SummaryRMgm-5189
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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) : 37314965 |
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-5188 |
Other information parent line | In the mutant the gama gene has been replaced by the selectable marker cassette containing the positive/negative selectable marker hdhfr/yfcu. This enables to introduce additional genes in the disrupted gama locus by the method of GIMO transfection |
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The mutant parasite was generated by | |
Name PI/Researcher | Jennison C, Vaughan AM |
Name Group/Department | Center for Global Infectious Disease Research |
Name Institute | Seattle Children’s Research Institute |
City | Seattle |
Country | USA |
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Name of the mutant parasite | |
RMgm number | RMgm-5189 |
Principal name | Pb(PfGAMA) |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not tested |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | (Strongly) reduced oocyst production. However, the defect in midgut infectivity appeared less severe than for ΔGAMA parasites |
Sporozoite | (Strongly) reduced oocyst production and strongly reduced numbers of midgut- and salivary gland sporozoites. Evidence is provided for a defect in the egress of sporozoites from oocysts. |
Liver stage | (Midgut) sporozoites showed a strongly reduced capacity to establish an infection in mice (after intravenous injection). |
Additional remarks phenotype | Mutant/mutation Protein (function) Phenotype The mutant with the P. berghei gama gene replaced with the P. falciparum gama gene showed the following phenotype: Conclusion: P. falciparum GAMA can only partially complement the function of P. berghei GAMA Additional information |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0701900 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0828800 | ||||||||||||||||||||||||||
Gene product | GPI-anchored micronemal antigen | ||||||||||||||||||||||||||
Gene product: Alternative name | GAMA, Putative Secreted Ookinete Protein 9, PSOP9 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | The P. berghei gama gene replaced by the P. falciparum gama gene | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
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 | No | ||||||||||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||||||||||
Additional remarks genetic modification | To generate the knock-out and P. falciparum GAMA knock-in parasite lines the Gene In Marker Out (GIMO) methodology was employed. In brief, the GIMO plasmid was modified with the addition of 5’ and 3’ homology arms corresponding with the 5’ and 3’ UTR of GAMA, flush to the ORF of GAMA, so that a KO parasite would be generated expressing the hdhfr/yfcu cassette in place of the GAMA gene. Transfections were performed in the marker-free, genome integrated GFPLuc expressing P. berghei parasite line Pb676, which was used as wild type throughout this study. Transfectant parasites obtained after negative selection with pyrimethamine were cloned by limiting dilution into naïve recipient mice to obtain clonal parasites. To generate the Pb PfGAMA parasite line, the full-length P. falciparum GAMA gene was amplified from P. falciparum genomic DNA and inserted via the KpnI and SacII restriction sites into the PbGAMA knockout GIMO plasmid. Transfections were performed on ∆GAMA parasites and integration of PfGAMA was achieved by negative selection with 5-fluorocytosine. Clonal parasites were obtained. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV) | ||||||||||||||||||
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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 | gfp (FACS) | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | FACS (flowsorting) | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | The GFP-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration. | ||||||||||||||||||
Additional remarks selection procedure | This reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence. | ||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1133300 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1357100 | ||||||||||||||||||
Gene product | elongation factor 1-alpha | ||||||||||||||||||
Gene product: Alternative name | eef1a | ||||||||||||||||||
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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 | 230p | ||||||||||||||||||
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