RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-505
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1433600; Gene model (P.falciparum): PF3D7_1218000; Gene product: thrombospondin-related apical membrane protein (PTRAMP)
PhenotypeNo phenotype has been described
Last modified: 9 February 2013, 16:03
  *RMgm-505
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 3
Reference (PubMed-PMID number) Reference 1 (PMID number) : 22184632
Reference 2 (PMID number) : 15003842
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 1037m1f1mocl1 (RMgm-32)
Other information parent lineP. berghei ANKA 1037m1f1mocl1 (1037cl1; RMgm-32) is a reference ANKA mutant line which expresses GFP-luciferase under control of a schizont-specific promoter (PubMed: PMID: 20019192). It does not contain a drug-selectable marker and has been selected by FACS sorting based on GFP expression
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherJ. Fonager, E.M. Pasini, C.J. Janse, B.M Franke-Fayard
Name Group/DepartmentLeiden Malaria Research Group
Name InstituteLeiden University Medical Center (LUMC)
CityLeiden
CountryThe Netherlands

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1433600
Gene Model P. falciparum ortholog PF3D7_1218000
Gene productthrombospondin-related apical membrane protein
Gene product: Alternative namePTRAMP
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 Asp718I/EcoRI
Partial or complete disruption of the geneUnknown
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationName of unsuccessful attempts: exp. 1250/1306/1486
The gene has been targeted for disruption/deletion using a construct for targeted gene disruption by double cross-over homologous recombination. The primers of the two target sequences of the gene are provided below.
The unsuccessful attempts to disrupt the gene might indicate an essential function during asexual blood stage development.
The gene has been selected for targeted gene disruption based on its presence in a proteome of membranes of infected red blood cells.

See also Thompson et al., 2004. Mol. Biochem Parasitol. 134: 225-32: PTRAMP; a conserved Plasmodium thrombospondin-related apical merozoite protein.

In this study the protein has been localised in the micronemes of P. berghei merozoites.

However, The P. falciparum ortholog (PfTRAMP) localizes in the rhoptries. See Siddiqui et al, 2013 Cell Microbiol).
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 1GCCGGTACCCGAAAAATTACCAACATTGGGTG
Additional information primer 1F1 primer
Sequence Primer 2GCCAAGCTTCCCCAATATTTCTTGGTGCAATG
Additional information primer 2R1 primer
Sequence Primer 3GCCGATATCGTAGCTGGCCGATTTATGTATG
Additional information primer 3F2 primer
Sequence Primer 4GCCGAATTCGCCCAAATTAAAGCCATTACTTTC
Additional information primer 4R2 primer
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6