SummaryRMgm-5157
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Introduction of a transgene, Introduction of a transgene |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 37143657 |
MR4 number | |
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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-1026 |
Other information parent line | The mutant expresses GFP under the control of the HSP70 promoter. The reporter gene is introduced into the silent 230p locus using the GOMO method of transfection and is drug-selectable marker-free. |
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The mutant parasite was generated by | |
Name PI/Researcher | Belhimeur S, Silvie S, Mecheri S |
Name Group/Department | Institut Pasteur, Université de Paris |
Name Institute | CNRS |
City | Paris |
Country | France |
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Name of the mutant parasite | |
RMgm number | RMgm-5157 |
Principal name | IL-6 Tg-PbANKA/LISP2 line1,2 |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | No |
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Phenotype | |
Asexual blood stage | Not tested |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | No blood-stage infection developed in mice after infecting with isolated sporozoites. Mutant sporozoites invade and develop in cultured hepatocytes (complete liver stage development and egress) but in mice arrest during liver stage development. In mice the mutant parasite liver load remained low over the course of infection, except at the 48-h time point, when the amount of mutant parasites rose but at levels significantly lower than those obtained for wild-type parasites. |
Additional remarks phenotype | Mutant/mutation Published in: bioRxiv preprint doi: https://doi.org/10.1101/2021.11.16.468835 |
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | murine IL6 | ||||||||||||||||||
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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 | ||||||||||||||||||
Promoter of the selectable marker | unknown | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | To generate transgenic P. berghei parasites expressing the murine IL6 transgene, we used a selection-linked integration strategy. We assembled a plasmid construct, named GFP-SLI-IL6, containing two cassettes. The first cassette includes a 3’ terminal sequence of the GFP coding sequence, fused to a 2A skip peptide and the human dihydrofolate reductase (hDHFR) gene, followed by the 3’ UTR of P. berghei calmodulin (CAM) gene. The second cassette corresponds to a codon-optimized version of murine IL6, under control of the promoter of P. berghei LISP2, and followed by the 3’ UTR of P. berghei DHFR. To ensure IL6 secretion, the peptide signal peptide of mIL6 was replaced by the signal peptide of P. berghei LISP2. | ||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1003000 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0405300 | ||||||||||||||||||
Gene product | liver specific protein 2, putative | sequestrin | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | LISP2 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
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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 | P230p | ||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | GFP | ||||||||||||||||||
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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 | To generate transgenic P. berghei parasites expressing the murine IL6 transgene, we used a selection-linked integration strategy. We assembled a plasmid construct, named GFP-SLI-IL6, containing two cassettes. The first cassette includes a 3’ terminal sequence of the GFP coding sequence, fused to a 2A skip peptide and the human dihydrofolate reductase (hDHFR) gene, followed by the 3’ UTR of P. berghei calmodulin (CAM) gene. The second cassette corresponds to a codon-optimized version of murine IL6, under control of the promoter of P. berghei LISP2, and followed by the 3’ UTR of P. berghei DHFR. To ensure IL6 secretion, the peptide signal peptide of mIL6 was replaced by the signal peptide of P. berghei LISP2. | ||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0818900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
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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 | P230p | ||||||||||||||||||
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