RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
Genetic modification not successful
TaggedGene model (rodent): PBANKA_0507500; Gene model (P.falciparum): PF3D7_1023300; Gene product: conserved protein, unknown function
Name tag: mCherry-3xMyc
PhenotypeNo phenotype has been described
Last modified: 23 September 2018, 20:25
Successful modificationThe gene/parasite could not be changed/generated by the genetic modification.
The following genetic modifications were attempted Gene tagging
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

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0507500
Gene Model P. falciparum ortholog PF3D7_1023300
Gene productconserved protein, unknown function
Gene product: Alternative name
Details of the genetic modification
Name of the tagmCherry-3xMyc
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(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
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPBANKA_0507500 encoding a hydrophobic polypeptide of 79 amino acid residues, remained refractory to endogenous tagging, most likely due to an interference of the fluorescent tag with essential protein functions

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. 5 proteins were shown to have a PV location in blood- and liver-stages.

For endogenous tagging of the PV candidates, a GFP(PV) co-localisation plasmid was constructed by fusing the bacterial backbone and adjacent mCherry-3xMyc tag sequence of the pBAT vector with the Plasmodium-specific fraction of the pGFP-PV plasmid using the KpnI and PvuII restriction sites. The sequences directly upstream of the stop codons were amplified from genomic DNA (ranging in size from 758 to 1,628 bp) and inserted into the GFP(PV) co-localisation plasmid using HpaI in combination with EcoRI or SacII. For double homologous recombination, 3′ fragments were amplified and cloned into the intermediate vectors as described for the gene deletion constructs.
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