RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
DisruptedGene model (rodent): PBANKA_0905900; Gene model (P.falciparum): PF3D7_1143100; Gene product: AP2 domain transcription factor AP2-O, putative (AP2-O, AP2 in ookinetes; ApiAP2)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 14 June 2010, 10:43
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 19220746
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/ResearcherM. Yuda, I. Kaneko
Name Group/DepartmentDepartment of Medical Zoology
Name InstituteMie University School of Medicine
Name of the mutant parasite
RMgm numberRMgm-207
Principal nameAP2-O (-)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteThe mutant parasites formed morphologically normal female and male gametocytes and exhibited normal exflagellation rates. However, they lacked the ability to infect mosquitoes as no oocysts or sporozoites were found in the mosquito. In culture, mutant parasites formed zygotes at normal conversion rates and differentiated into retort forms by 9 h after fertilization, as in wild type parasites. Subtle morphological differences between wild type and mutant parasites became visible 12 h after fertilization with a swelling protrusion in the retort forms. Mature ookinetes showed an aberrant morphology with an pear-shaped form, different from the banana (sword)-like form of wild type ookinetes.
OocystNo oocyst and sporozoite production
SporozoiteNo oocyst and sporozoite production
Liver stageNot tested
Additional remarks phenotype

The mutant lacks expression of  a transcription factor with AP2 domain(s) (AP2-O, AP2 in ookinetes)

Protein (function)
AP2-O belongs to the Apetala2 (AP2) family which encode transcription factors (TFs). The AP2 family TFs were first identified in plants. Plant AP2 family TFs are named for their possession of at least one common DNA-binding domain of approximately 60 amino acids, the AP2 domain. Bioinformatic analyses have revealed the presence of 26 AP2-related genes in the Plasmodium genome. The predicted Plasmodium AP2-related genes encode proteins with one to four AP2 domains. AP2-O has two conserved regions which include the AP2 domain near the C-terminus.

The phenotype analyses indicate an essential role of AP2-O during ookinete development. The phenotype analyses as described in 'Additional information' indicate that AP2-O is a transcription factor that binds to promoter regions of genes that are expressed during ookinete development and activates expression of genes, including genes involved in mosquito midgut invasion.

Additional information
In wild type parasites ap2-0 transcripts are present in both gametocytes and ookinetes. Protein expression was analysed using a mutant expressing a GFP-tagged form of AP2-O (RMgm-208). No expression was observed in the gametocyte stage indicating translational repression of the ap2-o transcripts in gametocytes. Weak GFP fluorescent signals were observed in retort-form ookinetes, beginning at 8h after fertilization. The signals were localized in the nucleus and signal intensities increased with ookinete development.

Cross-fertilization experiments mating mutant gametes with either fertile wild type female or fertile wild type male gametes indicated that the AP2-O defect is inherited from the female line. Mutant females when fertilized with wild type males produced deformed ookinetes. However, wild type females when fertilized by mutant males produced normal ookinetes. These results suggest that AP2-O transcripts in zygotes/ookinetes are derived from female gametocytes and are essential for zygote development into normal infective ookinetes.

Micro-array analysis of mutant retort-form ookinetes at 12h after fertilization showed decreased levels of transcripts of ookinete specific proteins. Evidence is presented that AP2-O binds to a six base sequence (TAGCTA) in the upstream promoter region of the genes whose transcripts are decreased in the absence of AP2-O. Evidence is presented that the TAGCTA sequence is a cis-acting element in the ookinete stage.

Other mutants:
RMgm-208: a mutant expressing a GFP-tagged form of AP2-O
RMgm-399: a mutant lacking expression of AP2-Sp (AP2 in sporozoites; PBANKA_132980;PF14_0633)
RMgm-398: a mutant expressing a GFP-tagged form of AP2-Sp (PBANKA_132980;PF14_0633)
RMgm-400: a mutant expressing a mutated form of AP2-O (PBANKA_090590; PF11_0442) whose AP2 domain had been swapped with that of AP2-Sp (PBANKA_132980;PF14_0633)

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0905900
Gene Model P. falciparum ortholog PF3D7_1143100
Gene productAP2 domain transcription factor AP2-O, putative
Gene product: Alternative nameAP2-O, AP2 in ookinetes; ApiAP2
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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 genePartial
Additional remarks partial/complete disruption The hdhfr selectable marker gene was integrated into the AP2-O locus, replacing a relative small part of the coding sequence
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTwo fragments of the AP2-O gene were amplified by PCR. The fragments were annealed to either side of the selectable marker gene (human DHFR) by PCR with primers 1 and 4.
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
Additional information primer 1Primer 1 (SacI)
Additional information primer 2Primer 2 (BamHI)
Additional information primer 3Primer 3 (XhoI)
Additional information primer 4Primer 4 (KpnI)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6