RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4883
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0719300; Gene model (P.falciparum): PF3D7_0417200; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS)
Details mutation: Pbdhfr/ts replaced with a mutated P. falciparum dhfr/ts (N51I, C59R, S108N, I164L)
Transgene
 Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
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: 28 October 2020, 15:22
  *RMgm-4883
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, 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 P. berghei ANKA 676m1cl1 (RMgm-29)
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
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The mutant parasite was generated by
Name PI/ResearcherKoonyosying P, Uthaipibull C
Name Group/DepartmentNational Center for Genetic Engineering and Biotechnology (BIOTEC)
Name InstituteNational Science and Technology Development Agency (NSTDA) 113 Thailand Science Park
CityKhlong Luang, Pathum Thani
CountryThailand
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Name of the mutant parasite
RMgm numberRMgm-4883
Principal namedhfr (N51I+C59R+S108N+I164L)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageblood stages resistant against pyrimethamine. While 30 mg/kg of pyrimethamine could not inhibit the transgenic parasite, P218 could inhibit the transgenic parasite with the ED50 of 0.11±0.02 mg/kg, a level similar to the P. falciparum in SCID mice model.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
In the mutant the endogenous P. berghei dhfr/ts gene has been replaced with a mutated P. falciparum dhfr/ts gene with amino acid mutations in dhfr at positions N51I, C59R, S108N and I164L, the same as the V1/S strain P. falciparum
Published in bioRxiv preprint doi: https://doi.org/10.1101/2020.09.30.281055

Protein (function)
Dihydrofolate reductase-thymidylate synthase (PfDHFR-TS) is an enzyme involved in folate metabolism and DNA synthesis

Phenotype
Blood stages resistant against pyrimethamine. While 30 mg/kg of pyrimethamine could not inhibit the transgenic parasite, P218 could inhibit the transgenic parasite with the ED50 of 0.11±0.02 mg/kg, a level similar to the P. falciparum in SCID mice model. These results demonstrated the validity of our model and showed that P218 was very potent against quadruple Pfdhfr-ts mutant parasite, in vivo.

Additional information

Other mutants

  Mutated: Mutant parasite with a mutated gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0719300
Gene Model P. falciparum ortholog PF3D7_0417200
Gene productbifunctional dihydrofolate reductase-thymidylate synthase
Gene product: Alternative nameDHFR-TS
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Details of the genetic modification
Short description of the mutationPbdhfr/ts replaced with a mutated P. falciparum dhfr/ts (N51I, C59R, S108N, I164L)
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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 parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationpY003V1S plasmid for P. berghei transfection was constructed to contain Pfdhfr-ts with amino acid mutations in dhfr at the positions N51I, C59R, S108N and I164L, the same as the V1/S strain P. falciparum, flanking by 5’ and 3’ untranslated region (UTR) of Pbdhfr-ts. The quadruple mutant Pfdhfr fragment was amplified from pCas.SgDHFR.HR.V1S plasmid (Posayapisit et al., 2020) and cloned into pY003 plasmid at NcoI and SpeI restriction sites. The P. berghei UTRs serve as sites for double crossover homologous recombination at the endogenous Pbdhfr-ts locus. To generate integrative transformant, the pY003V1S plasmid was linearized at the HindIII, KasI and ScaI site, before transfection experiments.
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 nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV)
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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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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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 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