RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-5259
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Last modified: 7 November 2022, 14:06
*RMgm-5259| 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 | 3 |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 36162149 |
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| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. yoelii |
| Parent strain/line | P. y. yoelii 17XNL |
| 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 | Kamil M, Aly ASI |
| Name Group/Department | Aly Lab, Beykoz Institute of Life Sciences and Biotechnology |
| Name Institute | Bezmialem Vakif University |
| City | Istanbul |
| Country | Turkey |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PY17X_0921400 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1129000 | ||||||||||||||||||||||||
| Gene product | spermidine synthase | ||||||||||||||||||||||||
| Gene product: Alternative name | SpdS | ||||||||||||||||||||||||
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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 | unknown | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | The 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 | |||||||||||||||||||||||||
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Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
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