RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5018
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1458300; Gene model (P.falciparum): PF3D7_1245100; Gene product: kinesin-13, putative (KLP8)
Details mutation: 'promoter swap mutant': the promoter of kinesin-1 replaced with the clag9 promoter (PBANKA_0836300).
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 Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 29 July 2022, 10:46
  *RMgm-5018
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 35900985
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, Queens Medical Centre
Name InstituteUniversity of Nottingham
CityNottingham
CountryUK
Name of the mutant parasite
RMgm numberRMgm-5018
Principal nameKinesin-13PTD
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteStrongly reduced male gamete formation (exflagellation)
Fertilization and ookineteStrongly reduced male gamete formation (exflagellation). Strongly reduced zygote/ookinete formation.
OocystStrongly reduced zygote/ookinete formation. No oocyst or sporozoite formation
SporozoiteNo oocyst or sporozoite formation
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
In the 'promoter swap mutant': the promoter of kinesin13 has been replaced with the asexual blood stage promoter clag9 ((PBANKA_0836300). This promoter is active in asexual blood stages but not in gametocytes.

Protein (function)
Kinesins are microtubule (MT)-based motor proteins that use energy from the hydrolysis of ATP and function in various cellular processes including intracellular transport, mitotic spindle formation and chromosome segregation during cell division, and the organisation of cell polarity and cytoskeleton associated with motility.
There are 14-16 kinesin subfamilies identified in eukaryotes, and categorised based on analysis of the motor domain, with similar biological roles established by in vitro studies, and in vivo phenotypes in these organisms . Kinesin subfamilies that regulate MT dynamics, such as kinesin-8 and -13, are found in most eukaryotes including primitive and evolutionarily divergent eukaryotes. 
In a bioinformatic analysis of kinesins in Apicomplexa, nine kinesins encoded in the Plasmodium berghei genome were identified, with members of three conserved kinesin subfamilies (kinesin-5, -8B, -8X and -13); kinesin-4, -15 and -20; and two Apicomplexa-specific kinesins: kinesin-X3 and -X4 (Zeeshan et al., 2019).

Phenotype
To investigate the function of Kinesin-13 in mosquito life cycle stages, we used a promoter trap double homologous recombination (PTD) approach to downregulate gene expression at the blood stage by placing the kinesin-13 gene under the control of the Clag9 promoter. Clag9 is known to be highly expressed in asexual blood stages, but not during sexual differentiation and in mosquito stages.

Knock-out of the kinesin-13 gene is not possible since it has an essential role in asexual blood stages (see RMgm-5010). In the promoter swap mutant Kinesin-13 is expressed in asexual blood stages but is down-regulated in gametocytes and mosquito life cycle stages.

Normal asexual blood stage growth/multiplication and production of gametocytes. 
Strongly reduced male gamete formation (exflagellation). Strongly reduced zygote/ookinete formation. No oocyst or sporozoite formation.

Evidence is presented that: 'High resolution ultrastructure analysis of kinesin-13PTD parasites indicates defects in the spindle assembly and axoneme microtubules (MT) of gametocytes, and the subpellicular MT of ookinetes'. 

Additional information
From the paper: 'we used an auxin-inducible degron (AID) system to try and study the effect of rapid kinesin-13 degradation in gametocytes. We tagged the endogenous kinesin-13 gene with an AID/HA epitope tag to degrade the fusion protein in presence of auxin (Philip and Waters, 2015), and successfully generated kinesin-13- AID parasite lines as shown by integration PCR but could not deplete kinesin-13 protein by auxin treatment'.

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1458300
Gene Model P. falciparum ortholog PF3D7_1245100
Gene productkinesin-13, putative
Gene product: Alternative nameKLP8
Details of the genetic modification
Short description of the mutation'promoter swap mutant': the promoter of kinesin-1 replaced with the clag9 promoter (PBANKA_0836300).
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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 parasitehdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe conditional knockdown construct kinesin-13PTD was derived from Pclag (pSS367) where kinesin-13 was placed under the control of the clag gene (PBANKA_083630) promoter, as described previously (Sebastian et al., 2012).
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 modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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