RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-4703
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*RMgm-4703| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Gene mutation, Introduction of a transgene |
| 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 | RMgm-4186 |
| Other information parent line | P. berghei ANKA TRAP/FlpL This line expresses FlpL recombinase under the control of the TRAP promoter, active in the mosquito oocyst stage, and selectively excises the DNA sequence flanked by FRT sites (S. Mishra, K.A. Kumar and P. Sinnis, unpublished data). The FlpL expression cassette is introduced into the silent p230p gene locus (by GIMO transfection using line 1596cl1; RMgm-687) resulting in line 1809 (RMgm-4186). The line has been generated in Leiden and cloned in the Sinnis lab. This line does not contain a drug-selectable marker. |
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| The mutant parasite was generated 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 |
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| Name of the mutant parasite | |
| RMgm number | RMgm-4703 |
| Principal name | Spatr cKO |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype | |
| Asexual blood stage | Not different from wild type |
| Gametocyte/Gamete | Not tested |
| Fertilization and ookinete | Not tested |
| Oocyst | Not different from wild type |
| Sporozoite | Not different from wild type |
| Liver stage | 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 phenotype | Mutant/Mutation (Part of) the spatr ORF is removed from the genome by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-269, RMgm-747). Removal of (part of) the FRTed ORF of spatr has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase in sporozoites that resulted in the excision of the (part of the) ORF of spatr which was flanked by FRT sequences. Protein (function) |
Mutated: Mutant parasite with a mutated 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 | |||||||||||||||||||||||||||
| Short description of the mutation | The mutant contains a FRTed (part of the) ORF of the spatr locus | ||||||||||||||||||||||||||
| Inducable system used | Flp/FRT | ||||||||||||||||||||||||||
| Short description of the conditional mutagenesis | (Part of) the ORF of spatr is removed in the sporozoite stage by the Flp/FRT system | ||||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||||
| Type of plasmid/construct | (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 | |||||||||||||||||||||||||||
| 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 | Flp recognition target (FRT) site was engineered in the only intron present in the gene. To generate Spatr conditional KO parasites, three fragments F5, F6 and F7 having a size of (1.2 Kb), (1.6 Kb) and (0.53 Kb) were amplified using primer pairs 1250/1252, 1253/1254 and 1255/1256 respectively. F6 was cloned at EcoRV site in the p3′TRAP‐hDHFR‐flirte vector. For the continuation of 3ʹUTR function, fragment F6 reverse primer also contained 12 bp of TRAP 3ʹUTR as described previously. Fragments F5 and F7 were subcloned sequentially at SacII/NotI and PstI/KpnI sites respectively. Final targeting cassette was separated from vector backbone by digestion with SacII/KpnI and used for transfection into P. berghei ANKA TRAP/FlpL line | ||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
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Transgene: Mutant parasite expressing a transgene| top of page | |||||||||||||||||||
| Type and details of transgene | |||||||||||||||||||
| Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
| Transgene name | FlpL recombinase of yeast (FlpL) | ||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||
| Type of plasmid/construct | (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 | |||||||||||||||||||
| 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 | The FlpL coding sequence was flanked by 1.58 kb and 0.55 kb of 5′ and 3′ regulatory sequences of trap, respectively. | ||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||
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| Other details transgene | |||||||||||||||||||
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| Promoter | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1335900 | ||||||||||||||||||
| Gene product | thrombospondin-related anonymous protein | sporozoite surface protein 2 | ||||||||||||||||||
| Gene product: Alternative name | TRAP; SSP2; SSP-2 | ||||||||||||||||||
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| 3'-UTR | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
| Gene product | sporozoite surface protein 2 thrombospondin-related anonymous protein | ||||||||||||||||||
| Gene product: Alternative name | sporozoite surface protein 2; SSP2; SSP-2 | ||||||||||||||||||
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| Insertion/Replacement locus | |||||||||||||||||||
| Replacement / Insertion | Replacement locus | ||||||||||||||||||
| Gene Model of Parasite | PBANKA_0306000 | ||||||||||||||||||
| Gene product | 6-cysteine protein | ||||||||||||||||||
| Gene product: Alternative name | 230p | ||||||||||||||||||
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