Summary

RMgm-4673
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0202700; Gene model (P.falciparum): PF3D7_0111000; Gene product: kinesin-8, putative (kinesin-8b)
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;
Last modified: 21 August 2019, 14:32
  *RMgm-4673
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) : 31409625
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-4673
Principal nameΔkinesin-8B
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNormal numbers of male/female gametocytes are formed. No male gamete formation (exflagellation).
Fertilization and ookineteNormal numbers of male/female gametocytes are formed. No male gamete formation (exflagellation). No ookinete formation.
OocystNo ookinete formation. No oocyst formation.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of kinesin-8B and expresses GFP under control of the constitutive ef1a promoter

Protein (function)
Proteomic analysis has revealed many potential regulators of axoneme assembly present in male gametes, including members of the kinesin superfamily. Kinesins are molecular motor proteins that have essential roles in intracellular transport, cell division, and motility. Some kinesin families are known to contribute to the ciliary structure and function, either by transporting ciliary components along axonemal  microtubules (MTs) by IFT such as kinesin-2s, or by destabilizing MTs, such as kinesin-9 and kinesin-13. Kinesin-8s are conserved from protozoa to mammals, and can be subclassified as kinesin-8A, -8B and -8X. In general, kinesin-8s are multitasking motors, able to move along MTs, cross-link and slide MTs, and influence MT dynamics at their ends.  Kinesin-8As are best characterised as regulators of spindle length and chromosome positioning during metaphase, whereas the mammalian kinesin-8B (Kif19) has a role in ciliary length control. The Plasmodium genome encodes two kinesin-8s (kinesin-8X and kinesin-8B), along with six or seven other kinesin proteins. Kinesin-8X is conserved across the Apicomplexa, whereas kinesin-8B is restricted to certain genera such as Toxoplasma, Eimeria, and Plasmodium that have flagellated gametes. 

Phenotype
Normal numbers of male/female gametocytes are formed. No male gamete formation (exflagellation). No ookinete and oocyst formation.

Additional information
Evidence is presented for the following:
- Kinesin-8B is not required for DNA replication in activated male gametocytes, but its deletion impairs male exflagellation
- Activated Δkinesin-8B gametocytes lack the 9+2 microtubules (MTs) axoneme architecture. Ddeletion of the kinesin-8B gene did not appear to affect the initiation, growth, or density of single and doublet MTs but completely prevented their organisation into 9+2 axonemes. In addition, there was a reduction in the number of basal bodies and, more markedly, in their association with the nuclear poles. This defect ultimately prevented exflagellation and the formation of viable male gametes.
- Spatiotemporal profiles of kinesin-8B and the kinetochore protein Ndc80 show that kinesin-8B is absent from the mitotic spindle. 
- Kinesin-8B was localised on cytoplasmic MTs rather than on spindle MTs during male gamete formation
- Kinesin-8B associates dynamically with basal bodies and growing axonemes 

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0202700
Gene Model P. falciparum ortholog PF3D7_0111000
Gene productkinesin-8, putative
Gene product: Alternative namekinesin-8b
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 markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe gene-deletion targeting vector for kinesin-8B (PBANKA_020270) was constructed using the pBS-DHFR plasmid, which contains polylinker sites flanking a Toxoplasma gondii dhfr/ts expression cassette conferring resistance to pyrimethamine, as described previously (Saini et al, 2017). PCR primers N1261 and N1262 were used to generate a 1,000-bp fragment of kinesin-8B 59 upstream sequence from genomic DNA, which was inserted into ApaI and HindIII restriction sites upstream of the dhfr/ts cassette of pBSDHFR.
A 1,240-bp fragment generated with primers N1263 and 1264 from the 39 flanking region of kinesin-8B 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