RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-281
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: ovalbumin (OVA)
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Insertion locus: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Phenotype Asexual bloodstage;
Last modified: 29 May 2009, 13:51
  *RMgm-281
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 18606696
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/ResearcherM. Miyakoda, K. Yui
Name Group/DepartmentDivision of Immunology, Department of Molecular Microbiology and Immunology
Name InstituteNagasaki University
CityNagasaki
CountryJapan
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Name of the mutant parasite
RMgm numberRMgm-281
Principal nameOVA-PbA
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageThe mutant constitutively express a truncated C-terminal fragment of OVA (aa 150–386) fused to the N-terminal sequence (aa 1–5) of the hsp70 gene of P. berghei. The expression of the recombinant OVA was confirmed by immunoblotting of infected RBC lysates with anti-OVA antibodies (see also 'Additional remarks phenotype').
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses the protein antigen ovalbumin (OVA) under the control of the regulatory sequences of hsp70 gene. The mutant constitutively express a truncated C-terminal fragment of OVA (aa 150–386) fused to the N-terminal sequence (aa 1–5) of the hsp70 gene of P. berghei.

Protein (function)

Phenotype
Mutants expressing OVA (OVA-PbA) were generated to investigate the parasite-specific T cell responses during blood stage infections in mice and specifically to investigate  whether Plasmodium-specific CD8+ T cells are activated during the erythrocyte stage of malaria and how they are involved in the pathogenesis of cerebral malaria. Using this model system, evidence is presented for Ag (OVA)-specific CD8+ T cells activation by TAP-dependent cross-presentation during infection with OVA-PbA leading to their expression of an activation phenotype and granzyme B and the development to functional CTL.

Additional information

Other mutants

 

  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameovalbumin (OVA)
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructPlasmid 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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationMutant parasites were engineered to constitutively express a truncated C-terminal fragment of OVA (aa 150–386) fused to the N-terminal sequence (aa 1–5) of the PbA heat shock protein (hsp) 70 gene.
The gene construct was based on pBluescript KS' (Stratagene) and contains the following elements: 1) PbA dihydrofolate reductase-thymidyltransferase-ts (DHFR-ts) gene; 2) PbA hsp70 5'untranslated region and N-terminal coding sequence; 3) coding sequence of C-terminal fragment of OVA; 4) PbA hsp70 3'-untranslated region and DHFR-ts 3'-untranslated region. The DHFR-ts gene contains a point mutation at position 110 of the DHFR gene causing a Ser3Asn transition conferring resistance to the antimalaria drug pyrimethamine. No sequences are provided of the OVA-gene and the regulatory sequences.
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 PBANKA_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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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P.berghei model of malaria
Technical design and realization: S. Mededovic and A. van Wigcheren