RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4874
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0807700; Gene model (P.falciparum): PF3D7_0317500; Gene product: kinesin-5 (EG5)
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)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 24 September 2020, 12:37
  *RMgm-4874
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) Not published (yet)
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/ResearcherZeeshan M, Tewari R
Name Group/DepartmentSchool of Life Sciences
Name InstituteUniversity of Nottingham
CityNottingham
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4874
Principal nameΔ kinesin-5
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
OocystThere was no significant difference in the number of Δkinesin-5 and wild type WT-GFP oocysts, and the size of the oocysts was similar for both parasites. However, we observed a 40 to 50% decrease in the number of sporozoites in each oocyst at days 14 and 21 post-infection in Δkinesin-5 parasites compared to WT-GFP parasites.
SporozoiteA significant decrease in the number of Δkinesin-5 sporozoites in salivary glands was also observed, but the shape, size, and motility of these sporozoites were indistinguishable from WT-GFP parasites. Normal infectivity of sporozoites.
Liver stageNormal infectivity of sporozoites as determined by blood stage infection in mice after infection by mosquito bite
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of kinesin-5 and expresses GFP under the constitutive eef1a promoter
( bioRxiv preprint doi:https://www.biorxiv.org/content/10.1101/2020.07.03.186031v2 , this version posted September 22, 2020)

Protein (function)
Kinesin-5 proteins are a family of molecular motors that is structurally and functionally conserved throughout eukaryotes. They are involved in spindle pole separation and are considered essential for mitosis in the vast majority of eukaryotes. Kinesin-5 is located at spindle MTs and spindle poles during cell division and is distributed diffusely in the cytoplasm during interphase in most eukaryotic cells. 
In Plasmodium berghei there are nine kinesin genes, including two kinesin-8 genes that are important in cell division and male gamete formation. There is a single Plasmodium kinesin-5.

Phenotype
Normal blood stage development, gametocyte and ookinete production. There was no significant difference in the number of Δkinesin-5 and wild type WT-GFP oocysts, and the size of the oocysts was similar for both parasites. However, we observed a 40 to 50% decrease in the number of sporozoites in each oocyst at days 14 and 21 post-infection in Δkinesin-5 parasites compared to WT-GFP parasites. A significant decrease in the number of Δkinesin-5  sporozoites in salivary glands was also observed, but the shape, size, and motility of these sporozoites were indistinguishable from WT-GFP parasites. Normal infectivity of sporozoites as determined by blood stage infection in mice after infection by mosquito bite


Additional information
From the Abstract: 'Deletion of kinesin-5 had little visible effect at any proliferative stage except sporozoite production in oocysts, resulting in a significant decrease in the number of motile sporozoites in mosquito salivary glands, which were able to infect a new vertebrate host. Live-cell imaging showed kinesin-5-GFP located on the spindle and at spindle poles during both atypical mitosis and meiosis. Fixed-cell immunofluorescence assays revealed kinesin-5 co-localized with α-tubulin and centrin-2 and a partial overlap with kinetochore marker NDC80 during early blood stage schizogony. Dual-colour live-cell imaging showed that kinesin-5 is closely associated with NDC80 during male gametogony, but not with kinesin-8B, a marker of the basal body and axonemes of the forming flagella.'

To quantify the expression of kinesin-5 at different stages of the parasite life cycle, we isolated RNA and performed qRT-PCR. Kinesin-5 is expressed constitutively throughout the blood and mosquito stages of parasite development, with the highest level in gametocytes, followed by schizonts and ookinetes

Analyses of a mutant expressing a C-terminal GFP-tagged version of kinesin-5 (RMgm-4875) provided evidence that: 
- Pbkinesin-5 is located at the spindle apparatus during mitotic stages of asexual blood stage schizogony
- Spatiotemporal dynamics of Pbkinesin-5 reveal its location on the spindle apparatus during male gametogony
- During meiosis in zygote to ookinete development, Pbkinesin-5 location follows spindle dynamics. Kinesin-5-GFP fluorescence was initially diffuse within the zygote nucleus, after 1.5 to 2 hours post fertilization the GFP signal coalesced to a single focal point adjacent to the DNA
- Pbkinesin-5-GFP exhibits multiple nuclear foci during oocyst development and in liver stage schizogony 

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0807700
Gene Model P. falciparum ortholog PF3D7_0317500
Gene productkinesin-5
Gene product: Alternative nameEG5
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 modificationThe gene-deletion targeting vector for Pbkinesin-5 (PBANKA_0807700) was constructed using the pBS-DHFR plasmid, which contains polylinker sites flanking a
T. gondii dhfr/ts expression cassette conferring resistance to pyrimethamine, as described previously (Tewari et al., 2010). PCR primers N1061 and N1062 were used to generate a 995 bp fragment of kinesin-5 5′ upstream sequence from genomic DNA, which was inserted into ApaI and HindIII restriction sites upstream of the dhfr/ts cassette of pBS-DHFR. A 1008 bp fragment generated with primers N1063 and N1064 from the 3′ flanking region of kinesin-5 was then inserted downstream of the dhfr/ts cassette using EcoRI and XbaI restriction sites. The linear targeting sequence was released using ApaI/XbaI.
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