RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-798
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1029800; Gene model (P.falciparum): PF3D7_1412800; Gene product: glycylpeptide N-tetradecanoyltransferase | peptide N-myristoyltransferase (NMT)
Details mutation: The nmt promoter modified to contain a tet-inducible promoter and TRAD4 activating domain
Details conditional mutagenesis: Downregulation of nmt expression by addition of the tetracycline derivative anhydrotetracycline(ATc)
Phenotype Asexual bloodstage;
Last modified: 19 December 2012, 20:09
  *RMgm-798
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-798
Principal namenmt-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 (see 'Additional information').
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 nmt locus was mutated as follows:

A double crossing over recombination strategy was used to position TRAD4 downstream of the endogenous P. berghei nmt promoter, while the coding sequence of nmt 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 nmt.

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)
N-myristoyltransferase (NMT) catalyzes the transfer of myristate from myristoyl-CoA to the amino-terminal glycine residue on numerous cellular proteins, many of  which are subsequently modified by palmitoylation.  Myristoylation of proteins is a crucial regulatory mechanism implicated in a great variety of biological processes, and consequently NMT is essential for viability in many different cell types.

Phenotype
NMT expression in nmt-iKO blood stages was downregulated by addition of the tetracycline derivative anhydrotetracycline (ATc).
Strongly reduced growth (parasitemia) of blood stages in mice treated with ATc (see 'Additional information').

Additional information
After intravenous injection of equal numbers of parasitized erythrocytes, ATc treatment of mice delayed the appearance of a detectable parasitaemia from day 2 to day 8 p.i. From day 8, only four out of ten treated mice developed a detectable and increasing parasitaemia, suggesting thata small fraction of parasites escaped the ATc treatment. By isolating these surviving parasites, it was found that they had lost regulation of HA-NMT expression by ATc, an in vivo phenomenon that has also been observed with the Tet system in T. brucei. The nmt-iKO parasites harvested from mice after 24 hr treatment with ATc were assayed for intraerythrocytic development in vitro. While the morphology of the ring stages collected from the mice was normal, the maturation was severely impaired, resulting in the appearance of aberrant trophozoites and schizonts.

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1029800
Gene Model P. falciparum ortholog PF3D7_1412800
Gene productglycylpeptide N-tetradecanoyltransferase | peptide N-myristoyltransferase
Gene product: Alternative nameNMT
Details of the genetic modification
Short description of the mutationThe nmt promoter modified to contain a tet-inducible promoter and TRAD4 activating domain
Inducable system usedTET-based (TRAD4)
Short description of the conditional mutagenesisDownregulation of nmt 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 nmt locus was mutated as follows:

A double crossing over recombination strategy was used to position TRAD4 downstream of the endogenous P. berghei nmt 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 nmt.

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