Back to search resultsSummaryRMgm-1172
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Successful modification | The 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 |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. yoelii |
Parent strain/line | P. y. yoelii 17X |
Name parent line/clone | Not applicable |
Other information parent line | |
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Attempts to generate the mutant parasite were performed by | |
Name PI/Researcher | Meibalan, E; Burns, J.M. |
Name Group/Department | Center for Molecular Parasitology |
Name Institute | Department of Microbiology and Immunology |
City | Drexel University, Philadelphia PA |
Country | USA |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_1339000 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1471100 | ||||||||||||||||||||||||
Gene product | exported protein 2 | ||||||||||||||||||||||||
Gene product: Alternative name | EXP2 | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (Linear) plasmid double cross-over | ||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | tgdhfr | ||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||
Additional remarks genetic modification | The 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
Primer information: Primers used for amplification of the target sequences
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