RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-797
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_0833000; Gene model (P.falciparum): PF3D7_0932200; Gene product: profilin (PFN)
Details mutation: The prf promoter modified to contain a tet-inducible promoter and TRAD4 activating domain
Details conditional mutagenesis: Downregulation of prf expression by addition of the tetracycline derivative anhydrotetracycline(ATc)
Phenotype Asexual bloodstage; Fertilization and ookinete;
Last modified: 19 December 2012, 20:08
  *RMgm-797
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) : 23245327
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherPino, P.; Billker, O.; Soldati-Favre, D.
Name Group/DepartmentDepartment of Microbiology and Molecular Medicine
Name InstituteUniversity of Geneva
CityGeneva
CountrySwitzerland
Name of the mutant parasite
RMgm numberRMgm-797
Principal nameprf-iKO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageStrongly reduced growth (parasitemia) in mice treated with ATc
Gametocyte/GameteNot different from wild type
Fertilization and ookineteReduced (50%) ookinete production in the presence of ATc
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
For tetracyclin (ATc)-dependent gene regulation the prf locus was mutated as follows:

A double crossing over recombination strategy was used to position TRAD4 downstream of the endogenous P. berghei prf promoter, while the coding sequence of prf was brought under the control of the inducible promoter containing the tet operator. Simultaneously, two HA epitope tags were placed at the N terminus of the resulting inducible copy of prf.

Transctivator TRAD4 was made by a gene fusion between TetRep and an AD (activating domains/transactivating regions)  of the ApiAP2 protein PFF0200c (= PfSIP2; fragment PfSIP2_7.3).

Protein (function)
Profilins are ubiquitous and essential actin monomer binding proteins, known to interact with proline-rich regions in a variety of proteins and to regulate actin filament formation. For fast actin polymerization at selected sites, profilin is an essential control element by recruiting actin monomers in a polymerizable form to actin polymerization machineries. Profilins are usually defined by their shared, highly conserved structure and by their ability to bind actin, proline-rich sequences.

Failure to disrupt the prf gene indicates that PRF is essential for blood stages (see RMgm-389).

Phenotype
Prf expression in prf-iKO blood stages was downregulated (90%) by addition of the tetracycline derivative anhydrotetracycline (ATc).
Strongly reduced growth (parasitemia) of blood stages in mice treated with ATc.

Additional information
Evidence is presented that invasion of erythrocytes by prf-iKO merozoites is reduced in the presence of ATc.
No effect of ATc treatment was observed on gametocyte production and exflagellation.
Evidence is presented that ATc treatment of prf-iKO parasites influences ookinete production.

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0833000
Gene Model P. falciparum ortholog PF3D7_0932200
Gene productprofilin
Gene product: Alternative namePFN
Details of the genetic modification
Short description of the mutationThe prf promoter modified to contain a tet-inducible promoter and TRAD4 activating domain
Inducable system usedTET-based (TRAD4)
Short description of the conditional mutagenesisDownregulation of prf expression by addition of the tetracycline derivative anhydrotetracycline(ATc)
Additional remarks inducable system
Click to view information
Click to hide information
Conditional expression system for stage-specific, tetracycline-dependent (ATc) gene regulation.

For in vivo treatment of mice, ATc (SIGMA) was dissolved in water + 5% sucrose at a concentration of 0.2 mg/ml. The drinking water bottle was wrapped in aluminum foil to prevent precipitation of ATc due to light and the solution changed every 48 hr. For in vitro treatments, ATc was diluted in culture medium at 1 mg/ml as previously described (Meissner et al., 2005).
Type of plasmid/constructPlasmid 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 modificationFor tetracyclin (ATc)-dependent gene regulation the prf locus was mutated as follows:

A double crossing over recombination strategy was used to position TRAD4 downstream of the endogenous P. berghei prf promoter, while the coding sequence of prf was brought under the control of the inducible promoter containing the tet operator. Simultaneously, two HA epitope tags were placed at the N terminus of the resulting inducible copy of prf.

Transctivator TRAD4 was made by a gene fusion between TetRep and an AD (activating domains/transactivating regions) of the ApiAP2 protein PFF0200c (= PfSIP2; fragment PfSIP2_7.3).
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