RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-5404
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0605900; Gene model (P.falciparum): PF3D7_1207400; Gene product: conserved Plasmodium protein, unknown function (S14)
Transgene
 Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: Not available; Gene product: Not available
Replacement locus: Gene model: PBANKA_0605900; Gene product: conserved Plasmodium protein, unknown function
Phenotype Sporozoite; Liver stage;
Last modified: 10 March 2024, 19:39
  *RMgm-5404
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) Not published (yet)
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/ResearcherGhosh A, Mishra S
Name Group/DepartmentDivision of Molecular Microbiology and Immunology
Name InstituteCSIR-Central Drug Research Institute
CityLucknow
CountryIndia
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Name of the mutant parasite
RMgm numberRMgm-5404
Principal nameS14 KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot tested
OocystNot different from wild type
SporozoiteEvidence for normal development inside oocysts and egress of sporozoites in the hemolymph. The number of salivary gland sporozoites strongly reduced. Sporozoites are non-motile (no gliding motility)
Liver stageSporozoites are unable to infect mice (by mosquito bite or intravenous injection) and unable to invade hepatocytes in vitro.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of S14 and expresses GFP under control of the constitutive eef1a promoter.

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2023.08.30.555469

Protein (function)
The gene encoding S14 is highly transcribed in sporozoites. the protein does not contain a transmembrane domain or signal peptide

Phenotype
Evidence for normal development inside oocysts and egress of sporozoites in the hemolymph. The number of salivary gland sporozoites strongly reduced. Sporozoites are non-motile (no gliding motility).
Sporozoites are unable to infect mice (by mosquito bite or intravenous injection) and unable to invade hepatocytes in vitro.

Additional information
Analysis of a mutant expressing a 3xHA-mCherry tagged version of S14 (RMgm-5405) showed the following:
- S14 expression was observed in sporozoites but not in the blood and liver stages
- Analysis of the mCherry pattern on sporozoites revealed localization on the membrane of the sporozoites
- IFA with anti-CSP and anti-mCherry antibodies confirmed S14 localization on the membrane
- S14 localizes on the inner membrane and is essential for parasite gliding motility

Evidence is presented that S14 associates with the glidosome-associated proteins MTIP (PBANKA_1459500) and GAP45 (PBANKA_1437600) and that S14 performs gliding-specific function and absence of S14 does not affect the organization (location/expression) of IMC and surface proteins (MTIP, GAP45, CSP, TRAP).


Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0605900
Gene Model P. falciparum ortholog PF3D7_1207400
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative nameS14
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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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationP. berghei S14 (PBANKA_0605900) was disrupted by double-crossover homologous recombination. For this, two fragments, F3 (0.73 kb) and F4 (0.637 kb), were amplified using P. berghei ANKA genomic DNA with primers 1003/1004 and 1005/1006, respectively. The fragments F3 and F4 were cloned sequentially into the pBC-GFP-hDHFR vector at XhoI/ClaI and NotI/AscI, respectively, and finally linearized with XhoI/AscI
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
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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 markereef1a
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_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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 / InsertionReplacement locus
Gene Model of Parasite PBANKA_0605900
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative name
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