Summary

RMgm-4095
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1205900; Gene model (P.falciparum): PF3D7_1007700; Gene product: AP2 domain transcription factor (ApiAP2)
PhenotypeNo phenotype has been described
Last modified: 15 January 2017, 13:00
  *RMgm-4095
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene disruption
Number of attempts to introduce the genetic modification 4
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28081440
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 676m1cl1 (RMgm-29)
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherModrzynska K, Billker O
Name Group/DepartmentWellcome Trust Sanger Institute
Name InstituteWellcome Trust Sanger Institute
CityHinxton, Cambridge
CountryUK

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1205900
Gene Model P. falciparum ortholog PF3D7_1007700
Gene productAP2 domain transcription factor
Gene product: Alternative nameApiAP2
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 usedYes
Name of PlasmoGEM construct/vector-
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 modificationThe unsuccessful attempts to delete this gene indicate an essential function during asexual blood stage development

From the paper: To target P. berghei ApiAP2 genes systematically, we succeeded in producing deletion vectors for all but one member of the family (PBANKA_1313200) and transfected each of them into a reporter line constitutively expressing GFP to facilitate phenotyping. Fourteen ApiAP2 genes resisted at least four disruption attempts with up to two different vector designs, providing tentative evidence that more than half of the genes in this family are potentially essential for asexual blood stage growth in vivo. For the remaining eleven, a KO line could be generated. These included six genes that had not previously been studied in Plasmodium.

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.
The paper presents a systematic knockout (KO) screen targeting the ApiAP2 family in the rodent malaria parasite P. berghei. Phenotyping of eleven viable ApiAP2 KO mutants reveals ten critical gene functions at different points in the life cycle.
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