RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5130
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1101300; Gene model (P.falciparum): PF3D7_0501300; Gene product: skeleton-binding protein 1 (SBP1)
Transgene
 Transgene not Plasmodium: GFP-Luciferase fusion protein
Promoter: Gene model: PBANKA_0915000; Gene model (P.falciparum): PF3D7_1133400; Gene product: apical membrane antigen 1 (ama1)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Asexual bloodstage;
Last modified: 14 December 2021, 13:44
  *RMgm-5130
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34843584
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone RMgm-4363
Other information parent lineThe mutant expresses a fusion protein of GFP and Luciferase under control of the schizont-specific ama1 promoter. The mutant lacks a drug selectable marker. The GFP-Luciferase expression casette has been introduced in a GIMO mother line using GIMO transfection (see above).
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The mutant parasite was generated by
Name PI/ResearcherPossemiers H, Van den Steen P
Name Group/DepartmentLaboratory of Immunoparasitology, Department of Microbiology, Immunology and Transplantation
Name InstituteRega Institute for Medical research, KU Leuven
CityLeuven
CountryBelgium
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Name of the mutant parasite
RMgm numberRMgm-5130
Principal name2559cl2
Alternative nameSBP-1 KO PbNK65
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageEvidence is presented for:
- reduced sequestration of blood stages in mice
- enhanced survival of C57BL/6 mice (reduced MA-ARDS; lung pathology)
- SBP-1-mediated sequestration is not essential for the development of pulmonary pathology but affects its resolution
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of SBP1 and expresses the fusion protein GFP-luciferase under control of the schizont-specific ama1 promoter

Protein (function)
SBP1 of P. falciparum is a Maurer's cleft protein and is part of the trafficking machinery that trafics PfEMP1 to the surface of infected red blood cells. SBP1 is a PEXEL-negative protein. Infected red blood cells with P. falciparum parasites lacking expression of SBP1 fail to cytoadhere to endothelial receptors of blood vessels, such as CD36 and ICAM1.

Phenotype
Evidence is presented for: 
- reduced sequestration of blood stages in mice 
- enhanced survival of C57BL/6 mice (reduced MA-ARDS; lung pathology) 
- SBP-1-mediated sequestration is not essential for the development of pulmonary pathology but affects its resolution

Additional information

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1101300
Gene Model P. falciparum ortholog PF3D7_0501300
Gene productskeleton-binding protein 1
Gene product: Alternative nameSBP1
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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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe sbp1 gene (PBANKA_1101300) was deleted using the construct as described in De Niz et al. (pL2109; pOBconGFP-PbΔSBP1)
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-Luciferase fusion protein
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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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationParasites of the PbNK65 GIMO 1995cl2 mother cloned line were transfected with construct pL1156 that contains a GFP-luciferase expression cassette and 230p targeting sequences. The gfp-luciferase gene is under control of the schizont-specific ama1 promoter. After transfection, negative selection was applied to select for parasites in which the hdhfr::yfcu selection cassette in the 230p locus is replaced by the expression cassette. The selected parasites (2168 cloned lines) were cloned by the method of limiting dilution and the 2168cl2 cloned line was genotyped by Southern blot analysis of pulsed field gel electrophoresis (PFGE) separated chromosomes
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0915000
Gene Model P. falciparum ortholog PF3D7_1133400
Gene productapical membrane antigen 1
Gene product: Alternative nameama1
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_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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