SummaryRMgm-5024
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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-5024 |
Principal name | kinesin-5::GFP |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Kinesin-5 was expressed throughout the parasite life cycle, including blood stage schizogony, and was located on the mitotic spindle in both asexual and sexual stages. Kinesin-5GFP was restricted to the nucleus and not detected in mature extracellular parasites such as merozoites, male gametes and sporozoites. |
Gametocyte/Gamete | Kinesin-5 was expressed throughout the parasite life cycle, including blood stage schizogony, and was located on the mitotic spindle in both asexual and sexual stages. Kinesin-5GFP was restricted to the nucleus and not detected in mature extracellular parasites such as merozoites, male gametes and sporozoites. |
Fertilization and ookinete | Kinesin-5 was expressed throughout the parasite life cycle, including blood stage schizogony, and was located on the mitotic spindle in both asexual and sexual stages. Kinesin-5GFP was restricted to the nucleus and not detected in mature extracellular parasites such as merozoites, male gametes and sporozoites. |
Oocyst | Kinesin-5 was expressed throughout the parasite life cycle, including blood stage schizogony, and was located on the mitotic spindle in both asexual and sexual stages. Kinesin-5GFP was restricted to the nucleus and not detected in mature extracellular parasites such as merozoites, male gametes and sporozoites. |
Sporozoite | Kinesin-5 was expressed throughout the parasite life cycle, including blood stage schizogony, and was located on the mitotic spindle in both asexual and sexual stages. Kinesin-5GFP was restricted to the nucleus and not detected in mature extracellular parasites such as merozoites, male gametes and sporozoites. |
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. Kinesin-5 was expressed throughout the parasite life cycle, including blood stage schizogony, and was located on the mitotic spindle in both asexual and sexual stages. Kinesin-5GFP was restricted to the nucleus and not detected in mature extracellular parasites such as merozoites, male gametes and sporozoites.
Kinesin-5 is a nuclear spindle kinesin associated with the kinetochore (NDC80) that bind centromeres.
Live cell imaging showed that the location of kinesin-5, was restricted to the nucleus throughout the life cycle stages. We crossed parasite lines expressing kinesin-5-GFP with lines expressing NDC80-Cherry, a kinetochore protein in the nucleus, and kinesin-8B-Cherry, an axonemal protein in the cytoplasm, and compared protein location by live-cell imaging. Kinesin-5 (green) was co-localised with NDC80 (red) suggesting a role in mitotic spindle function and chromosome segregation. On the other hand, kinesin-5 showed no overlap with kinesin-8B (red) during male gametogenesis confirming their restricted location in the nucleus.
We analysed further the binding of these kinesin-5 at the centromere DNA. We performed ChIP-seq experiments with parasites undergoing gametogenesis (6 min post-activation [mpa]), using kinesin-5GFP tagged parasites and GFP-specific antibodies. Strong ChIP-seq peaks for each chromosome were observed with this kinesin, indicating its binding sites. Binding was restricted to a region close to the previously annotated centromeres of all 14 chromosomes. Together, live cell imaging and ChIP-seq analysis support the notion that kinesin-5 associates with kinetochores assembled at centromeres. 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_0807700 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0317500 | ||||||||||||||||||||||||||
Gene product | kinesin-5 | ||||||||||||||||||||||||||
Gene product: Alternative name | EG5 | ||||||||||||||||||||||||||
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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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