RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5186
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1218500; Gene model (P.falciparum): PF3D7_0320000; Gene product: protein phosphatase inhibitor 2 (PbI2, I2)
Name tag: mCherry
Transgene
 Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
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 (P230p)
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 28 March 2022, 17:36
  *RMgm-5186
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 35162991
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-1026
Other information parent lineA drug-selectable marker free reporter line expressing GFP under the constitutive hsp70 promoter
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The mutant parasite was generated by
Name PI/ResearcherWitte de C, Khalife J
Name Group/DepartmentCenter for Infection and Immunity of Lille
Name InstituteUniv. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille
CityLille
CountryFrance
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Name of the mutant parasite
RMgm numberRMgm-5186
Principal namePbI2-mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageGenotyping analysis showed the correct integration of the tagged PbI2 and the expression of mCherry–PbI2 was checked by immunoblot using anti-RFP antibody.
Gametocyte/GameteGenotyping analysis showed the correct integration of the tagged PbI2 and the expression of mCherry–PbI2 was checked by immunoblot using anti-RFP antibody.
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal mCherry-tagged version of PbI2.

Protein (function)
Phosphatase type 1 is a key enzyme playing diverse and essential roles in cell survival. Its dephosphorylation activity/specificity is governed by the interaction of its catalytic subunit (PP1c) with regulatory proteins. Among these, inhibitor-2 (I2) is one of the most evolutionarily ancient PP1 regulators. In vivo studies in various organisms revealed a defect in chromosome segregation and cell cycle progression when the function of I2 is blocked.
A database search with Inhibitor-1 (I1) and Inhibitor-2 (I2), known to be powerful regulators of PP1c, identified one open reading frame in the P. falciparum genome (PlasmoDB gene identifier: PF3D7_0320000) encoding a potential protein with identity to known I2.

Phenotype
Genotyping analysis showed the correct integration of the tagged PbI2 and the expression of mCherry–PbI2 was checked by immunoblot using anti-RFP antibody.

Additional information
From the Abstract: 'In this study, we used Plasmodium berghei transgenic parasite strains stably expressing PP1c or its inhibitor 2 (I2) tagged with mCherry, combined with the mCherry affinity pulldown of proteins from asexual and sexual stages, followed by mass spectrometry analyses. Mapped proteins were used to identify interactomes and to cluster functionally related proteins'

Other mutants

  Tagged: Mutant parasite with a tagged gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1218500
Gene Model P. falciparum ortholog PF3D7_0320000
Gene productprotein phosphatase inhibitor 2
Gene product: Alternative namePbI2, I2
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Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single 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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe Plasmodium berghei strain used in this study was a PbGFP ANKA line kindly provided by O. Silvie (Université Pierre et Marie Curie, Paris, France). Plasmid pL1886 was given by B. Franke-Fayard (Leiden University Medical Center, Leiden, The Netherlands.
The generation of a P. berghei line expressing mCherry-tagged PP1c has previously been reported. As for PbI2, a parasite line expressing mCherry-tagged I2 from its endogenous locus was generated in the same strain used for PbPP1c.
A C-terminal mCherry-tagged PbI2 was generated by single homologous recombination of a 1436 pb region of PbI2 without the stop codon (Pr3–Pr2) inserted into the pL1886 vector and NdeI-linearized before transfection into the PbGFP ANKA strain
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 parasiteNo selectable marker
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThe parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting.
1) Transfected parasites are first selected in a mouse by pyrimethamine treatment
2) GFP+mCherry+ parasites are selected by FACS sorting and used to infect a mice
3) This mouse is treated with 5-FC to select for parasites that have the selectable marker removed
4) GFP+mCherry- and marker free parasites are selected by FAC sorting and used to infect a mouse
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_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 nameP230p
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