SummaryRMgm-5025
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Successful modification | The 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 | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone | P. berghei ANKA 2.34 |
Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). |
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The mutant parasite was generated by | |
Name PI/Researcher | Zeeshan, M; Tewari R |
Name Group/Department | University of Nottingham |
Name Institute | School of Life Sciences |
City | Nottingham |
Country | UK |
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Name of the mutant parasite | |
RMgm number | RMgm-5025 |
Principal name | kinesin-X3::GFP |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Kinesin-X3::GFP expression in female gametocytes. |
Fertilization and ookinete | Kinesin-X3::GFP expression in female gametocytes, zygotes, ookinetes and later stages of sporogony |
Oocyst | Kinesin-X3::GFP expression in female gametocytes, zygotes, ookinetes and later stages of sporogony |
Sporozoite | Kinesin-X3::GFP expression in female gametocytes, zygotes, ookinetes and later stages of sporogony |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Phenotype
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 From the paper: |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_060950 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1211000 | ||||||||||||||||||||||||||
Gene product | kinesin-X3, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | X3 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | GFP | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid single cross-over | ||||||||||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | The 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 | |||||||||||||||||||||||||||
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