RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5129
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0204600; Gene model (P.falciparum): PF3D7_0109000; Gene product: photosensitized INA-labeled protein PHIL1 (PHIL1)
Name tag: GFP
Phenotype Asexual bloodstage; Fertilization and ookinete; Sporozoite;
Last modified: 12 April 2022, 14:24
  *RMgm-5129
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number)
MR4 number 35403820
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherKehrer J, Frischknecht F
Name Group/DepartmentIntegrative Parasitology, Center for Infectious Diseases
Name InstituteHeidelberg University Medical School
CityHeidelberg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-5129
Principal namePhil1-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagelocalization of the GFP-tagged PHIL1 protein at the periphery of merozoites, ookinetes and sporozoites.
Gametocyte/GameteNot tested
Fertilization and ookinetelocalization of the GFP-tagged PHIL1 protein at the periphery of merozoites, ookinetes and sporozoites.
OocystNot tested
Sporozoitelocalization of the GFP-tagged PHIL1 protein at the periphery of merozoites, ookinetes and sporozoites.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of PHIL1

Protein (function)
PhIL has been identified in Toxoplasma gondii as an inner membrane complex (IMC) protein. PhIL1 is localized mainly with IMC and the apical cap of T. gondii tachyzoites, which is an apical structure linked to the IMC membrane that covers the parasite’s apical protrusion.

Phenotype
Localization of the GFP-tagged PHIL1 protein at the periphery of merozoites, ookinetes and sporozoites.

Additional information
Concavin-GFP expressing sporozoites showed a rapid recovery of the fluorescence signal after a bleached spot was introduced by a high energy laser suggesting high lateral diffusion of concavin-GFP. In contrast, in bleached PHIL1-GFP sporozoites there was no detectable recovery as expected from a protein anchored in the sub-pellicular network. These data suggest that concavin-GFP is not associated to the subpellicular network or another stable cytoskeletal structure.
Next, we performed super-resolution (STED) co-localization experiments with antibodies against concavin-GFP, CSP and PHIL1-GFP. STED imaging showed that the signals of anti-GFP antibodies detecting PHIL1-GFP and antibodies against CSP were spatially separated. In contrast, anti-GFP antibodies detecting concavin-GFP co-localized with anti-CSP antibodies. Similarly, antibodies recognizing CSP but stained with two different colours also co-localized. This suggests that concavin-GFP is localized closer to the plasma membrane than PHIL1, probably within the alveolar space between IMC and plasma membrane or at the inner leaflet of the plasma membrane. 

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0204600
Gene Model P. falciparum ortholog PF3D7_0109000
Gene productphotosensitized INA-labeled protein PHIL1
Gene product: Alternative namePHIL1
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPhIL1-GFP parasites with GFP at the C-terminus were generated via single homologous recombination. A region of the PhIL1 gene 54 bp downstream of the ATG start codon and lacking the stop codon was amplified using primers P969 and P970. The PCR product was digested using EcoR1 and BamH1 enzymes and ligated into a vector containing the TgDHFR selection cassette as a positive selection marker. For transfection the final vector was linearized using BsaB1
Additional remarks selection procedure
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6