SummaryRMgm-999
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene tagging |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 24594931 |
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 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 | Brochet, M; Billker, O. |
Name Group/Department | Wellcome Trust Sanger Institute, Hinxton |
Name Institute | Wellcome Trust Sanger Institute, Hinxton |
City | Hinxton, Cambridge |
Country | UK |
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Name of the mutant parasite | |
RMgm number | RMgm-999 |
Principal name | PKG-HA |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | No |
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Phenotype | |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not different from wild type |
Fertilization and ookinete | Mutant parasites show normal development throughout the complete life cycle. Expression of HA-tagged PKG in ookinetes |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Not different from wild type |
Additional remarks phenotype | Mutant/mutation Work in Toxoplasma gondii and Eimeria tenella, coccidian parasites that are related to Plasmodium, identified PKG as the primary target for two structurally distinct anticoccidal compounds, the trisubstituted pyrrole compound 1 (C1) and the imidazopyridine-based inhibitor compound 2 (C2). Both compounds achieve high selectivity over PKG of humans by exploiting an unusually small gatekeeper residue within the active site of all apicomplexan PKG enzymes. Mutating the threonine gatekeeper residue of apicomplexan PKG to a larger residue renders parasites resistant to both inhibitors. Gliding motility of ookinetes expressing PKG-HA was strongly inhibited by the inhibitor C2 (imidazopyridine-based inhibitor compound 2) with a half-maximal effect of ~100 nM. Expression of PKG(T619Q-HA), in contrast, conferred complete resistance to C2 up to at least 5 mM, demonstrating that PKG is the critical target for C2 and essential for ookinete gliding. Inhibition of gliding motility by C2 was as potent as disrupting cGMP production genetically by deleting guanylyl cyclase beta (GCβ; PBANKA_113670). In contrast, interfering with degradation of cyclic nucleotides through complete deletion of phosphodiesterase delta, putative (PDEδ; PBANKA_133370) had the opposite effect, resulting in a marked increase in average gliding speed |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1008200 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1436600 | ||||||||||||||||||||||||||
Gene product | cGMP-dependent protein kinase | ||||||||||||||||||||||||||
Gene product: Alternative name | PKG | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | triple-HA | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | Plasmid double cross-over | ||||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | Yes | ||||||||||||||||||||||||||
Name of PlasmoGEM construct/vector | - | ||||||||||||||||||||||||||
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 | No details are provided for the PlasmoGEM vector used The mutant does not contain a selectable marker. The positive/negative selectable marker cassette has been removed by negative selection with 5-FC. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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