SummaryRMgm-139
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene disruption, Introduction of a transgene |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 18312843 |
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 | Not applicable |
Other information parent line | The mutant is the result of a crossing between the transgenic GFP-expressing mutant RMgm-136 (ConF) and the mutant RMgm-138 that lacks expression of SPECT. Both mutants have been generated in an ANKA background parent line. |
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The mutant parasite was generated by | |
Name PI/Researcher | R. Amino, R. Menard |
Name Group/Department | Unité de Biologie et Génétique du Paludisme |
Name Institute | Institut Pasteur |
City | Paris |
Country | France |
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Name of the mutant parasite | |
RMgm number | RMgm-139 |
Principal name | SpectF |
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 different from wild type |
Fertilization and ookinete | Not different from wild type |
Oocyst | Not different from wild type |
Sporozoite | Normal numbers of midgut- and salivary gland sporozoites are formed. Sporozoites showed wild type gliding motility. Sporozoites had lost cell passage ability (membrane-damaging capacity) as shown in the cell-wounding assay. Compared to wild type sporozoites, the mutant sporozoites were rapidly immobilized in the dermis. Mutant sporozoites were shown to be arrested by and invade dermal fibroblasts. |
Liver stage | No difference was noticed between wild type and mutant parasites in number, size, and fluorescence intensity of EEF at 4, 12, 24, or 48 hr in rat or mouse primary hepatocytes. Sporozoites show in vitro a 'rapid invader' phenotype, as a result of the lack of cell traversal. Mutant sporozoites show an adhesion/attachment phenotype (to CRL-2017 cells in vitro) which is comparable to wild type sporozoites. |
Additional remarks phenotype | Mutant/mutation Protein (function) Phenotype Other mutants See also RMgm-135 and RMgm-137 for mutants lacking the expression of SPECT2/PPLP1 which show a comparable defect in cel traversal capacity, whereas the capabilty to invade hepatocytes is not affected. |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1355600 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1342500 | ||||||||||||||||||||||||
Gene product | sporozoite protein essential for cell traversal | ||||||||||||||||||||||||
Gene product: Alternative name | SPECT, SPECT1 | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (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 | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | pbdhfr | ||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||
Additional remarks genetic modification | In addition to a disrupted spect gene, this mutant contains gfp stably integrated into the genome (see below). The mutant is the result of a crossing between the transgenic GFP-expressing mutant RMgm-136 (ConF) and the mutant RMgm-138 that lacks expression of SPECT. | ||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||
Primer information: Primers used for amplification of the target sequences
Primer information: Primers used for amplification of the target sequences
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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 | pbdhfr | ||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | No PB gene model exists for SPECT, sporozoite protein essential for cell traversal, MAL13P1.212, hypothetical protein, conserved). The P. berghei GenBank Accession No. is AB125370. In addition to a stably integrated gfp gene, this mutant contains a disrupted spect gene (see above). The mutant is the result of a crossing between the transgenic GFP-expressing mutant RMgm-136 (ConF) and the mutant RMgm-138 that lacks expression of SPECT. ConF parasites were generated using a construct that integrates by double crossover integration of the gfp expression cassette at the dhfr-ts locus. The gfp coding region was fused to the upstream and downstream regions of the hsp70 gene and the resulting expression cassette was inserted between the pyrimethamine-resistant, mutant dhfr-ts gene and its downstream region (targeting construct). | ||||||||||||||||||
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_0711900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
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