RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4962
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0715500; Gene model (P.falciparum): PF3D7_0413500; Gene product: phosphoglucomutase-2 (PGM2)
Name tag: triple-HA
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0914400; Gene model (P.falciparum): PF3D7_0831700; Gene product: heat shock protein 70 (HSP70x, HSP70-3, UIS24)
3'UTR: Gene model: PBANKA_0914400; Gene product: heat shock protein 70 (HSP70x, HSP70-3, UIS24)
Insertion locus: Gene model: PBANKA_0715500; Gene product: phosphoglucomutase-2 (PGM2)
Phenotype Asexual bloodstage;
Last modified: 17 February 2021, 13:24
  *RMgm-4962
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) Not published (yet)
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 cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
The mutant parasite was generated by
Name PI/ResearcherEsveld SL, Kooij TWA, Huynen MA
Name Group/DepartmentCenter for Molecular and Biomolecular Informatics, Radboud Institute for Molecular Life Sciences
Name InstituteRadboudumc
CityNijmegen
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-4962
Principal name3xHA-PBANKA_0715500
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageEvidence for mitochondrial location of the tagged protein
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a 3xHA-tagged PBANKA_0715500 and expresses the mitochondrial marker HSP70-3.
Tagging of target protein with a 3xHA tag (or a combined fluorescent (mOrange) and epitope (3xHA) tag) and simultaneously introducing a mitochondrial targeted GFP marker by fusion of the promoter and N-terminus of HSP70-3 (PBANKA_0914400).
 
Published in: bioRxiv preprint doi: https://doi.org/10.1101/2021.01.22.427784;

Protein (function)
To obtain a comprehensive, prioritized list of Plasmodium falciparum mitochondrial proteins, we rigorously analyzed and integrated eight datasets using Bayesian statistics into a predictive score per protein for mitochondrial localization. At a corrected false discovery rate of 25%, we identified 295 proteins with a sensitivity of 65% and a specificity of 98%. They include proteins that have not been identified as mitochondrial in other eukaryotes but have characterized homologs in bacteria that are involved in metabolism or translation. 


Phenotype
Mitochondrial localization of seven out of 13 Plasmodium berghei orthologs (see below) was confirmed by epitope labeling and co-localization with a mitochondrial marker protein.
 
PBANKA_0715500 3xHA tag: mitochondrial
PBANKA_0109600 3xHA tag: mitochondrial
PBANKA_1024800 3xHA tag: mitochondrial
PBANKA_0310100 mOrange-3xHA tag: mitochondrial (3xHA tag; fixed blood stages)
PBANKA_0708900 3xHA tag: mitochondrial
PBANKA_1459800 3xHA tag: no subcellular location could be assigned
PBANKA_0814400 3xHA tag: no subcellular location could be assigned
PBANKA_1203200 3xHA-tagging: mitochondrial (3xHA tag; fixed blood stages)
PBANKA_0827900 3xHA tag: mitochondrial
PBANKA_1103500 3xHA tag: possible mitochondrial
PBANKA_0927100 3xHA tag: no subcellular location could be assigned
PBANKA_0914000 3xHA tag: possible mitochondrial
PBANKA_1350000 3xHA tag: possible mitochondrial 

Additional information
No mitochondrial location of mOrange-tagged proteins could be detected in live parasites using mOrange fluorescence microscopy. Therefore, proteins were tagged with a 3xHA tag only

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0715500
Gene Model P. falciparum ortholog PF3D7_0413500
Gene productphosphoglucomutase-2
Gene product: Alternative namePGM2
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 usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTagging of target protein with a 3xHA tag (or combined fluorescent (mOrange) and epitope (3xHA) tag) and simultaneously introducing a mitochondrial targeted GFP marker by fusion of the promoter and N-terminus of HSP70-3 (PBANKA_0914400).
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
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 parasitehdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0914400
Gene Model P. falciparum ortholog PF3D7_0831700
Gene productheat shock protein 70
Gene product: Alternative nameHSP70x, HSP70-3, UIS24
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_0914400
Gene productheat shock protein 70
Gene product: Alternative nameHSP70x, HSP70-3, UIS24
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 / InsertionInsertion locus
Gene Model of Parasite PBANKA_0715500
Gene productphosphoglucomutase-2
Gene product: Alternative namePGM2
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