SummaryRMgm-4540
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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 | Unknown |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 30232409 |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
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 | Matz JM, Matuschewski K |
Name Group/Department | Department of Molecular Parasitology |
Name Institute | Institute of Biology, Humboldt University |
City | Berlin |
Country | Germany |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0826700 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0925900 | ||||||||||||||||||||||||
Gene product | conserved Plasmodium protein, unknown function | ||||||||||||||||||||||||
Gene product: Alternative name | parasitophorous vacuole protein 5, PV5 | ||||||||||||||||||||||||
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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 | hdhfr | ||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||
Additional remarks genetic modification | Repeated 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
Primer information: Primers used for amplification of the target sequences
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