Back to search resultsSummaryRMgm-4132
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene mutation |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 28205319 |
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 | |
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The mutant parasite was generated by | |
Name PI/Researcher | Ghosh S; de Koning-Ward TF |
Name Group/Department | School of Medicine |
Name Institute | Deakin University |
City | Waurn Ponds |
Country | Australia |
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Name of the mutant parasite | |
RMgm number | RMgm-4132 |
Principal name | PbRAP1 iKD |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Blood stage parasites were analysed either with knock-down of RAP1 or knockdown of RAP2/3 (see RMgm-4133). Knockdown of RAP1 and RAP2 in PbRAP1 iKD and PbRAP2 iKD blood stages was performed by addition of the tetracycline derivative anhydrotetracycline (ATc) to (short-term) cultures of ringforms. Parasites of these cultures were analysed directly or after infection of mice. Analyses of both mutants showed the following: - Knockdown of RAPs does not alter rhoptry morphology or maturation of merozoites - Trafficking of RAP2/3 but not other rhoptry proteins is affected by knockdown of RAP1 - The RAP complex is not essential for erythrocyte invasion - Knocking down RAP expression affects parasite growth in vivo and the development of parasites post-invasion - Electron microscopy of RAP2/3 knockdown parasites reveals a PVM defect - RAP1 persists after erythrocyte invasion and is peripherally bound to the PVM These observation provide evidence for a role of these proteins the formation of the parasitophorous vacuole membrane (PVM) (and not in invasion of red blood cells). |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1032100 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1410400 | ||||||||||||||||||||||||||
Gene product | rhoptry-associated protein 1 | ||||||||||||||||||||||||||
Gene product: Alternative name | RAP1 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | The rap1 promoter modified to contain a tet-inducible promoter and TRAD4 activating domain | ||||||||||||||||||||||||||
Inducable system used | TET-based (TRAD4) | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Downregulation of RAP expression by addition of the tetracycline derivative anhydrotetracycline(ATc) | ||||||||||||||||||||||||||
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 | ||||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | The targeting vector for inducible knockdown of PbRAP1 and PbRAP2 was based on pPRF-TRAD4-Tet07-HAPRFhDHFR (Pino et al., 2012) that had been modified to include a NheI restriction site downstream of the profiling coding sequence and a BssHII restriction enzyme site between the profilin 5′ untranslated region and the TRAD4 sequence (Elsworth et al., 2014). This enabled cloning of the first 1.5 kilobases (kb) of the RAP1 coding sequence including its signal sequence (amplified with 91F/75R) into the PstI and NheI sites and 1.3 kb of the RAP1 5′ untranslated region (amplified using oligonucleotides 76F/77R) into the NheI and BssHII sites. For the RAP2 inducible knockout construct, the first 1.67 kb of the RAP2 cds (amplified with 147F/83R) and 1.15 kb of the RAP2 5′ untranslated region (amplified with 80F/156R) was cloned into the PstI/NheI and NheI/BssHII sites of the modified pPRF-TRAD4-Tet07-HAPRFhDHFR vector, respectively. Prior to transfection, the constructs were linearized with NheI. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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