RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-309
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*RMgm-309| Successful modification | The parasite was generated by the genetic modification | ||||||||||||||||
| The mutant contains the following genetic modification(s) | Gene disruption, Gene disruption | ||||||||||||||||
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 19779564 | ||||||||||||||||
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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 2.34 | ||||||||||||||||
| Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). | ||||||||||||||||
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| The mutant parasite was generated by | |||||||||||||||||
| Name PI/Researcher | R.W. Moon; D.A. Baker; O. Billker | ||||||||||||||||
| Name Group/Department | Department of Cell and Molecular Biology | ||||||||||||||||
| Name Institute | Imperial College London | ||||||||||||||||
| City | London | ||||||||||||||||
| Country | UK | ||||||||||||||||
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| Name of the mutant parasite | |||||||||||||||||
| RMgm number | RMgm-309 | ||||||||||||||||
| Principal name | gcβ pdeδ double KO | ||||||||||||||||
| 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 | Normal fertilisation rates and ookinete production. Mutant ookinetes had a greatly reduced gliding motility (speed) comparable to the reduced motility of ookinetes of a mutant lacking only GCß (RMgm-307). The morphology of ookinetes was comparable to that of wild type ookinetes and no aberrant ookinetes were observed as described for a mutant lacking only PDEδ (RMgm-308). | ||||||||||||||||
| Oocyst | Not tested | ||||||||||||||||
| Sporozoite | Not tested | ||||||||||||||||
| Liver stage | Not tested | ||||||||||||||||
| Additional remarks phenotype | Mutant/mutation The following different genes encoding proteins with homology to cyclic nucleotide phosphodiesterase have been identified in Plasmodium
Analyses of a mutant lacking expression of PDEδ (RMgm-308) indicate that PDEδ has a crucial role in ookinete development and motility. Phenotype The mutant lacking expression of both PDEδ and GCß produced ookinetes that were affected in motility, comparable to the reduced motility of ookinetes of a mutant lacking only GCß (RMgm-307). However, the morphology of ookinetes was comparable to that of wild type ookinetes and no aberrant ookinetes were observed as described for a mutant lacking only PDEδ (RMgm-308). The suppression in the 'double knock-out mutant' of the phenotype of aberrant ookinete morphology in the mutant lacking expression of only PDEδ supports a model in which both PDEδ and GCß operate in the same cGMP dependent pathway (see 'Additional information').
In the paper evidence is presented that in ookinetes GCß is the main source for cGMP and that a cyclic GMP signalling module exists that regulates gliding motility of ookinetes. This evidence is partly based on the analysis of mutants lacking expression PDEδ (RMgm-308) and GCβ (RMgm-307) and the mutant described here lacking expression of both GCβ and PDEδ. These analyses indicate that PDEδ is the relevant cGMP degrading enzyme. Signalling via a cGMP-dependent protein kinase (PKG; PF14_0346; PB000726.02.0) may regulate ookinete differentiation and motility. | ||||||||||||||||
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1136700 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1360500 | ||||||||||||||||||||||||
| Gene product | guanylyl cyclase beta | ||||||||||||||||||||||||
| Gene product: Alternative name | GCβ, guanylyl cyclase beta | ||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||
| Type of plasmid/construct used | 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 | ClaI | ||||||||||||||||||||||||
| Partial or complete disruption of the gene | Partial | ||||||||||||||||||||||||
| Additional remarks partial/complete disruption | |||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | tgdhfr | ||||||||||||||||||||||||
| Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | The gcβ gene has been disrupted using a construct that integrates by single cross-over integration (insertion vector) that contains the tgdhfr selectable marker. The disadvantage of using an insertion construct is that the construct can be removed from the genome, thereby restoring the wild type genotype. This mutant has been described as mutant RMgm-307. The pdeδ gene has been disrupted using a construct that contains the tgdhfr selectable marker and that integrates by double cross-over recombination. This mutant has been described as mutant RMgm-308. The gcβ pdeδ double KO mutant was produced by crossing (in the mosquito) the gcβ KO mutant (RMgm-307) with the pdeδ KO mutant (RMgm-308). Multiple clones were genotyped to identify and select the gcβ pdeδ double KO mutants. | ||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||
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Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
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Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1333700 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1470500 | ||||||||||||||||||||||||
| Gene product | phosphodiesterase delta, putative | ||||||||||||||||||||||||
| Gene product: Alternative name | PDEδ, Phosphodiesterase delta | ||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||
| Type of plasmid/construct used | 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 | KpnI, BamHI | ||||||||||||||||||||||||
| Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
| Additional remarks partial/complete disruption | |||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | tgdhfr | ||||||||||||||||||||||||
| Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | The gcβ gene has been disrupted using a construct that integrates by single cross-over integration (insertion vector) that contains the tgdhfr selectable marker. The disadvantage of using an insertion construct is that the construct can be removed from the genome, thereby restoring the wild type genotype. This mutant has been described as mutant RMgm-307. The pdeδ gene has been disrupted using a construct that contains the tgdhfr selectable marker and that integrates by double cross-over recombination. This mutant has been described as mutant RMgm-308. The gcβ pdeδ double KO mutant was produced by crossing (in the mosquito) the gcβ KO mutant (RMgm-307) with the pdeδ KO mutant (RMgm-308). Multiple clones were genotyped to identify and select the gcβ pdeδ double KO mutants. | ||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||
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Primer information: Primers used for amplification of the target sequences
Primer information: Primers used for amplification of the target sequences
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