RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4526
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0614000; Gene model (P.falciparum): PF3D7_0716300; Gene product: conserved protein, unknown function
Name tag: mCherry-3xMyc
Transgene
 Transgene not Plasmodium: GFP fused to the signal peptide of PBANKA_081890
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: Not available; Gene product: Not available
Insertion locus: Gene model: PBANKA_0614000; Gene product: conserved protein, unknown function (conserved protein, unknown function)
Phenotype Asexual bloodstage;
Last modified: 23 September 2018, 20:26
  *RMgm-4526
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) : 30232409
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/ResearcherMatz JM, Matuschewski K
Name Group/DepartmentDepartment of Molecular Parasitology
Name InstituteInstitute of Biology, Humboldt University
CityBerlin
CountryGermany
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Name of the mutant parasite
RMgm numberRMgm-4526
Principal namePBANKA_0614000-tagged
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageExpression in blood stages. No evidence for parasitophorous vacuole location.
PBANKA_0614000, encoding a hypothetical protein, localised to a distinct structure, suggestive of a parasite organelle of yet unknown identity.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a mCherry tagged version of PBANKA_0614000 and expresses GFP under the constitutive hsp70 promoter. GFP is fused to the signal peptide of PBANKA_081890 and consequently GFP is localized to the parasitophourous vacuole  (see RMgm-1333).

Protein (function)
The protein was selected in a screen for putative PV (parasitophorous vacuole) proteins.
Proteins are targeted to the PV by means of default protein secretion, which is initiated by the recognition and cleavage of an amino-terminal signal peptide (SP). To identify novel PV candidates, we searched the Plasmodium genome database (PlasmoDB) for SP-containing proteins and sequentially removed proteins containing predicted transmembrane domains, export motifs, apicoplast targeting peptides, endoplasmic reticulum (ER) retention signals and GPI anchors, which could potentially redirect the protein to other locations. Twelfe proteins were selected. 5 proteins were shown to have a PV location in blood- and liver-stages.

Phenotype
Expression in blood stages. No evidence for parasitophorous vacuole location.
PBANKA_0614000, encoding a hypothetical protein, localised to a distinct structure, suggestive of a parasite organelle of yet unknown identity.

Additional information


Other mutants

  Tagged: Mutant parasite with a tagged gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0614000
Gene Model P. falciparum ortholog PF3D7_0716300
Gene productconserved protein, unknown function
Gene product: Alternative name
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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
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor endogenous tagging of the PV candidates, a GFP(PV) co-localisation plasmid was constructed by fusing the bacterial backbone and adjacent mCherry-3xMyc tag sequence of the pBAT vector with the Plasmodium-specific fraction of the pGFP-PV plasmid using the KpnI and PvuII restriction sites. The sequences directly upstream of the stop codons were amplified from genomic DNA (ranging in size from 758 to 1,628 bp) and inserted into the GFP(PV) co-localisation plasmid using HpaI in combination with EcoRI or SacII. For double homologous recombination, 3′ fragments were amplified and cloned into the intermediate vectors as described for the gene deletion constructs.
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
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP fused to the signal peptide of PBANKA_081890
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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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
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Other details transgene
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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
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3'-UTR
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative name
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_0614000
Gene productconserved protein, unknown function
Gene product: Alternative nameconserved protein, unknown function
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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren