RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5397
Malaria parasiteP. yoelii
Genotype
Transgene
 Transgene not Plasmodium: mCherry
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)
Replacement locus: Gene model: PY17X_0306600; Gene product: 6-cysteine protein (P230p; 230p)
Phenotype Asexual bloodstage; Oocyst; Sporozoite; Liver stage;
Last modified: 22 February 2024, 17:35
  *RMgm-5397
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) Not published (yet)
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone RMgm-688
Other information parent lineGIMO-Py17X (RMgm-688; line 1923cl1) contains as a selectable marker (SM) the fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PY04774) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter. This reference line of P. yoelii 17XNL line is used for rapid introduction of transgenes free of drug-resistance genes (PubMed: PMID: 22216235).
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The mutant parasite was generated by
Name PI/ResearcherFranke-Fayard B, C.J. Janse
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-5397
Principal name2537cl1
Alternative namemCherry(hsp70)
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stage(High) mCherry expression in blood stages
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
Oocyst(High) mCherry expression in oocysts
Sporozoite(High) mCherry expression in sporozoites
Liver stage(High) mCherry expression in liver stages
Additional remarks phenotype

Mutant/mutation
The mutant expresses mCherry under the control of the P. berghei hsp70 promoter. The mutant does not contain a drug-selectable marker.

The mutant has been generated and selected using the GIMO transfection method ('gene insertion/marker out') of transfection of a GIMO mother line that contains the hdhfr::yfcu selectable marker into the silent 230p locus.

GIMO-Py17X (RMgm-688; line 1923cl1) contains as a selectable marker (SM) the fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PY04774) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter. This reference line of P. yoelii 17XNL line is used for rapid introduction of transgenes free of drug-resistance genes (PubMed: PMID: 22216235).

Protein (function)

Phenotype
(High) mCherry expression in multiple life cycle stages throughout the life cycle.
This parasite mutant shows normal development of blood stages, oocysts, sporozoites and liver stages, comparable to wild type parasites.

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 namemCherry
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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) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modification
Additional remarks selection procedureThis reporter mutant expressing mCherry does not contain a drug-selectable marker.
The mutant has been generated in the reference line GIMOPbANKA (RMgm-688). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice.
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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 / InsertionReplacement locus
Gene Model of Parasite PY17X_0306600
Gene product6-cysteine protein
Gene product: Alternative nameP230p; 230p
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