RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4660
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1444500; Gene model (P.falciparum): PF3D7_1229800; Gene product: myosin J (MyoJ)
Transgene
Transgene not Plasmodium: GFP (gfp-mu3)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
PhenotypeNo phenotype has been described
Last modified: 8 August 2019, 14:30
  *RMgm-4660
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 31283102
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 507cl1 (RMgm-7)
Other information parent lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherWall RJ, Tewari R
Name Group/DepartmentSchool of Life Sciences, Queens Medical Centre
Name InstituteUniversity of Nottingham
CityNottingham
CountryLondon
Name of the mutant parasite
RMgm numberRMgm-4660
Principal name∆MyoJ
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 ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
The mutants lacks expression of MyoJ (mutants were made in the P. berghei ANKA and P. berghei ANKA GFPcon background parasites).

Protein (function)
The myosin superfamily is comprised of molecular motors present during early eukaryotic cell evolution. In unicellular parasites, they perform a wide variety of cellular functions that require movement, including differentiation, host interactions, and cell invasion. The myosin molecule contains three main domains: the N‐terminal head domain, which hydrolyses ATP and binds actin filaments; the neck domain/lever arm, which has an α‐helical structure containing up to six IQ motifs; and a tail region, which is required for cargo binding. There are six P. berghei myosins, two of these (MyoA and MyoB) have no tail region, and the remainder have a tail, which in the case of MyoF contains five WD40 repeats.
Quantitative reverse transcription polymerase chain reaction (qRT‐PCR) analyses of blood stages, ookinetes and sporozoites revealed that the Class XIV myosins MyoA (PBANKA_0135570), MyoB (PBANKA_0110300), and MyoE (PBANKA_0112200) were strongly transcribed in the invasive stages with an abundance of MyoE transcript in developing merozoites within schizonts. MyoF (PBANKA_1344100) was transcribed at all stages and was second only to MyoA in abundance. In contrast, low levels of transcription of MyoK (PBANKA_0908500) and MyoJ (PBANKA_1444500) could be seen throughout these life cycle stages.

Phenotype
No phenotype detected in blood stages, mosquito stages (ookinetes, oocysts, sporozoites) and liver stages (length of prepatent period in mice after bite by infected mosquitoes)

Additional information
We investigated the presence and location of the six myosin proteins throughout the life cycle, by adding a C‐terminal GFP tag to each via single homologous recombination at the corresponding myosin gene locus. MyoA‐GFP and MyoB‐GFP have been generated previously. In addition, MLC-B (PBANKA_0929400 myosin light chain B, putative) was C-terminally tagged with GFP and MyoA was C-terminally tagged with mCherry.

We used live cell imaging to detect expression of each protein throughout the life cycle and to examine their location. The Class XIV myosins were detected predominantly in the invasive stages (developing merozoites in schizonts and merozoites, ookinetes, and developing sporozoites within oocysts and sporozoites). As shown previously, MyoA‐GFP was associated with the surface pellicle of each invasive stage, whereas MyoB‐GFP was localised as an apical end dot in merozoites, mature (>20 hr) ookinetes, and late stage sporozoites. MyoE‐GFP was detected as a dot at the basal end of ookinetes and sporozoites and as a dot in merozoites. MyoE‐GFP was also expressed in liver stages, but as with merozoites, it was not possible to clearly identify the basal end of these stages. MyoF‐GFP was most abundant in the insect stages, particularly ookinetes and oocysts. In mature ookinetes, MyoF‐GFP was restricted to the apical end, whereas in oocysts, it was more evenly distributed throughout the developing sporozoites. In contrast, MyoJ‐GFP was only observed in mature oocysts, located at the junction between the differentiating sporozoites and the oocyst bodies. Following oocyst rupture, MyoJ‐GFP remained associated with the oocyst body and was not present in sporozoites. Finally, MyoK‐GFP was found exclusively associated with the nucleus of gametocytes, appearing as an arc across the cell, and in zygotes/early ookinetes, as two distinctive dots. These dots were either absent or only remnants were seen in mature ookinetes. 

Analyses of mutants expressing tagged myosins provide evidence for the following:
- distinct spatio‐temporal profiles for each myosin during ookinete development
- Colocalisation of myosins with MyoA and the apical marker, ISP1 (PBANKA_1209400; inner membrane complex sub-compartment protein 1)

Other mutants

 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1444500
Gene Model P. falciparum ortholog PF3D7_1229800
Gene productmyosin J
Gene product: Alternative nameMyoJ
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (gfp-mu3)
Details of the genetic modification
Inducable system usedNo
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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the 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