RMgmDB - Rodent Malaria genetically modified Parasites
Back to search resultsSummaryRMgm-4704
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Last modified: 23 January 2020, 12:03
*RMgm-4704| 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) : 31755154 |
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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 | Gupta R, Mishra S |
| Name Group/Department | Division of Molecular Parasitology and Immunology |
| Name Institute | CSIR-Central Drug Research Institute |
| City | Lucknow |
| Country | India |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_0309500 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0212600 | ||||||||||||||||||||||||
| Gene product | secreted protein with altered thrombospondin repeat domain | ||||||||||||||||||||||||
| Gene product: Alternative name | SPATR | ||||||||||||||||||||||||
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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/yfcu | ||||||||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | Multiple attempts to delete the spatr gene were unsuccessful indicating an essential role for multiplication/growth of asexual blood stages For the generation of Spatr knockout, two independent constructs were generated. For this, two fragments F1 (0.672 Kb) and F2 (0.564 Kb) were amplified using primer pairs 1239/1240 and 1241/1242 respectively. Both fragments were cloned in pBC‐GFP‐hDHFR vector at XhoI/ClaI for F1 and NotI/AscI for F2. Similar gene disruption vector was prepared with long homology regions (F3 and F4 with the size of 1.12 and 1.02 Kb, respectively) for double‐crossover recombination. F3 and F4 were amplified using primer pairs 1245/1240 and 1241/1246 respectively. Integration fragment was separated from vector backbone by digesting with XhoI/AscI and transfected into P. berghei ANKA WT line Secreted protein with altered thrombospondin repeat (SPATR) is a protein conserved in apicomplexan parasites and shown to be a micronemal protein in T. gondii. Plasmodium SPATR is a 30 kDa protein and is highly expressed during merozoite, gametocyte and sporozoite stages See RMgm-4703 for conditional knock-out of spatr in sporozoites. The following was found: Spatr cKO sporozoites glide and invade hepatocyte normally. Spatr cKO parasites develop normally into hepatic merozoites. SPATR is not required for hepatic merozoite egress from the parasitophorous vacuole while being essential for blood stage infection. Spatr cKO hepatic merozoites do not establish blood infection | ||||||||||||||||||||||||
| 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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