RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4409
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1003900; Gene model (P.falciparum): PF3D7_0406200; Gene product: sexual stage-specific protein precursor (Pfs16)
Name tag: mCherry
PhenotypeNo phenotype has been described
Last modified: 14 December 2018, 11:15
  *RMgm-4409
Successful modificationThe 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) : 29307698
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/ResearcherDeligianni, E; Siden-Kiamos, I.
Name Group/DepartmentInstitute of Molecular Biology and Biotechnology
Name InstituteFORTH
CityHeraklion
CountryGreece
Name of the mutant parasite
RMgm numberRMgm-4409
Principal name100390-mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
In the mutant the PBANKA_1003900 gene has been (C-terminally) tagged with mCherry.
This gene is a 'syntenic ortholog' of sexual stage-specific protein precursor (Pfs16; PF3D7_0406200) of P. falciparum.
The similarity of P. falciparum Pfs16 and the syntenic P. berghei gene (Pbs16?) is below threshold.

To investigate protein expression a construct encoding PBANKA_1003900 fused at the C-terminus to mCherry was integrated by single cross-over in the endogenous locus and a clonal line was generated (the mCherry-tagged gene is under control of the 3'UTR of the PPPK-DHPS gene (used in the pBAT-SIL6 vector). Preliminary experiments revealed mCherry signal in the periphery of gametocytes Immunofluorescence analysis using an anti-mCherry antibody detected a diffuse signal in the cytoplasm of the parasite with no clear localization to the PVM (data not shown). We were thus unable to definitely localize the protein to the PVM. Western blot analysis revealed that a protein of the expected molecular weight was present in gametocytes. In ookinetes samples a very weak band was only seen after exposure for a very long time compared to the strong signal of the gametocyte sample at short exposure time. Most likely these traces of protein are derived from gametocytes persisting in the ookinete culture.

See also RMgm-968 for a mutant with a disrupted PBANKA_100390) gene locus
See the link PBANKA_1003900 for other mutants

 


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1003900
Gene Model P. falciparum ortholog PF3D7_0406200
Gene productsexual stage-specific protein precursor
Gene product: Alternative namePfs16
Details of the genetic modification
Name of the tagmCherry
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single 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/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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