RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4543
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1315700; Gene model (P.falciparum): PF3D7_1452000; Gene product: rhoptry neck protein 2 (RON2)
Name tag: c-myc
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; Sporozoite;
Last modified: 17 October 2018, 17:40
  *RMgm-4543
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) : 30307699
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 line
The mutant parasite was generated by
Name PI/ResearcherIshino T, Torii M
Name Group/DepartmentDivision of Molecular Parasitology
Name InstituteProteo-Science Center, Ehime University
CityToon, Ehime
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-4543
Principal nameRON2-c-Myc
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageRON2-c-Myc expression in blood stages.
Evidence (by immuno-transmission electron microscopy) is presented for localisation of RON2 in the rhoptry neck of rhoptries of merozoites while in rhoptries of sporozoites RON2 is distributed throughout the rhoptry body.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteRON2-c-Myc expression in sporozoites
Evidence (by immuno-transmission electron microscopy) is presented for localisation of RON2 in the rhoptry neck of rhoptries of merozoites while in rhoptries of sporozoites RON2 is distributed throughout the rhoptry body.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal cmyc-tagged version of RON2.

Protein (function)
RON2 is a rhoptry neck protein expressed in rhoptries of merozoites and ookinetes. Unsuccessful attempts to knock-out the ron2 gene (RMgm-214) indicates an essential function in blood stages. A number of rhoptry neck proteins (RONs), including RON2, RON4, and RON5 are secreted and inserted into the target cellular membrane as a complex. RON2 interacts with the micronemal protein AMA1.

Phenotype
RON2-c-Myc expression in blood stages and sporozoites
Evidence (by immuno-transmission electron microscopy) is presented for localisation of RON2 in the rhoptry neck of rhoptries of merozoites while in rhoptries of sporozoites RON2 is distributed throughout the rhoptry body.

Additional information

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1315700
Gene Model P. falciparum ortholog PF3D7_1452000
Gene productrhoptry neck protein 2
Gene product: Alternative nameRON2
Details of the genetic modification
Name of the tagc-myc
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 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 modificationTo generate RON2-c-Myc expressing transgenic parasites, the native ron2 coding region in GFP parasites was replaced by single crossover homologous recombination with an expression cassette of RON2 fused with a c-Myc tag at the C-terminus. A Toxoplasma dihydrofolate reductase (DHFR) expressing cassette (TgDHFR) was also integrated to confer pyrimethamine drug resistance in transgenic parasites. The transgenic DNA vector was a modification of the pL0033 plasmid, obtained from BEI Resource. The RON2 partial coding region (5009-6244 nt from the first methionine) was amplified using Pb genome DNA as a template and specific primers (Table S1), then cloned into pL0033 vector using SacII and NcoI restriction sites.
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) plasmid 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