RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5590
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: FRb-Cre60 (DiCre).
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_0306000; Gene product: 6-cysteine protein (230p)
Transgene
 Transgene not Plasmodium: Cas9 from Streptococcus pyogenes
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: Not available; Gene product: Not available (small subunit ribosomal rna gene (c/d-type unit))
Phenotype Asexual bloodstage;
Last modified: 11 January 2025, 13:06
  *RMgm-5590
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-4870
Other information parent lineThis mutant parasite (RMgm-4870) expresses Cas9 from Streptococcus pyogenes, introduced into the c/d-ssu-rrna gene. It does not contain a drug-selectable marker that has been removed by negative selection.
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The mutant parasite was generated by
Name PI/ResearcherNishi T, Yuda M
Name Group/DepartmentDepartment of Medicine
Name InstituteMie University
CityTsu
CountryJapan
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Name of the mutant parasite
RMgm numberRMgm-5590
Principal namePbDiCre
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageSee mutant RMgm-5591 for the use of this line to conditional knockout PbSIP2 in P. berghei blood stage schizonts (pbsip2-cKO). In the pbsip2 gene (PbANKA_0102900) two loxP sequences were inserted in the 5' and 3' UTR regions. This mutant will lose the entire open reading frame of pbsip2 in the presence of rapamycin.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a dimerizable Cre recombinase (DiCre), constitutively expressing Cre59 (Thr19-Asn59) fused with FKBP12 and Cre60 (Asn60-Asp343) fused with FRB and it expresses CAS9 (see mutant RMgm-4870)
The DiCre expression cassette is introduced into the silent 230p locus into mutant RMgm-4870 that contains the cas9 in the silent c/d-ssu-rRNA gene unit and does not contain a drug-selectable marker that has been removed by negative selection.

Published in: bioRxiv preprint doi: https://doi.org/10.1101/2024.08.02.606280

Protein (function)
The mutant expresses a dimerizable Cre recombinase (DiCre), constitutively expressing Cre59 (Thr19-Asn59) fused with FKBP12 and Cre60 (Asn60-Asp343) fused with FRB and it expresses CAS9 (see mutant RMgm-4870).

Phenotype
See mutant RMgm-5591 for the use of this line to conditional knockout PbSIP2 in P. berghei blood stage schizonts (pbsip2-cKO). In the pbsip2 gene (PbANKA_0102900) two loxP sequences were inserted in the 5' and 3' UTR regions. This mutant will lose the entire open reading frame of pbsip2 in the presence of rapamycin.

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 nameFRb-Cre60 (DiCre).
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructCRISPR/Cas9 construct: integration through double strand break repair
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 parasiteNo selectable marker
Promoter of the selectable markerNo
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationWe first developed a parasite line constitutively expressing Cre59 (Thr19-Asn59) fused with FKBP12 and Cre60 (Asn60-Asp343) fused with FRB (PbDiCre), using the Cas9 expressing parasite, PbCas9 (RMgm-4870). The DiCre expression cassette is introduced into the silent 230p locus into mutant RMgm-4870 that contains the cas9 in the silent c/d-ssu-rRNA gene unit and does not contain a drug-selectable marker that has been removed by negative selection.
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_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 nameCas9 from Streptococcus pyogenes
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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 parasiteNo selectable marker
Promoter of the selectable markerN.A.
Selection (positive) procedureN.A.
Selection (negative) procedureN.A.
Additional remarks genetic modificationWe first developed a parasite line constitutively expressing Cre59 (Thr19-Asn59) fused with FKBP12 and Cre60 (Asn60-Asp343) fused with FRB (PbDiCre), using the Cas9 expressing parasite, PbCas9 (RMgm-4870). The DiCre expression cassette is introduced into the silent 230p locus into mutant RMgm-4870 that contains the cas9 in the silent c/d-ssu-rRNA gene unit and does not contain a drug-selectable marker that has been removed by negative selection.
Additional remarks selection procedure
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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 Not available
Gene productNot available
Gene product: Alternative namesmall subunit ribosomal rna gene (c/d-type unit)
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