RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5583
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0932300; Gene model (P.falciparum): PF3D7_1115500; Gene product: AP2 domain transcription factor, putative ApiAP (Ap2-D (AP2-dispensable))
Name tag: 3xHA-mCherry
Phenotype Asexual bloodstage; Gametocyte/Gamete;
Last modified: 13 November 2024, 13:17
  *RMgm-5583
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 38752809
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/ResearcherNirdosh, Mishra S
Name Group/DepartmentDivision of Molecular Microbiology and Immunology
Name InstituteCSIR-Central Drug Research Institute
CityLucknow
CountryIndia
Name of the mutant parasite
RMgm numberRMgm-5583
Principal nameAp2-D-HA-mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stagemCherry expression was detectable in the blood stages of the parasite. The Ap2-D-specific mCherry signal colocalized with cytoplasmic and nuclear localization patterns. No fluorescence was detected in the mosquito or liver stages of the parasites
Gametocyte/GametemCherry expression was detectable in the blood stages of the parasite. The Ap2-D-specific mCherry signal colocalized with cytoplasmic and nuclear localization patterns. No fluorescence was detected in the mosquito or liver stages of the parasites
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal 3xHA-mCherry-tagged version of Ap2-D

Protein (function)
Most sequence-specific transcription factor families found in other eukaryotes seem to be absent from Plasmodium. Instead, an expansion of a protein family containing one or more apetala2 (AP2) DNA-binding domains was observed across the phylum apicomplexa. In total, 27 members of this family have been found in the human malaria parasite Plasmodium falciparum (although a possible 28th member of the family may be present. In total, 26 of these have syntenic orthologs in rodent malaria species, each with its unique stage-specific expression profile.

Phenotype
mCherry expression was detectable in the blood stages of the parasite. The Ap2-D-specific mCherry signal colocalized with cytoplasmic and nuclear localization patterns. No fluorescence was detected in the mosquito or liver stages of the parasites

Analysis of a mutant lacking expression of Ap2-D (RMgm-5582) showed the following:
No phenotype detected throughout the complete lifecycle different from wild type parasites.(see also P. berghei mutant RMgm-2638 for successful knocking out AP2-D and P. berghei/P. yoelii mutants RMgm-4100 and RMgm-4372 for unsuccessful attempts to knockout this gene).

Additional information
From the paper:
Using a conserved domain (NCBI CDD) search, we found that amino acids ranging from glycine 185 to isoleucine 228 at the C-terminus have an Ap2 DNA binding domain.

See also P. berghei mutant RMgm-2638 for successful knocking out AP2-D and P. berghei/P. yoelii mutants RMgm-4100 and RMgm-4372 for unsuccessful attempts to knockout this gene.

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0932300
Gene Model P. falciparum ortholog PF3D7_1115500
Gene productAP2 domain transcription factor, putative ApiAP
Gene product: Alternative nameAp2-D (AP2-dispensable)
Details of the genetic modification
Name of the tag3xHA-mCherry
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) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTwo fragments, F1 and F2, encompassing the Ap2-D gene C-terminus (0.58 kb) and 3′UTR (0.57 kb) were amplified using the primers 2040/2041 and 2042/1941, respectively. Both fragments were cloned into the pBC-3XHAmCherry-hDHFR vector at XhoI/BglII and NotI/AscI, respectively. The plasmid was linearized using XhoI/AscI and transfected into WT P. berghei schizonts.
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