RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-5092
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Last modified: 1 November 2021, 16:49
*RMgm-5092| 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) : 34673764 |
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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 | Hart KJ, Lindner SE |
| Name Group/Department | Department of Biochemistry and Molecular Biology, The Huck Center for Malaria Research |
| Name Institute | Pennsylvania State University |
| City | Pennsylvania |
| Country | USA |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PY17X_1027900 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1417200 | ||||||||||||||||||||||||
| Gene product | NOT family protein, putative | ||||||||||||||||||||||||
| Gene product: Alternative name | NOT1 | ||||||||||||||||||||||||
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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 | eef1a | ||||||||||||||||||||||||
| 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. Additional information: 'In agreement with the critical/essential role of NOT1 to eukaryotic RNA metabolism, our attempts to delete py17x_1027900 were largely unsuccessful. However, in 1 of 2 technical duplicates of 6 independent transfection attempts, we were able to obtain a 100% transgenic population with an extremely slow growth phenotype. It was only possible to produce this parasite line because the population was entirely transgenic, as the presence of even a small number of wild-type parasites with de novo resistance to pyrimethamine would likely have rapidly outgrown these transgenic parasites in the mouse. These data align with results from both PlasmoGEM (P. berghei) and piggyBac (P. falciparum) genetic screens that noted the importance of this gene to asexual blood-stage growth. Due to the severe asexual blood-stage defect, which closely aligned with observations of NOT1-related phenotypes in other eukaryotes, we propose that this “NOT Family Protein” is truly the NOT1 protein of Plasmodium parasites. Finally, it is notable that in contrast to other eukaryotes, PyNOT1 can be deleted and indicates that it is not strictly essential.' 'We identified a protein annotated only as “NOT Family Protein” (PY17X_1027900) that was found to be associated with CCR4-1. Bioinformatic analyses using PlasmoDB and BLASTp identified that the protein encoded by PY17X_1027900 mostly closely matched eukaryotic NOT1 proteins but lacked a bioinformatically predictable TTP-binding domain that is present in the gene currently annotated as NOT1 (PY17X_0945600). We concluded that these 2 not1 genes were paralogues due to the high degree of sequence conservation in specific domains at both the DNA and protein levels (DNA: CAF1-binding domain 72% identity, NOT4-binding domain 66% identity; Protein: CAF1-binding domain 41% identical/63% positive, CAF40-binding domain 33%/57%, NOT4-binding domain 48%/62%, NOT1 domain 50%/73%). Moreover, these genes are highly conserved, syntenic, and only present across Plasmodium species and 2 other species of the Aconoidasida class (Genera: Theileria, Babesia) but not in other apicomplexans, model eukaryotes, or humans.' | ||||||||||||||||||||||||
| 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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