SummaryRMgm-5325
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene tagging, Gene tagging |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 37704606 |
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 | School of Life Sciences |
Name Institute | University of Nottingham |
City | Nottingham |
Country | UK |
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Name of the mutant parasite | |
RMgm number | RMgm-5325 |
Principal name | Kinesin-8b::mCherry/ARK2-GFP |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not tested |
Gametocyte/Gamete | To investigate the association of ARK2 and the mitotic spindle during male gametogony, its location was compared with that of the kinetochore marker NDC80 and cytoplasmic axonemal protein kinesin-8B (examined by live cell imaging of both markers). One to two minutes after gametocyte activation, ARK2-GFP was observed close to the DNA and adjacent to, but not overlapping, the kinesin-8B-mCherry tetrad. ARK2-GFP remained distributed on spindles, while there was duplication of kinesin-8B-mCherry labelled tetrads. In later stages of male gametogony, ARK2-GFP remained associated with spindles and spindle poles, while kinesin-8B-mCherry showed a distinct cytoplasmic axonemal location. |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Kinesin-8b: 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. ARK2 is expressed in the nucleus throughout the P. berghei life cycle. To investigate the association of ARK2 and the mitotic spindle during male gametogony, its location was compared with that of the kinetochore marker NDC80 and cytoplasmic axonemal protein kinesin-8B. The ARK2-mCherry mutant has been used to cross with a mutant expressing GFP-tagged NDC80 (see mutant RMgm-4843) resulting in mutant ARK2-mCherry/NDC80-GFP (RMgm-5323). Analysis of this mutant showed the following: Parasite lines expressing ARK2-mCherry and NDC80-GFP were crossed (RMgm-5323), and the progeny were analysed by live-cell imaging to establish the spatiotemporal relationship of the two tagged proteins. The location of both ARK2-mCherry and NDC80-GFP was next to the stained DNA, and with a partial overlap, although NDC80-GFP was always closer to the DNA. In addition the ARK2-mCherry mutant has been used to cross with a mutant expressing GFP-tagged EB1 (see mutant RMgm-5319) resulting in mutant EB1-GFP/ARK2-mCherry (RMgm-5326). Analysis of this mutant showed the following: 'To further resolve the location of EB1 with respect to the kinetochore and basal body at higher resolution, 3D-SIM was performed on EB1-GFP/NDC80-mCherry (RMgm-RMgm-5327), EB1-GFP/ARK2-mCherry (RMgm-RMgm-5326) and EB1-mCherry/SAS4-GFP (RMgm-RMgm-5328) fixed gametocytes. The 3D-SIM images of gametocytes expressing EB1-GFP/NDC80-mCherry showed EB1 bridge(s) across the nucleus with NDC80 distributed like beads on the bridge, each bead presenting a kinetochore. The 3D-SIM images of gametocytes expressing EB1-GFP/ARK2-mCherry showed EB1 bridge(s) across the nucleus with ARK2, overlapping each other. The bridged pattern of spindles for EB1 were restricted to the nucleus as shown by 3D-SIM images of gametocytes expressing EB1-mCherry/SAS4-GFP; whereby SAS4 was located in the cytoplasm but aligned with the EB1 bridge in the nucleus'. Since ARK2 is expressed in male gametocytes and parasite development is affected after fertilization, we investigated whether the defect is due to inheritance from the male gamete. We performed genetic crosses between Pclag-ark2 parasites and other mutants deficient in production of either male (Δhap2) or female (Δdozi) gametocytes. Crosses between Pclag-ark2 and Δdozi mutants produced some normal-sized oocysts that were able to sporulate, showing a partial rescue of the Pclag-ark2 phenotype. In contrast, crosses between Pclag-ark2 and Δhap2 did not rescue the Pclag-ark2 phenotype. These results reveal that a functional ark2 gene copy from a male gamete is required for subsequent oocyst development. Analysis of a mutant expressing GFP-tagged ARK2 (RMgm-5321) showed the following: Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0407400 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0309200 | ||||||||||||||||||||||||||
Gene product | serine/threonine protein kinase ARK2, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | ARK2 | ||||||||||||||||||||||||||
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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 | unknown | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | To generate the GFP-tag line, a region of the ark2 gene downstream of the ATG start codon was amplified, ligated to p277 vector, and transfected. The p277 vector contains the human dhfr cassette, conveying resistance to pyrimethamine. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0202700 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0111000 | ||||||||||||||||||||||||||
Gene product | kinesin-8B, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | mCherry | ||||||||||||||||||||||||||
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 mCherry sequence by single crossover homologous recombination at the 3’end of the gene. To generate the tagged 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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