RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4922
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: Ovalbumin (OVA ) fused to HEP17/EXP1
Promoter: Gene model: PBANKA_0926700; Gene model (P.falciparum): PF3D7_1121600; Gene product: exported protein 1 | circumsporozoite-related antigen | parasitophorous vacuole membrane antigen QF 116 (HEP17; EXP1)
3'UTR: Gene model: PBANKA_0926700; Gene product: exported protein 1, putative circumsporozoite-related antigen (HEP17; EXP1)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Transgene
 Transgene not Plasmodium: GFP-Luciferase
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_1206800; Gene product: zinc finger (CCCH type) protein, putative (s1)
Phenotype Asexual bloodstage;
Last modified: 23 November 2020, 15:59
  *RMgm-4922
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene, 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 RMgm-4920
Other information parent lineOVA::HEP17(hep17)-s1GIMO (RMgm-4920; 3256cl2) expresses a fusion protein of OVA (ovalbumin) and HEP17/EXP1 (PBANKA_092670) under the hep17 promoter and contains the positive/negative selectable marker cassette hdhfr::yfcu in the silent s1 locus (s1-GIMO line).
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The mutant parasite was generated by
Name PI/ResearcherFranke-Fayard B, Janse C.J
Name Group/DepartmentLeiden Malaria Research Group, Parasitology
Name InstituteLeiden University Medical Centre
CityLeiden
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-4922
Principal nameOVA::HEP17(hep17)-GFP-Luc(eef1a)
Alternative name3316cl1, cl2, cl3
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageOVA, GFP and luciferase expression in blood stages. Normal blood stage growth/development/multiplication
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a fusion protein of OVA (ovalbumin) and HEP17/EXP1  (PBANKA_092670) under the hep17 promoter (introduced into the silent p230p locus) and expresses the fusion gene GFP-luciferase under control of the eef1a promoter (introduced into the silent s1 locus by GIMO transfection). The mutant does not contain a drug-selectable marker

The mutant has been generated in the the OVA::HEP17(hep17)-s1GIMO line (RMgm-4920; 3256cl2) that expresses a fusion protein of OVA (ovalbumin) and HEP17/EXP1  (PBANKA_092670) under the hep17 promoter and contains the positive/negative selectable marker cassette hdhfr::yfcu in the silent s1 locus (s1-GIMO line).

Protein (function)

Phenotype
OVA, GFP and luciferase expression in blood stages. Normal blood stage growth/development/multiplication

Additional information

Other mutants
Click on Ovalbumin for more rodent malaria mutants expressing OVA

  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 ) fused to HEP17/EXP1
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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 modificationwe replaced the positive-negative SM in the OVA::hep17(hep17) s1-GIMO genome with a GFP-luciferase expression cassette by GIMO transfection, using construct pL2366. To generate pL2366 we first PCR amplified and sequenced the GFP-luciferase fusion gene of pl0063 and this GFP-Luc fragment was cloned using SpeI and XhoI into pL1928, replacing the selectable marker cassette in pL1928. The construct was linearized using ApaI, BstXI and AhdI before transfection and used to transfect parasites of the OVA::hep17(hep17) s1-GIMO line using standard methods of GIMO-transfection. Transfected parasites were selected in mice by applying negative selection by providing 5-fluorocytosine (5-FC) in the drinking water of mice. Negative selection results in selection of transgenic parasites where the hdhfr::yfcu SM in the s1 locus of OVA::hep17(hep17) s1-GIMO line is replaced by the GFP-Luc expression cassette, resulting in line 3316. Transfected parasites of line 3316 were cloned by limiting dilution, resulting in the OVA::HEP17(hep17)-GFP-Luc(eef1a) line (line 3316cl1).
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0926700
Gene Model P. falciparum ortholog PF3D7_1121600
Gene productexported protein 1 | circumsporozoite-related antigen | parasitophorous vacuole membrane antigen QF 116
Gene product: Alternative nameHEP17; EXP1
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_0926700
Gene productexported protein 1, putative circumsporozoite-related antigen
Gene product: Alternative nameHEP17; EXP1
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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  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
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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 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 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_1206800
Gene productzinc finger (CCCH type) protein, putative
Gene product: Alternative names1
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