RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5592
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PbANKA_0102900; Gene model (P.falciparum): PF3D7_0604100; Gene product: SIP2
Name tag: GFP
Phenotype Asexual bloodstage;
Last modified: 13 January 2025, 11:49
  *RMgm-5592
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
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 Not applicable
Other information parent line
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-5592
Principal namePbSIP2::GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNo 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.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of SIP2

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

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.

Analysis of a mutant after conditional knockdown of SIP2 (RMgm-5591) showed the following:
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

Other mutants


  Tagged: Mutant parasite with a tagged 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
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
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
Promoter of the selectable markereef1a
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationFor tagging PbSIP2 with GFP, the DNA sequences of the two homologous regions were cloned into the gfp-fusion vector, which has an hdhfr expression cassette next to gfp as a pyrimethamine-selectable marker, to fuse pbsip2 in frame with gfp. The plasmid was linearized by XhoI and NotI digestion before use in transfection experiments
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