SummaryRMgm-5137
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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) : 36761022 |
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 | Fernandes P, Silvie O |
Name Group/Department | Sorbonne Université, INSERM, CNRS, Centre d’Immunologie et des Maladies Infectieuses |
Name Institute | CIMI-Paris |
City | Paris |
Country | France |
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Name of the mutant parasite | |
RMgm number | RMgm-5137 |
Principal name | PbΔb9 |
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 | Not tested |
Sporozoite | Not different from wild type |
Liver stage | C57BL/6 mice injected with 10,000 PbΔb9 did not develop a patent blood infection. Dramatic reduction in the number of PbΔb9 exoerythrocytic forms (EEFs) in vitro cultured HepG2 cells. Evidence presented that PbΔb9 were not able to form productive vacuoles in hepatocytes. A cell wound-repair assay indicated that the cell traversal activity of PbΔb9 sporozoites is not different to wild-type sporozoites, in both HepG2 and HepG2/CD81 cells. |
Additional remarks phenotype | Mutant/mutation Protein (function) Phenotype Evidence is presented that: Other mutants |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0808100 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0317100 | ||||||||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||||||||
Gene product: Alternative name | B9 | ||||||||||||||||||||||||
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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 | hdhfr/yfcu | ||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||||||||
Additional remarks genetic modification | The parasites have been generated and selected using the GOMO method of transfection (see RMgm-1026 for more details for this transfection method). For each target gene, a 5’ fragment and a 3’ fragment were amplified by PCR from P. berghei (ANKA) or P. yoelii (17XNL) WT genomic DNA and inserted into SacII/NotI and XhoI/KpnI restriction sites, respectively, of the GOMO-GFP vector, using the In-Fusion HD Cloning Kit (Clontech). The resulting targeting constructs were linearized with SacII and KpnI before transfection. | ||||||||||||||||||||||||
Additional remarks selection procedure | The parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting. 1) Transfected parasites are first selected in a mouse by pyrimethamine treatment 2) GFP+mCherry+ parasites are selected by FACS sorting and used to infect a mice 3) This mouse is treated with 5-FC to select for parasites that have the selectable marker removed 4) GFP+mCherry- and marker-free parasites are selected by FAC sorting and used to infect a mouse | ||||||||||||||||||||||||
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 | hdhfr/yfcu | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||
Additional remarks selection procedure | The parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting. 1) Transfected parasites are first selected in a mouse by pyrimethamine treatment 2) GFP+mCherry+ parasites are selected by FACS sorting and used to infect a mice 3) This mouse is treated with 5-FC to select for parasites that have the selectable marker removed 4) GFP+mCherry- and marker free parasites are selected by FAC sorting and used to infect a mouse | ||||||||||||||||||
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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_0808100 | ||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | B9 | ||||||||||||||||||
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