Back to search resultsSummaryRMgm-962
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene disruption |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 24244852 |
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 | B. Poulin; R. Tewari |
Name Group/Department | Centre for Genetics and Genomics |
Name Institute | School of Life Sciences, Queens Medical Centre, University of Nottingham |
City | Nottingham |
Country | UK |
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Name of the mutant parasite | |
RMgm number | RMgm-962 |
Principal name | Δisp3 |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not different from wild type |
Fertilization and ookinete | Not different from wild type |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Not different from wild type |
Additional remarks phenotype | Mutant/mutation ISP1-GFP was only detected at low levels by fluorescence microscopy in live asexual blood stages, and by IFA. ISP1-GFP showed faint cytosolic fluorescence in activatedmale and female gametocytes, and in addition there was strong peripheral membrane localisation in activated male gametocytes and a strong peripheral and polarised localisation in activated female gametocytes. In zygotes, this polarised area expanded to a crescent shape and the ISP1-GFP signal was reduced in the parasite cytosol. In contrast, ISP3-GFP was clearly evident in schizonts and merozoites, while in female gametocytes and zygotes it showed a pattern similar to ISP1-GFP with both a diffuse and localised distribution. The localisation of ISP3-GFP in developing schizonts resembles an IMC-like pattern. A striking difference between ISP1-GFP and ISP3-GFP was observed in ookinetes: while ISP1-GFP localisation was strong and largely limited to the apical tip of the ookinete, ISP3-GFP was distributed along the parasite periphery. Similarly, sporozoites isolated from mid-gut oocysts showed strong ISP1-GFP fluorescence at their apical tip, while ISP3-GFP was distributed throughout the cell but concentrated at the periphery of the parasite. Intriguingly, in sporozoites isolated from mosquito salivary glands, the distinctive apical localisation of ISP1-GFP was lost and instead the protein was distributed throughout the cell and concentrated at the periphery of the sporozoite, reminiscent of ISP3-GFP. While ISP1-GFP and ISP3-GFP fluorescence was distributed continuously along the length of extracellular sporozoites, it was disrupted at 5 h post-infection in the central protruding region of transforming intracellular sporozoites. At later stages of liver stage development (24 and 48 h), ISP1-GFP and ISP3-GFP appeared to be restricted to a small area at the parasite periphery.ISP1-GFP and ISP3- GFP expression increased towards the end of liver stage development, where both proteins were localised in internal structures inside the parasite. Liver merozoites released in culture appeared to display a circumferential distribution of ISP1 and ISP3. Evidence is presented that in the absence of ISP3, the apical localisation and polarity of ISP1 (PBANKA_120940) is altered. Evidence is presented that ISP1-GFP and ISP3-GFP are myristoylated proteins and are are phosphorylated in both asexual (schizont) and sexual stages (activated gametocytes)
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1324300 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1460600 | ||||||||||||||||||||||||
Gene product | inner membrane complex sub-compartment protein 3 | ||||||||||||||||||||||||
Gene product: Alternative name | ISP3 | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | Plasmid double 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 | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
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 | |||||||||||||||||||||||||
Primer information: Primers used for amplification of the target sequences
Primer information: Primers used for amplification of the target sequences
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