different distribution, two clear dots are observed at either side of the nucleus. |
| Liver stage | Not tested |
| Additional remarks phenotype | Mutant/mutation Phenotype Additional information The GFP expression pattern of PRP2-GFP in sporozoites is clearly different from the fluorescence pattern of the GFP-tagged rhoptry protein RAP2/3 in sporozoites that shows an apical location (see RMgm-209). The location of PRP2 has only been analysed by fluorescence microscopy of the GFP-tagged PRP2. Determination of the exact location in both blood stages and in sporozoites awaits confirmation by additional analyses, for example immuno-electron microscopy. Evidence has been presented that the P. falciparum ortholog (PF3D7_1320000) is NOT located in rhoptries but is localizes to the Golgi Apparatus (PMID 26375591). Other mutants |
Tagged: Mutant parasite with a tagged gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1418300 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1320000 | ||||||||||||||||||||||||||
| Gene product | golgi protein 1 | rhoptry protein 2, putative | ||||||||||||||||||||||||||
| Gene product: Alternative name | PRP2, putative rhoptry protein 2 | ||||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||||
| Name of the tag | EGFP | ||||||||||||||||||||||||||
| Details of tagging | C-terminal | ||||||||||||||||||||||||||
| Additional remarks: tagging | The chosen integration strategy for the construct results in integration at the target locus by a single cross-over homologous recombination event. | ||||||||||||||||||||||||||
| Commercial source of tag-antibodies | |||||||||||||||||||||||||||
| Type of plasmid/construct | (Linear) plasmid single cross-over | ||||||||||||||||||||||||||
| PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||||
| Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
| Plasmid/construct map | |||||||||||||||||||||||||||
| Plasmid/construct sequence | |||||||||||||||||||||||||||
| Restriction sites to linearize plasmid | PacI | ||||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | tgdhfr | ||||||||||||||||||||||||||
| Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||||
| Additional remarks genetic modification | |||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
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