RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1415700; Gene model (P.falciparum): PF3D7_1317200; Gene product: AP2 domain transcription factor AP2-FG, putative (AP2-FG, ApiAP2)
Name tag: mNeonGreen
Phenotype Gametocyte/Gamete;
Last modified: 6 August 2019, 14:17
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) : 31231888
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/ResearcherYuda M, Kato T
Name Group/DepartmentDepartment of Medical Zoology
Name InstituteMie University School of Medicine
CityTsu, Mie
Name of the mutant parasite
RMgm numberRMgm-4651
Principal nameAP2-FG::mNeonGreen
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteAP2-FG was expressed in the nucleus of female gametocytes, but not in other blood stages, including male gametocytes
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a C-terminal mNeonGreen-tagged version of AP2-FG

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.

To detect the weak expression of AP2‐FG more effectively (see also mutant RMgm-4650 expressing GFP-tagged AP2-FG), we used transgenic P. berghei parasites that produce mNeonGreen‐tagged AP2‐FG; mNeonGreen is a green fluorescent protein that produces a more intense signal than GFP. Specific expression of AP2‐FG in female gametocytes was also observed in these parasites. We transfected these AP2‐FG::mNeonGreen parasites with a centromere plasmid, which confers male‐ or female‐specific expression of the mCherry gene, and performed the fluorescent microscopic study. In all 100 females examined in this study, the expression of AP2‐FG was observed in the nucleus. In contrast, in all 100 males examined, the expression of AP2‐FG was not observed in the nucleus or in the cytoplasm. These results again confirmed the female‐specific expression of AP2‐FG and its localization in the nucleus.
To investigate the expression profile of AP2‐FG, rats were synchronously infected with AP2‐FG::mNeonGreen parasites by the intravenous inoculation of mature schizonts and the blood smears were examined at 2‐h intervals under a fluorescent microscope. AP2‐FG expression was not observed in mature schizonts before inoculation into the rats (0 hpi). At 14 hpi, weak expression was first observed in a small number of the parasites. Then, the fluorescent signals gradually increased with their development and reached a steady state at around 22–24 hpi. This result indicated that AP2‐FG expression begins before gametocyte‐specific features appear and then reaches a peak in accordance with their manifestation of sexual dimorphism. This expression profile strongly suggested that AP2‐FG is involved in female development from the early phase of gametocytogenesis.

Analyses of a mutant lacking AP2-FG showed the following (see RMgm-4649): Normal production of male gametocytes (and exflagellation). No production of mature female gametocytes. Evidence provided that immature female gametocytes are produced.Strongly reduced numbers of ookinetes in in vitro cultures. All ookinetes show aberrant morphology. No oocyst production

Additional information
See also other mutants in the database targeting this gene PF3D7_1317200

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1415700
Gene Model P. falciparum ortholog PF3D7_1317200
Gene productAP2 domain transcription factor AP2-FG, putative
Gene product: Alternative nameAP2-FG, ApiAP2
Details of the genetic modification
Name of the tagmNeonGreen
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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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