RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1259
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0941300; Gene model (P.falciparum): PF3D7_1105600; Gene product: translocon component PTEX88
TaggedGene model (rodent): PBANKA_1145400; Gene model (P.falciparum): Not available; Gene product: Plasmodium exported protein, unknown function (PHIST)
Name tag: mCherry
Phenotype Asexual bloodstage;
Last modified: 30 June 2016, 13:09
  *RMgm-1259
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 25820521
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/ResearcherMatz JM; kooij TW
Name Group/DepartmentParasitology Unit
Name InstituteMax Planck Institute for Infection Biology
CityBerlin
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-1259
Principal namePBANKA_114540-mCherry X ptex88–
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageThe m-Cherry-tagged protein is exported to red blood cell cytoplasm
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expressses a C-terminal mCherry-tagged version of PBANKA_114540 and lacks expression of PTEX88, a component of the putative export machinery termed PTEX (Plasmodium Translocon of EXported protein). In addition it expresses GFP under the control of the HSP70 promoter (transgene introduced in the the PTEX88 locus.

This mutant was generated by (mosquito) crossing the mutant expressing the mCherry-tagged mutant with the mutant lacking expression of PTEX88 and expressing GFP (RMgm-942)

Protein (function)

Phenotype
The m-Cherry-tagged protein is exported to red blood cell cytoplasm

Additional information
The protein was exported into the red blood cell in the absence of PTEX88 (PBANKA_094130), a component of the putative export machinery termed PTEX (Plasmodium Translocon of EXported protein).

Fom Moreira et al 2016 Plos One:
In the P. berghei genome sequence  3 phist genes (namely, PBANKA_1145400, PBANKA_1229000 and PBANKA_0700800) were identified via BLAST analysis using PHIST domains and pssm profiles as queries. This result differs from the single P. berghei phist gene described in the original annotation. Orthologs of PBANKA_1145400 and PBANKA_1229000 were identified in the genome nucleotide sequence databases for other rodent malaria parasites; namely, P. yoelii (PY00289 and PY01786, respectively) and P. chabaudi (PCAS_1144900 and PCAS_1229700, respectively). Additional phist genes were not identified in rodent malaria parasites. BLAST analyses using the PHIST domain as queries, identify as reciprocal best hits PBANKA_1229000, the P. vivax PHIST protein PvPHIST/CVC-8195 (PVX_093680), and P. falciparum PF3D7_0801000. This indicates possible orthologous (vertically inherited) relationships and is supported by the observed synteny of adjacent genes, including the ookinete-expressed gene warp, together composing a locus which is conserved across the Plasmodium genus. PBANKA_1229000 is internally localized in the P. berghei genome, but the locus appears to be sub-telomeric in species other than rodent malaria parasites.
The P. berghei PHIST proteins PBANKA_1145400 and PBANKA_1229000 possess similar features to P. falciparum PHIST proteins; namely, signal peptides and PEXEL/HT trafficking motifs; and single PHIST domains, which are divergent in aa sequence with respect to each other. Within the single predicted ORF of PBANKA_0700800 we were unable to identify a signal peptide sequence, and there were no attractive upstream ORFs suggestive of an erroneous gene model. We thus propose that PBANKA_0700800 is a pseudogene. The remaining P. berghei phist genes possess the typical 2-exon gene structure, in which the signal peptide is encoded on the first exon and the PEXEL/HT motif is located within the second exon.

Other mutants
See link

See PHIST


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0941300
Gene Model P. falciparum ortholog PF3D7_1105600
Gene producttranslocon component PTEX88
Gene product: Alternative name
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markerpbdhfr
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

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1145400
Gene Model P. falciparum ortholog Not available
Gene productPlasmodium exported protein, unknown function
Gene product: Alternative namePHIST
Details of the genetic modification
Name of the tagmCherry
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 markerpbdhfr
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