RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4132
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1032100; Gene model (P.falciparum): PF3D7_1410400; Gene product: rhoptry-associated protein 1 (RAP1)
Details mutation: The rap1 promoter modified to contain a tet-inducible promoter and TRAD4 activating domain
Details conditional mutagenesis: Downregulation of RAP expression by addition of the tetracycline derivative anhydrotetracycline(ATc)
Phenotype Asexual bloodstage;
Last modified: 16 April 2017, 12:35
  *RMgm-4132
Successful modificationThe 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
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherGhosh S; de Koning-Ward TF
Name Group/DepartmentSchool of Medicine
Name InstituteDeakin University
CityWaurn Ponds
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-4132
Principal namePbRAP1 iKD
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageBlood 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/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
For tetracyclin (ATc)-dependent gene regulation the rap1 locus was modified to contain a tet-inducible promoter and TRAD4 activating domain

Protein (function)
In P. falciparum a low-molecular-weight protein complex, consisting of three non-covalently linked members, RAP1, RAP2 and RAP3 localises to the rhoptries (rhoptry bulb) of merozoites. RAP2 and RAP3 are encoded by adjacent genes, organised in a head to tail fashion. Both rap2 and rap3 show a high homology with the single copy, syntenic gene, rap2/3 of P. berghei.

Phenotype

In this study blood stage parasites were analysed either with knock-down of RAP1 or knockdown of RAP2/3 (see RMgm-4133). Knockdown of RAP1 and RAP2/3 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).

Additional information
Unsuccessful attempts to disrupt P. berghei rap1 and rap2/3 indicate an essential function during blood stage growth/development/invasion.  

Other mutants
See RAP1 and RAP2/3


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1032100
Gene Model P. falciparum ortholog PF3D7_1410400
Gene productrhoptry-associated protein 1
Gene product: Alternative nameRAP1
Details of the genetic modification
Short description of the mutationThe rap1 promoter modified to contain a tet-inducible promoter and TRAD4 activating domain
Inducable system usedTET-based (TRAD4)
Short description of the conditional mutagenesisDownregulation 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 usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe 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
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6