Summary

RMgm-4540
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_0826700; Gene model (P.falciparum): PF3D7_0925900; Gene product: conserved Plasmodium protein, unknown function (parasitophorous vacuole protein 5, PV5)
PhenotypeNo phenotype has been described
Last modified: 23 September 2018, 21:30
  *RMgm-4540
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 Unknown
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30232409
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
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherMatz JM, Matuschewski K
Name Group/DepartmentDepartment of Molecular Parasitology
Name InstituteInstitute of Biology, Humboldt University
CityBerlin
CountryGermany

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0826700
Gene Model P. falciparum ortholog PF3D7_0925900
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative nameparasitophorous vacuole protein 5, PV5
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 usedNo
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
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationRepeated attempts to delete the gene encoding PV5 were unsuccessful, indicating an essential function for blood stage growth/multiplication.

For the generation of gene deletion constructs, 3′ fragments were amplifed from genomic DNA (ranging in size from 837 to 1,020 bp) and cloned into the pBAT-G624 or the pGFP-PV plasmid using the XhoI and KpnI restriction sites. Subsequently, 5′ fragments (ranging in size from 808 to 1,003bp) were amplifed and cloned into the intermediate vectors using SacII in combination with EcoRI or PvuII. Prior to transfection into wild-type ANKA parasites, knockout vectors were digested with XmnI and SalI.

The protein was selected in a screen for putative PV (parasitophorous vacuole) proteins.
Proteins are targeted to the PV by means of default protein secretion, which is initiated by the recognition and cleavage of an amino-terminal signal peptide (SP). To identify novel PV candidates, we searched the Plasmodium genome database (PlasmoDB) for SP-containing proteins and sequentially removed proteins containing predicted transmembrane domains, export motifs, apicoplast targeting peptides, endoplasmic reticulum (ER) retention signals and GPI anchors, which could potentially redirect the protein to other locations. Twelfe proteins were selected. Five (PV1-5) proteins were shown to have a PV location in blood- and liver-stages.

Five (PV1-5; PBANKA_0919100; PBANKA_1441700; PBANKA_1350500; PBANKA_1349200; PBANKA_0826700) proteins were shown to have a PV location in blood- and liver-stages.
No phenotype was detected for blood and liver stages that lack expression of PV1, PV2, PV3 and PV4.
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