RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-989
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0943500; Gene model (P.falciparum): PF3D7_1103400; Gene product: FeS cluster assembly protein SufD (SufD)
Name tag: mCherry-3xMyc
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (Dhfs-fpgs)
Replacement locus: Gene model: PBANKA_0943500; Gene product: FeS cluster assembly protein SufD (SufD)
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 27 July 2015, 18:52
  *RMgm-989
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) : 24586983
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/ResearcherHaussig, J.M.; Kooij, T.W.A.
Name Group/DepartmentParasitology Unit
Name InstituteMax Planck Institute for Infection Biology
CityBerlin
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-989
Principal namesufd::tag
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageExpression of the mCherry-3xMyc-tagged version of SuFD(apicoplast location)
Gametocyte/GameteExpression of the mCherry-3xMyc-tagged version of SufD(apicoplast location)
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal  mCherry-3xMyc-tagged version of PBANKA_094350 (iron-sulfur assembly protein, putative; SufD) and GFP under control of the constitutiive hsp70 promoter.

Protein (function)
Iron-sulfur [Fe-S] clusters are small inorganic cofactors that are present in most organisms. Proteins containing [Fe-S] clusters are involved in numerous biological processes, ranging from mitochondrial oxidative phosphorylation and photosynthesis to DNA replication, DNA repair, ribosome biogenesis, and regulation of gene expression. Bacteria harbor the sulfur utilization factor (SUF) and ironsulfur cluster (ISC) systems for assembly of [Fe-S] clusters. In eukaryotes, [Fe-S] biogenesis machineries are thought to have evolved from their bacterial counterparts that had been acquired by endosymbiosis.
In Plasmodium components of the SUF and ISC systems have been identified by bioinformatic analyses. Like in other eukaryotes, the ISC system is predicted to localize to the mitochondrion and the SUF system to the apicoplast.
In Plasmodium, all components of the SUF system are nuclear-encoded, except for SUFB, which is encoded in the small circular apicoplast genome
An systematic experimental genetics analysis of the P. berghei apicoplast SUF system shows that four of five nuclear encoded P. berghei SUF genes (SUFC, D, E, S) are refractory to gene deletion and, hence, can be considered likely essential for blood stage proliferation. Endogenous tagging confirmed accessibility of the loci to targeted gene modification and the resulting fluorescent fusion proteins showed co-localizations with an apicoplast resident protein.
Targeted deletion was successful of SUFA, indicative of a dispensable role during asexual blood stage growth in vivo. Furthermore, no abnormalities were observed during Plasmodium life cycle progression in the mosquito. In the liver a reduced (50%) merosome formation was observed.

Phenotype
Expression of the mCherry-3xMyc-tagged version of SufD (apicoplast location) in asexual blood stages and gametocytes

Additional information

Other mutants
Search for text term iron-sulfur (or SUF) for additional unsuccesfull attempts to dirupt SUF genes and for mutants expressing tagged-versions of SUF genes.


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0943500
Gene Model P. falciparum ortholog PF3D7_1103400
Gene productFeS cluster assembly protein SufD
Gene product: Alternative nameSufD
Details of the genetic modification
Name of the tagmCherry-3xMyc
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/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationAn intermediate pBAT-derived vector was used (see RMgm-757)
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/yfcu
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationAn intermediate pBAT-derived vector was used (see RMgm-757)
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative nameDhfs-fpgs
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_0943500
Gene productFeS cluster assembly protein SufD
Gene product: Alternative nameSufD
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