SummaryRMgm-684
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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, Introduction of a transgene |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 22177563 |
MR4 number | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei NK65 |
Name parent line/clone | P. berghei NK65 TRAP/FlpL(-)F |
Other information parent line | TRAP/FlpL(-)F is a P. berghei NK65 mutant (RMgm-683) that expresses the yeast FlpL recombinase under the control of the trap promoter. In addition it expresses GFP under the control of the constitutive hsp70 promoter. The mutant contains the dhfr/ts gene of P. berghei as a drug-selectable marker (PubMed: PMID: 21886105). |
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The mutant parasite was generated by | |
Name PI/Researcher | D. Giovannini; 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-684 |
Principal name | AMA1/Cond |
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 | In the absence of AMA1 expression, sporozoites were infective to mice as determined by measuring liver loads by qPCR after intravenous injection of sporozoites. In addition, 'AMA1-silenced' sporozoites showed normal infectivity (traversal and invasion) to hepatocytes in vitro. 'AMA1-silenced' sporozoites were not able to produce blood stage infections |
Liver stage | In the absence of AMA1 expression, sporozoites were infective to mice as determined by measuring liver loads by qPCR after intravenous injection of sporozoites. In addition, 'AMA1-silenced' sporozoites showed normal infectivity (traversal and invasion) to hepatocytes in vitro. 'AMA1-silenced' sporozoites were not able to produce blood stage infections |
Additional remarks phenotype | Mutant/mutation In the mutant described here the Flp/FRT site-specific recombination (SSR) system of yeast has been used to silence AMA1 expression specifically in sporozoites. Removal of the FRTed 3' UTR sequence of ama1 has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase in sporozoites that resulted in the excision of the ama1 sequence which was flanked by FRT sequences. In the same study a conditional knock-out mutant' of RON4 has been generated using the same approach as described here (see RMgm-685). It is shown that in contrast to AMA1, RON4 is essential for sporozoite invasion of hepatocytes. |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0915000 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1133400 | ||||||||||||||||||||||||||
Gene product | apical membrane antigen 1 | ||||||||||||||||||||||||||
Gene product: Alternative name | AMA1 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | The mutant contains a mutated ama1 gene with two FRT sites | ||||||||||||||||||||||||||
Inducable system used | Flp/FRT | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | The FRTed sequence of ama1 is removed in the sporozoite stage by the Flp/FRT system | ||||||||||||||||||||||||||
Additional remarks inducable system |
A conditional mutagenesis approach was utilized that uses stage-specific expression of a temperature-sensitive variant of the yeast recombinase, flippase (FlpL) to catalyze the excision of DNA placed between two Flp Recognition Target sequences (FRT) (see RMgm-683). FlpL catalyzes site-specific DNA recombination at a temperature range of 25°-30°C. The ama1 locus was modified by replacing the 3' UTR of ama1 with a 3' UTR of trap which contains the two FRT sites. This mutant has been generated by replacement of the 3'UTR of the endogenous ama1 gene by an 'FRTed' ama1 gene in mutant RMgm-683 that expresses FlpL. | ||||||||||||||||||||||||||
Type of plasmid/construct | 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 | ||||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
Selection (positive) procedure | WR99210 | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | FlpL recombinase of yeast (FlpL) | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | Flp/FRT | ||||||||||||||||||
Additional remarks inducable system | The mutant expresses the yeast FlpL recombinase under the control of the promoter of trap. The mutant does not contain a selectable marker. This marker (the hdhfr gene) has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the hdhfr gene has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences. | ||||||||||||||||||
Type of plasmid/construct | Plasmid single 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 | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | The FlpL coding sequence was flanked by 1.5 kb and 0.6 kb of 5′ and 3′ regulatory sequences of trap, respectively, and associated with a FRTed hdhfr selectable marker containing its own (eef1a) promoter and terminator sequences. The plasmid was linearized in the 5′ TRAP regulatory sequence to direct gap repair and plasmid integration by single crossover recombination at the homologous chromosomal locus, leaving the endogenous trap gene intact. The plasmid contained the hdhfr selectable marker that is flanked with the 34bp FRT sequences. In the resulting mutant (TRAP/Flp(-), by integrating at the DHFR-TS locus, a gfp gene under control of the hsp70 promoter was integrated into the dhfr/ts locus, using a construct that contains a pyrimethamine resistant pbdhfr-ts gene. | ||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1335900 | ||||||||||||||||||
Gene product | thrombospondin-related anonymous protein | sporozoite surface protein 2 | ||||||||||||||||||
Gene product: Alternative name | TRAP; SSP2; SSP-2 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
Gene product | sporozoite surface protein 2 thrombospondin-related anonymous protein | ||||||||||||||||||
Gene product: Alternative name | sporozoite surface protein 2; SSP2; SSP-2 | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Insertion locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
Gene product | sporozoite surface protein 2 thrombospondin-related anonymous protein | ||||||||||||||||||
Gene product: Alternative name | sporozoite surface protein 2; SSP2; SSP-2 | ||||||||||||||||||
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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 | 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 | The FlpL coding sequence was flanked by 1.5 kb and 0.6 kb of 5′ and 3′ regulatory sequences of trap, respectively, and associated with a FRTed hdhfr selectable marker containing its own (eef1a) promoter and terminator sequences. The plasmid was linearized in the 5′ TRAP regulatory sequence to direct gap repair and plasmid integration by single crossover recombination at the homologous chromosomal locus, leaving the endogenous trap gene intact. The plasmid contained the hdhfr selectable marker that is flanked with the 34bp FRT sequences. In the resulting mutant (TRAP/Flp(-), by integrating at the DHFR-TS locus, a gfp gene under control of the hsp70 promoter was integrated into the dhfr/ts locus, using a construct that contains a pyrimethamine resistant pbdhfr-ts gene. To make a GFP expression cassette driven by the hsp70 promoter, the 1.2 kb upstream region and 1.0 kb downstream region of hsp70 were ligated to either side of the GFP coding sequence. To make a targeted insertion vector, this expression cassette was ligated into the downstream region of the pyrimethamine-resistant pbdhfr-ts gene | ||||||||||||||||||
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 | Insertion locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts; pbdhfr/ts | ||||||||||||||||||
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