RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4688
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0407300; Gene model (P.falciparum): PF3D7_0309100; Gene product: OMD protein, putative (ookinete motility deficient, OMD)
Phenotype Fertilization and ookinete; Oocyst; Sporozoite; Liver stage;
Last modified: 27 October 2019, 19:55
  *RMgm-4688
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 31553751
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/ResearcherCurrĂ  C, Mair GR
Name Group/DepartmentInstitute of Molecular Biology and Biotechnology
Name InstituteFORTH
CityHeraklion
CountryGreece
Name of the mutant parasite
RMgm numberRMgm-4688
Principal nameomd(-)cl1 and omd(-)cl2
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteOokinete formation did not reveal a significant difference between mutant and WT parasite development with ookinete morphologies similar as revealed by Giemsa-stained smears and scanning electron microscopy analysis. Ookinetes were also formed in vivo as revealed by Giemsa stained smears of mosquito midguts formed 24 h after feeding on an infected mouse.
Mature omd(-) ookinetes were completely devoid of productive motility; at most they displayed stretching of the ookinete.
In vivo transmission experiments, where mosquitoes are allowed to blood feed on mice infected with the omd(-)cl1 mutant, did not result in the establishment of oocysts in three independent feedings; WT parasites on the other hand transmitted normally.
OocystIn vivo transmission experiments, where mosquitoes are allowed to blood feed on mice infected with the omd(-)cl1 mutant, did not result in the establishment of oocysts in three independent feedings; WT parasites on the other hand transmitted normally.
SporozoiteNo oocyst and sporozoite formation
Liver stageNo oocyst and sporozoite formation. No transmission to mice by bite of infected mosquitoes.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of OMD

Protein (function)
MD was as highlighted in a group of motility and invasion-related genes such as gap45, tlp1, celtos and spect2, whose transcription was rapidly downregulated during sporozoite to liver stage development.
The gene has five exons and encodes a 176 amino acids long protein with a predicted N-terminal signal peptide. The protein is highly conserved within the Plasmodium genus.

Phenotype
Ookinete formation did not reveal a significant difference between mutant and WT parasite development  with ookinete morphologies similar as revealed by Giemsa-stained smears and scanning electron microscopy analysis. Ookinetes were also formed in vivo as revealed by Giemsa stained smears of mosquito midguts formed 24 h after feeding on an infected mouse.
Mature omd(-) ookinetes were completely devoid of productive motility; at most they displayed stretching of the ookinete.
In vivo transmission experiments, where mosquitoes are allowed to blood feed on mice infected with the omd(-)cl1 mutant, did not result in the establishment of oocysts in three independent feedings; WT parasites on the other hand transmitted normally. No oocyst and sporozoite formation. No transmission to mice by bite of infected mosquitoes.

Additional information
Analysis of a mutant expressing a C-terminal GFP-tagged version of OMD (RMgm-4689) showed the following:
Consistent with the RT-PCR data, fluorescence was apparent in gametocytes, ookinetes and oocysts, as well as in midgut and salivary gland sporozoites, but never detected in asexuals of the omd::gfp line. The OMD::GFP signal appeared mostly uniform and cytoplasmic. Western blot analysis showed that the fusion protein had the expected molecular weight of 48 kDa . We verified that omd::gfp parasites transmitted readily into the mosquito vector, showing that the tag did not interfere with the normal function of the protein; the omd::gfp parasite line produced an average of 6500 salivary gland sporozoites (n = 20) compared to 9450 (n = 25) in the WT control infection.

Evidence is presented for absence of expression of OMD in asexual blood stages and in liver stages.

Evidence is presented that  the failure of ookinete motility in the omd(-) mutant is not due to gross mis-localization of either micronemal proteins or components of the motility machinery.

Other mutants
a mutant expressing a C-terminal GFP-tagged version of OMD (RMgm-4689)


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0407300
Gene Model P. falciparum ortholog PF3D7_0309100
Gene productOMD protein, putative
Gene product: Alternative nameookinete motility deficient, OMD
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
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