RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-916
Malaria parasiteP. berghei
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PBANKA_1334300; Gene model (P.falciparum): PF3D7_1471100; Gene product: exported protein 2 (EXP2)
PhenotypeNo phenotype has been described
Last modified: 30 July 2013, 08:48
  *RMgm-916
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) : 23869529
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA cl15cy1
Other information parent lineA reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255).
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Attempts to generate the mutant parasite were performed by
Name PI/ResearcherMatthews, K; de Koning-Ward, T.F.
Name Group/DepartmentSchool of Medicine
Name InstituteDeakin University
CityWaurn Ponds, Victoria
CountryAustralia
  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1334300
Gene Model P. falciparum ortholog PF3D7_1471100
Gene productexported protein 2
Gene product: Alternative nameEXP2
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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 geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedureThe unsuccessful attempts to disrupt the exp2 gene indicates an essential role of EXP2 in blood stages.

Plasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX (Plasmodium Translocon of EXported protein). HSP101 (PbANKA_094120), PTEX150 (PbANKA_100850), EXP2 (PbANKA_133430), PTEX88 (PbANKA_094130) and TRX2 (PbANKA_135800) have been identified as members of the PTEX complex.
These proteins are also expressed in early gametocytes, mosquito and liver stages. Although amenable to genetic tagging, HSP101, PTEX150, EXP2 and PTEX88 could not be genetically deleted in P. berghei, in keeping with the obligatory role this complex is postulated to have in maintaining normal blood-stage growth. In contrast, the putative thioredoxin-like protein TRX2 could be deleted, with knockout parasites displaying reduced grow-rates, both in vivo and in vitro.
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 1tagggcccGTGGTTAGCCTTAGAATGTTGTT
Additional information primer 1S9F: PbEXP2 5’ UTR (ApaI)
Sequence Primer 2gttccgcggGTTGTATGCTTATAGGTCGCATA
Additional information primer 2S10R: PbEXP2 5’ UTR (SacII)
Sequence Primer 3gcgatatcGCAAGAAACTGATTCTAATGAGGC
Additional information primer 3SF11: PbEXP2 3’ UTR (EcoRV)
Sequence Primer 4tacccgggCATTTAAGCACAAAGTGGCTGTA
Additional information primer 4S12R: PbEXP2 3’ UTR (SmaI)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren