RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. yoelii
Genetic modification not successful
DisruptedGene model (rodent): PY17X_0921400; Gene model (P.falciparum): PF3D7_1129000; Gene product: spermidine synthase (SpdS)
PhenotypeNo phenotype has been described
Last modified: 7 November 2022, 14:06
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) : 36162149
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
Attempts to generate the mutant parasite were performed by
Name PI/ResearcherKamil M, Aly ASI
Name Group/DepartmentAly Lab, Beykoz Institute of Life Sciences and Biotechnology
Name InstituteBezmialem Vakif University

  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0921400
Gene Model P. falciparum ortholog PF3D7_1129000
Gene productspermidine synthase
Gene product: Alternative nameSpdS
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 parasitehdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood stage growth/multiplication

The deletion or knock-in/tagging of PySpdS gene was accomplished by double-crossover homologous recombination.
For the Knock-out construct, the PCR amplified sequences were inserted into AA20 transfection plasmid between SacII-BamHI and HindIII-KpnI restriction enzyme sites for the 3'UTR and 5'UTR sequences, respectively. For the knock-in/tagging construct, the 5'UTR region cloned into SacII-BamHI in the knockout construct was replaced by the coding sequence of SpdS which was PCR amplified by primer pair and inserted into plasmid between SacII-EcoRI. The fragments cloned for SpdS were designed not to interfere with the coding sequences of any upstream or downstream neighboring genes and the makeup of those fragments was checked by sequencing to ensure that any unknown promoter or termination signals for the neighboring genes were not altered. Both knockout and knock-in vectors were linearized with SacII-KpnI before transfection to P. yoelii 17X-NL. Pilot experiments (that were repeated twice) only with the knockout construct to target SpdS for deletion with the supplementation of varying concentrations (from 50 mg/kg and up to 100 mg/kg per mouse per day; starting one day before transfection) of Spermidine and Spermine into mice receiving transfected parasites. Positive drug selection of resistant parasites with pyrimethamine and cloning of transfected parasites were done as described.
The first several attempts to generate SpdS-deficient parasites were not successful, despite the daily supplementation of Spermidine and Spermine to mice carrying the transfected parasites. To rule out any technical shortcoming or genetic accessibility in the inability to delete the PySpdS gene, we used a knock-in/knockout gene targeting strategy. In this strategy, the knock-in construct replaces the whole coding sequence or part of the coding sequence with an identical copy followed by eGFP sequence, and then followed by a constitutive 3' UTR. The knockout construct shares the same right homology arm as the knock-in construct, however, the left homology arm of the knockout construct was designed to delete or replace the gene with its own copy. The PCR genotyping confirms only the integration of the knock-in construct in the chromosomal locus (PySpdS-eGFP), however, the knockout construct for PySpdS did not integrate into the genome.

To find out the cellular localization of SpdS in mouse and mosquito stages of the parasites, blood-stages and sporozoites of the eGFP tagged PySpdS parasites were co-stained with the mitochondrial staining dye Mito-tracker Red. The eGFP signals of PySpdS were clearly co-localized with the mitochondrial marker dye Mito-tracker Red at both blood-stages and sporozoites. These results confirm the mitochondrial localization of the malaria parasite SpdS.
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