SummaryRMgm-5026
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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-5026 |
Principal name | kinesin-X4::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-X4::GFP expression during male gametogenesis with cytosolic location. |
Fertilization and ookinete | Kinesin-X4::GFP expression during male gametogenesis with cytosolic location. |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
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-X4::GFP expression during male gametogenesis with cytosolic location. The expression and location of kinesin-X4 (green) was compared by live-cell imaging during male gametogenesis with that of axonemal protein kinesin-8B (red), which is located on basal bodies and axonemes, tracking the process of male gametogenesis (Zeeshan et al., 2019a). Live cell imaging showed a diffuse cytosolic distribution of kinesin-X4 during early stages of male gametogenesis (1-3 mpa) and no strong signal on the basal body tetrads labelled with kinesin-8B (red). However, in later stages (4-6 mpa) the kinesin-X4 signal changed in distribution to exhibit linear structures, which maintained in the later stages (8-10 mpa) and show co-localization with kinesin-8B.
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_0902400 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1146700 | ||||||||||||||||||||||||||
Gene product | kinesin-X4, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | X4 | ||||||||||||||||||||||||||
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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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