RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4116
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1404100; Gene model (P.falciparum): PF3D7_1305600; Gene product: site-2 protease S2P, putative (S2P)
Name tag: triple-HA
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Asexual bloodstage; Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 28 January 2017, 21:19
  *RMgm-4116
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28107409
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherKoussis K; Loukeris TG; Siden-Kiamos I
Name Group/DepartmentInstitute of Molecular Biology and Biotechnology
Name InstituteFORTH
CityHeraklion
CountryGreece
Name of the mutant parasite
RMgm numberRMgm-4116
Principal namePbS2P-HA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageLocalisation of PbS2PHA in proximity to the nucleus in schizonts/ merozoites and ookinetes. Double-labelling experiments with the Golgi marker ERD2 in schizonts revealed close association of S2P with ERD2, although there was not complete overlap of the signals.
Gametocyte/GameteNot tested
Fertilization and ookineteLocalisation of PbS2PHA in proximity to the nucleus in schizonts/ merozoites and ookinetes. Double-labelling experiments with the Golgi marker ERD2 in schizonts revealed close association of S2P with ERD2, although there was not complete overlap of the signals.
OocystLocalisation of PbS2PHA in proximity to the nucleus in schizonts/ merozoites and ookinetes. Double-labelling experiments with the Golgi marker ERD2 in schizonts revealed close association of S2P with ERD2, although there was not complete overlap of the signals. A signal was detected in mature oocysts (d12-d14) after mosquito infection; and in mature salivary gland sporozoites. In the latter, the protease was localised close to the nucleus.
SporozoiteLocalisation of PbS2PHA in proximity to the nucleus in schizonts/ merozoites and ookinetes. Double-labelling experiments with the Golgi marker ERD2 in schizonts revealed close association of S2P with ERD2, although there was not complete overlap of the signals. A signal was detected in mature oocysts (d12-d14) after mosquito infection; and in mature salivary gland sporozoites. In the latter, the protease was localised close to the nucleus.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal (triple) HA-tagged version of S2P and expresses GFP under control of the eefia constitutive promoter.

Protein (function)
Plasmodium berghei encodes a single member of the M50 family of proteases (PBANKA_ 1404100), henceforth termed PbS2P.The protein is predicted to have 7 transmembrane domains and a molecular weight of 39 kDa. In silico searches in PlasmoDB (http://plasmoDB.org) and EupathDB (http://eupathdb.org) identified genes encoding M50 metalloproteases in all Plasmodium species and in most organisms of the Apicomplexa phylum, with the notable exceptions of Babesia and Cryptosporidium species. All proteins contain the characteristic HExxH motif and only one (Eimeriatenella) does not contain the NPDG motif. PbS2P has an overall amino acid identity of more than 92% with other rodent malarial parasite S2Ps; a value that drops to 70% identity when compared to human parasite orthologues and to less than 30% in S2Ps of related Apicomplexa, such as Toxoplasma and Neospora, and other eukaryotic organisms or bacteria.

Phenotype
Localisation of PbS2PHA in proximity to the nucleus in schizonts/ merozoites and ookinetes. Double-labelling experiments with the Golgi marker ERD2 in schizonts revealed close association of S2P with ERD2, although there was not complete overlap of the signals. A signal was detected in mature oocysts (d12-d14) after mosquito infection;  and in mature salivary gland sporozoites. In the latter, the protease was localised close to the nucleus.

Analyses of a mutant lacking S2P (RMgm-1133) showed the following:

Reduced growth of asexual blood stages. Normal numbers of salivary gland sporozoites. Reduced infectivity of sporozoites to mice. Evidence is presented for normal liver infection but affected late liver development. Mutant liver stages in culture showed a small, albeit significant, size reduction at 48h but the number of EEFs was very similar to wild type cultures. Mutant infected hepatoma cells produced two-fold fewer merosomes as compared to wild type.

Additional information

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1404100
Gene Model P. falciparum ortholog PF3D7_1305600
Gene productsite-2 protease S2P, putative
Gene product: Alternative nameS2P
Details of the genetic modification
Name of the tagtriple-HA
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vector-
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) PCR construct double 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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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