Summary

RMgm-1172
Malaria parasiteP. yoelii
Genotype
Genetic modification not successful
DisruptedGene model (rodent): PY17X_1339000; Gene model (P.falciparum): PF3D7_1471100; Gene product: exported protein 2 (EXP2)
PhenotypeNo phenotype has been described
Last modified: 13 March 2015, 18:37
  *RMgm-1172
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 ≥ 5
Reference (PubMed-PMID number) Reference 1 (PMID number) : 25662767
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17X
Name parent line/clone Not applicable
Other information parent line
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherMeibalan, E; Burns, J.M.
Name Group/DepartmentCenter for Molecular Parasitology
Name InstituteDepartment of Microbiology and Immunology
CityDrexel University, Philadelphia PA
CountryUSA

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1339000
Gene Model P. falciparum ortholog PF3D7_1471100
Gene productexported protein 2
Gene product: Alternative nameEXP2
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe unsuccessful attempts to disrupt the exp2 gene indicates an essential role of EXP2 in blood stages.

The exp2 gene was targeted for disruption in both P. yoelii 17X (5 times) and 17XNL (2 times).

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). EXP2 has have been identified as a member of the PTEX complex.

A double cross-over homologous recombination strategy was used in an effort to knock out the pyexp2 gene. The pB3D-3M plasmid (obtained from Dr. Lawrence Bergman, Drexel University College of Medicine, PA) containing the pyrimethamine resistant Toxoplasma gondii dhfr-ts (Tg-dhfr-ts) cassette as a selectable marker was used to generate the targeting construct. Two fragments 292 bp and 331 bp flanking the 5’ end and 3’ end of the pyexp2 gene respectively were PCR amplified from P. yoelii 17X genomic DNA. The 5’ fragment was cloned into KpnI and HindIII sites while the 3’ fragment was cloned into BamHI and NotI sites to generate the plasmid pB3D-3M-ΔExp2. The plasmid DNA was isolated using the Qiagen Plasmid Mega kit (Qiagen) and digested with KpnI and NotI to release the fragment containing the drug resistant cassette flanked by 5’end and 3’end sequences. Ten micrograms of the linear DNA fragment were used for transfection of P. yoelii parasites.
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