RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5582
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0932300; Gene model (P.falciparum): PF3D7_1115500; Gene product: AP2 domain transcription factor, putative ApiAP2 (Ap2-D (AP2-dispensable))
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_0711900; Gene product: heat shock protein 70 (HSP70)
Replacement locus: Gene model: PBANKA_0932300; Gene product: AP2 domain transcription factor, putative ApiAP2 (Ap2-D (AP2-dispensable))
PhenotypeNo phenotype has been described
Last modified: 13 November 2024, 12:59
  *RMgm-5582
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
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-5582
Principal nameAp2-D KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
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 lacks expression of Ap2-D and expresses GFP under the constitutive hsp promoter (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).

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
No phenotype detected throughout the complete lifecycle different from wild type parasites

Additional information
Analysis of a a mutant expressing a C-terminal 3xHA-mCherry-tagged version of Ap2-D (RMgm-5583) showed the following:
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

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


  Disrupted: Mutant parasite with a disrupted 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 ApiAP2
Gene product: Alternative nameAp2-D (AP2-dispensable)
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo disrupt the Ap2-D gene, two fragments, F3 (0.6 kb) and F4 (0.57 kb), consisting of the 5′ and 3′ UTRs of the gene, respectively, were amplified using primers 1938/1939 and 1940/1941 and cloned into the pBC-GFP-hDHFR-yFCU vector at XhoI/SalI and NotI/AscI, respectively. The final construct was linearized using XhoI/AscI restriction digestion 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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) PCR construct 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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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 PBANKA_0932300
Gene productAP2 domain transcription factor, putative ApiAP2
Gene product: Alternative nameAp2-D (AP2-dispensable)
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