SummaryRMgm-5191
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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) | Not published (yet) |
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Other information parent line | In the mutant the gama gene has been replaced by the selectable marker cassette containing the positive/negative selectable marker hdhfr/yfcu.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-5191 |
Principal name | (CTRP)GAMA |
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 | Wild type numbers of oocyst. Reduced numbers of salivary gland sporozoites |
Sporozoite | Wild type numbers of oocyst. Reduced numbers of salivary gland sporozoites. |
Liver stage | (Midgut) sporozoites showed a strongly reduced capacity to establish an infection in mice (after intravenous injection). |
Additional remarks phenotype | Mutant/mutation In the manuscript two additional promoter swap mutants are described. In these mutants the promoter of the gama gene has been replaced by the promoter of cap380 gene (PBANKA_1218100) and the trap gene (PBANKA_1349800), respectively. Published in: bioRxiv preprint doi: https://doi.org/10.1101/2022.02.24.481744 Phenotype The promoter swap mutants, (ctrp)gama, (cap380)gama and (trap)gama showed the following phenotype: 2. (cap380)gama 3.(trap)gama In conclusion: in none of the promoter-swap mutants, the promoter swap could rescue (completely) the phenotype(s) observed in the mutant lacking expression of GAMA (∆GAMA; RMgm-5188) 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 promoter of the gama gene replaced by the promoter of the ctrp gene (PBANKA_0412900) | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||||
Type of plasmid/construct | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||||||||||
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 | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | A CRISPR/Cas9 methodology was used to generate promoter switch parasites. To generate promoter switch parasites, guides were identified in the 188 bp region just upstream of the GAMA ORF. Homology arms corresponding to the 758 bp upstream of this region (5’ flank), or the first 768 bp of the GAMA gene (3’ flank) were amplified from genomic DNA and ligated into the pYC-L2 plasmid (PMID: 24987097) via restriction cloning. Stage-specific promoters were amplified from genomic DNA and ligated between the homology arms; CTRP = 781bp, CAP380 = 1065 , TRAP = 946. Single stranded guide primers were annealed in a 50 μL volume (10 μL of each 100 μM primer with 25 μL water and 5 μL cutsmart buffer), by heating to 95 ºC for 5 minutes and allowing to cool to room temperature. Annealed guides were inserted into the pYC-L2 plasmid, via restriction cloning with the enzyme Esp3I. Guide integration was confirmed via sanger sequencing with the primer CJ059. | ||||||||||||||||||||||||||
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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