RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5591
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PbANKA_0102900; Gene model (P.falciparum): PF3D7_0604100; Gene product: SIP2
Details mutation: The pbsip2 gene contains two loxP sequences, inserted in the 5' and 3' UTR regions.
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: 13 January 2025, 11:38
  *RMgm-5591
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-5590
Other information parent lineThis mutant parasite (RMgm-5590) 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. 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.
The mutant parasite was generated by
Name PI/ResearcherNishi T, Yuda M
Name Group/DepartmentDepartment of Medicine
Name InstituteMie University
CityTsu
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-5591
Principal namepbsip2-cKO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageTo investigate the role of PbSIP2 during schizont development, we performed a conditional knockout of pbsip2 using a dimerizable Cre recombinase (DiCre) system. A parasite mutant (PbDiCre; RMgm-5590) was generated constitutively expressing Cre59 (Thr19-Asn59) fused with FKBP12 and Cre60 (Asn60-Asp343) fused with FRB, using the Cas9 expressing parasite, PbCas9 (RMgm-4870). Subsequently, two loxP sequences were inserted on 5’ and 3’ sides of pbsip2, arranged in the same direction (pbsip2-cKO). This parasite will lose the entire open reading frame of pbsip2 in the presence of rapamycin.

In culture, pbsip2-cKORapa+ developed as comparable to pbsip2-cKORapa− until 24 hpi. At 26 hpi, approximately 20% of pbsip2-cKORapa− became mature schizonts (number of nuclei>10), and some have already formed merozoites. In contrast, most schizonts of pbsip2-cKORapa+ had less than ten nuclei at 26 hpi. The number ofpbsip2-cKORapa− schizonts with six–ten nuclei increased at 28 hpi, mature schizonts were barely produced in pbsip2-cKORapa+, and no merozoite formation was observed. These results indicate that PbSIP2 plays an essential role in schizont development.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
In this mutant the pbsip2 gene contains two loxP sequences that 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.
The loxP sequences in pbsip2 were introduced in a mutant (RMgm-5590) that 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. In this mutant 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)
SIP2 is an AP2 transcription factor expressed during the schizont stage. AP2 transcription factors are the major family of transcription factors found in Plasmodium. Members in this family contain sequence-specific DNA binding domains with three anti-parallel β-sheets and one α-helix, called AP2 domain. In P. falciparum, SIP2 (PF3D7_0604100) has been reported to bind to the subtelomeric var promoter element 2 (SPE2) and has been proposed to be involved in chromosome end biology.
PbSIP2 contains two tandem AP2 domains at its N-terminus and a putative nuclear localization signal (NLS) on the C-terminal side of the AP2 domains. Protein-protein BLAST (BLASTP) search using the AA sequence of its AP2 domains detected proteins in Hepatocystis, Babesia, and Theileria species but not from other apicomplexan species. These species belong to the Haemosporida or Piroplasmida, which constitute a group of parasites that infect host RBCs.

Phenotype

To investigate the role of PbSIP2 during schizont development, we performed a conditional knockout of pbsip2 using a dimerizable Cre recombinase (DiCre) system. A parasite mutant (PbDiCre; RMgm-5590) was generated constitutively expressing Cre59 (Thr19-Asn59) fused with FKBP12 and Cre60 (Asn60-Asp343) fused with FRB, using the Cas9 expressing parasite, PbCas9 (RMgm-4870). Subsequently, two loxP sequences were inserted on 5’ and 3’ sides of pbsip2, arranged in the same direction (pbsip2-cKO). This parasite will lose the entire open reading frame of pbsip2 in the presence of rapamycin.

In culture, pbsip2-cKORapa+ developed as comparable to pbsip2-cKORapa− until 24 hpi. At 26 hpi, approximately 20% of pbsip2-cKORapa− became mature schizonts (number of nuclei>10), and some have already formed merozoites. In contrast, most schizonts of pbsip2-cKORapa+ had less than ten nuclei at 26 hpi. The number ofpbsip2-cKORapa− schizonts with six–ten nuclei increased at 28 hpi, mature schizonts were barely produced in pbsip2-cKORapa+, and no merozoite formation was observed. These results indicate that PbSIP2 plays an essential role in schizont development.

Additional information
It was first assessed whether DiCre-mediated recombination in pbsip2-cKO occurs quickly enough to disrupt pbsip2 before PbSIP2 expression begins. The whole blood from mice infected with synchronized pbsip2-cKO was split into two cultures at 6 hpi, and the one was treated with 10 nM rapamycin (pbsip2-cKORapa+) and the other with dimethyl sulfoxide as a control (pbsip2-cKORapa−). At 18 hpi (12 h of culture), when no parasites had started nuclear division, recombination at the pbsip2 locus was assessed by genotyping PCR. The assay showed that the pbsip2 locus was almost completely excised from the genome in pbsip2-cKORapa+ while no recombination was detected in pbsip2-cKORapa−. Notably, using the parental PbDiCre parasite, it was confirmed that schizont development was not affected by rapamycin.

Evidence is presented for the following:
- PbSIP2 binds to the upstream of genes related to merozoite formation.
- PbSIP2 functions as a transcriptional activator
- PbSIP2 binds to the GTGCA motif through its tandem AP2 domains.
- Th domain RGTGCA is essential for PbSIP2 binding and transcription of the downstream gene
To assess whether RGTGCA is essential for the DNA binding of PbSIP2 and functions as a cis-regulatory element for the downstream gene, we introduced a mutation into the motif within the ChIP peak upstream of rop14 (PBANKA_0111600; PF3D7_0613300). We developed parasites expressing GFP-fused PbSIP2 using PbCas9 (PbSIP2::GFPCas9) and then introduced a mutation upstream of rop14, altering GGTGCA to ttTGCA (PbSIP2::GFPCas9_cismut). ROP14 is a functionally uncharacterized rhoptry protein that localizes to the rhoptry bulbs of merozoites. Rop14 was selected as the target in this experiment because it is not essential for the asexual blood-stage development in P. berghei. Using these parasite lines, we examined whether the disruption of the RGTGCA motif affected the binding of PbSIP2 to the genome by ChIP-qPCR analysis.
- PbSIP2 recognize the bipartite motif upstream of rhoptry genes.

Analysis of a mutant expressing a C-terminal GFP-tagged version of SIP2 (RMgm-5592) showed the following: No fluorescent signals were observed in the ring or trophozoite stages. Fluorescent signals first appeared in the maturing schizonts with four nuclei (at 22 hpi). The signals continued until the schizonts had eight nuclei and then faded in later stages.

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PbANKA_0102900
Gene Model P. falciparum ortholog PF3D7_0604100
Gene productSIP2
Gene product: Alternative name
Details of the genetic modification
Short description of the mutationThe pbsip2 gene contains two loxP sequences, inserted in the 5' and 3' UTR regions.
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo investigate the role of PbSIP2 during schizont development, we performed a conditional knockout of pbsip2 using a dimerizable Cre recombinase (DiCre) system. A parasite mutant (PbDiCre; RMgm-5590) was generated constitutively expressing Cre59 (Thr19-Asn59) fused with FKBP12 and Cre60 (Asn60-Asp343) fused with FRB, using the Cas9 expressing parasite, PbCas9 (RMgm-4870). Subsequently, two loxP sequences were inserted on 5’ and 3’ sides of pbsip2, arranged in the same direction (pbsip2-cKO). This parasite will lose the entire open reading frame of pbsip2 in the presence of rapamycin.
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameFRb-Cre60 (DiCre).
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
Other details transgene
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
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameCas9 from Streptococcus pyogenes
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
Other details transgene
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
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