Summary

RMgm-208
Malaria parasiteP. berghei
Genotype
TaggedGene 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)
Name tag: GFP
Phenotype Fertilization and ookinete;
Last modified: 14 June 2010, 10:46
  *RMgm-208
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) : 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
CityMie
CountryJapan
Name of the mutant parasite
RMgm numberRMgm-208
Principal nameAP2-O::GFP
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 ookineteNo expression was observed in the gametocyte stage. Weak GFP fluorescent signals were observed in retort-form ookinetes beginning 8 h after fertilization. The signals were localized in the nucleus and signal intensities increased with ookinete development.
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a GFP-tagged form 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. See also mutant RMgm-207 lacking expression of AP2-O. Phenotype analyses of this mutant indicate an essential role of AP2-O during ookinete development and 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.

Phenotype
In wild type parasites ap2-0 transcripts are present in both gametocytes and ookinetes. No GFP 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. See also mutant RMgm-207 lacking expression of AP2-O.

Additional information

Other mutants
RMgm-207: A mutant lacking expression 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)


  Tagged: Mutant parasite with a tagged 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
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: taggingTo construct AP2-O::GFP parasites, the targeting vector containing a marker gene, human DHFR (hDHFR), was integrated into the AP2-O locus by double-cross-over recombination, adding the GFP gene to the C-terminal portion of AP2-O and conferring pyrimethamine resistance to the AP2-O::GFP parasites
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid 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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe targeted insertion vector was constructed in a pBluescript plasmid (Stratagene, La Jolla, CA, USA). For the targeted insertion construct, a DNA fragment containing the 3' part of the AP2-O coding region was amplified by PCR and inserted into the pBluescript XhoI/NheI site in frame with the GFP gene. The downstream region of the AP2-O gene was also amplified by PCR and inserted into the pBluescript BamHI/NotI site. Plasmids containing the construct were separated from plasmids without the construct by digestion with XhoI and NotI. AP2-O::GFP parasites were obtained by inserting the construct into the AP2-O locus by homologous recombination.
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