RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_060950; Gene model (P.falciparum): PF3D7_1211000; Gene product: kinesin-X3, putative (X3)
Name tag: GFP
Phenotype Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 29 July 2022, 11:03
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
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 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherZeeshan, M; Tewari R
Name Group/DepartmentUniversity of Nottingham
Name InstituteSchool of Life Sciences
Name of the mutant parasite
RMgm numberRMgm-5025
Principal namekinesin-X3::GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteKinesin-X3::GFP expression in female gametocytes.
Fertilization and ookineteKinesin-X3::GFP expression in female gametocytes, zygotes, ookinetes and later stages of sporogony
OocystKinesin-X3::GFP expression in female gametocytes, zygotes, ookinetes and later stages of sporogony
SporozoiteKinesin-X3::GFP expression in female gametocytes, zygotes, ookinetes and later stages of sporogony
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a C-terminal GFP-tagged version of kinesin-X3 

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).

The kinesin-GFP parasite line completed the full life cycle with no detectable phenotypic  change resulting from the GFP tagging.

Apicomplexa-specific kinesins (kinesin-X3 and X4) have discrete locations during
pellicle formation and axoneme assembly in sexual stage parasites.

Kinesin-X3::GFP expression in female gametocytes, zygotes, ookinetes and later stages of sporogony
The live cell imaging of kinesin-X3 and kinesin–X4 revealed a stage-specific expression during sexual development with a distinct location. During ookinete differentiation, kinesin-X3 expression was restricted to one side of the cell in the early stages of development (stage IIII), suggesting an involvement in pellicle formation. In later stages (stage IV-VI), the kinesin-X3 location became more distinct around the periphery of the ookinete. Monoclonal antibody 13.1 conjugated with cy3 (red), which recognizes the protein P28 on the surface of zygote and ookinete stages, was used to stain these stages and showed colocalization with kinesin-X3 (green), although kinesin-X3 was not present at the apical and basal ends of the developing ookinete

Additional information
The different kinesins in the P. berghei genome are:

Kinesin-4: PBANKA_1208200
Kinesin-5: PBANKA_0807700; PF3D7_0317500
Kinesin-8B: PBANKA_0202700; PF3D7_0111000
Kinesin-8X: PBANKA_0805900; PF3D7_0319400
Kinesin-13: PBANKA_1458300; PF3D7_1245100
Kinesin-15: PBANKA_1458800; PF3D7_1245600
Kinesin-20: PBANKA_0622400; PF3D7_0724900

X3: PBANKA_060950; PF3D7_1211000
X4: PBANKA_0902400; PF3D7_1146700

From the paper: 
'We elucidate the subcellular location of each kinesin using a protein endogenously tagged at the C-terminus with GFP, showing a differential localisation of kinesins in the mitotic and meiotic stages and a pellicular and polar location in certain invasive stages.  Eight of the nine kinesins are required only for parasite transmission through the mosquito vector during the sexual and asexual sporogony stages. Only kinesin-13 is likely essential during blood stage schizogony. An in-depth analysis of kinesin-13 and kinesin-20 revealed a distinct subcellular location and function in MT assembly during spindle formation, axoneme assembly and cell polarity. Kinesin-20 was associated with a striking ring-like structure during zygote to ookinete differentiation and deletion of the gene revealed a function in the morphology and motility of the ookinete'.

Other mutants

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_060950
Gene Model P. falciparum ortholog PF3D7_1211000
Gene productkinesin-X3, putative
Gene product: Alternative nameX3
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid single 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 C-terminus was tagged with green fluorescent protein (GFP) sequence by single crossover homologous recombination at the 3’end of the gene. To generate the GFP-tag line, a region of these genes downstream of the ATG start codon was amplified, ligated to p277 vector, and transfected as described previously (Guttery et al., 2012). The p277 vector contains the human dhfr cassette, conveying resistance to pyrimethamine.
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