RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-32
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Last modified: 8 October 2015, 11:38
*RMgm-32| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Introduction of a transgene |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 20019192 |
| 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 | P. berghei ANKA cl15cy1 |
| Other information parent line | A reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255). |
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| The mutant parasite was generated by | |
| Name PI/Researcher | B. Franke-Fayard; C.J. Janse; A.P. Waters |
| Name Group/Department | Leiden Malaria Research Group |
| Name Institute | Leiden University Medical Center |
| City | Leiden |
| Country | The Netherlands |
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| Name of the mutant parasite | |
| RMgm number | RMgm-32 |
| Principal name | 1037cl1 |
| Alternative name | 1037m1f1mocl1; 1037m1f1m1cl1; PbGFP-Luc(ama1); PbGFP-Lucama1; wt-GFP-Luc(schiz); ANKA-GFP-Luc(schiz) |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype | |
| Asexual blood stage | GFP-Luciferase expression in mature schizonts and young ring forms. |
| Gametocyte/Gamete | Not different from wild type |
| Fertilization and ookinete | Not tested |
| Oocyst | GFP-Luciferase expression in maturing (late-stage) oocysts |
| Sporozoite | GFP-Luciferase expression in salivary gland sporozoites |
| Liver stage | GFP-Luciferase expression in maturing liver stages (liver merozoites) |
| Additional remarks phenotype | Mutant/mutation Phenotype Unpublished observations: GFP-Luciferase expression in maturing (late-stage) oocysts. (Relatively strong) GFP-Luciferase expression in salivary gland sporozoites and GFP-Luciferase expression in maturing liver stages (liver merozoites). Additional information In comparison with mutants expressing only GFP under the same promoter (ama1 promoter), the GFP-fluorescence intensity of this reporter mutant is lower as a result of the GFP-Luciferase fusion. This mutant is used for in vitro antimalarial drug screening in mice using the ITDL assay (Franke-Fayard et al., 2008, Int. J. Parasitol. 38, 1651-62). The in vitro drug luminescence (ITDL) assay, measured the success of schizont maturation in the presence of candidate drugs quantifying luciferase activity in mature schizonts only (ama-1 promoter). The ITDL assay generated drug-inhibition curves and EC(50) values comparable to those obtained with standard in vitro drug-susceptibility assays. See also the luciferase expressing mutant RMgm-29 which is used for in vivo antimalarial drug screening. Other mutants |
Transgene: Mutant parasite expressing a transgene| top of page | |||||||||||||||||||
| 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 |
  
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| Plasmid/construct sequence |
1 aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
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| Restriction sites to linearize plasmid | KspI (SacII) | ||||||||||||||||||
| Selectable marker used to select the mutant parasite | gfp (FACS) | ||||||||||||||||||
| Promoter of the selectable marker | ama-1 | ||||||||||||||||||
| 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_0915000 | ||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1133400 | ||||||||||||||||||
| Gene product | apical membrane antigen 1 | ||||||||||||||||||
| Gene product: Alternative name | |||||||||||||||||||
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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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