SummaryRMgm-4666
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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) : 31283102 |
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 | Wall RJ, Tewari R |
Name Group/Department | School of Life Sciences, Queens Medical Centre |
Name Institute | University of Nottingham |
City | Nottingham |
Country | London |
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Name of the mutant parasite | |
RMgm number | RMgm-4666 |
Principal name | MyoF-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 | Not different from wild type |
Fertilization and ookinete | MyoF‐GFP was most abundant in the insect stages, particularly ookinetes and oocysts. In mature ookinetes, MyoF‐GFP was restricted to the apical end, whereas in oocysts, it was more evenly distributed throughout the developing sporozoites. |
Oocyst | MyoF‐GFP was most abundant in the insect stages, particularly ookinetes and oocysts. In mature ookinetes, MyoF‐GFP was restricted to the apical end, whereas in oocysts, it was more evenly distributed throughout the developing sporozoites. |
Sporozoite | MyoF‐GFP was most abundant in the insect stages, particularly ookinetes and oocysts. In mature ookinetes, MyoF‐GFP was restricted to the apical end, whereas in oocysts, it was more evenly distributed throughout the developing sporozoites. |
Liver stage | Not different from wild type |
Additional remarks phenotype | Mutant/mutation Phenotype We used live cell imaging to detect expression of each protein throughout the life cycle and to examine their location. The Class XIV myosins were detected predominantly in the invasive stages (developing merozoites in schizonts and merozoites, ookinetes, and developing sporozoites within oocysts and sporozoites). As shown previously, MyoA‐GFP was associated with the surface pellicle of each invasive stage, whereas MyoB‐GFP was localised as an apical end dot in merozoites, mature (>20 hr) ookinetes, and late stage sporozoites. MyoE‐GFP was detected as a dot at the basal end of ookinetes and sporozoites and as a dot in merozoites. MyoE‐GFP was also expressed in liver stages, but as with merozoites, it was not possible to clearly identify the basal end of these stages. MyoF‐GFP was most abundant in the insect stages, particularly ookinetes and oocysts. In mature ookinetes, MyoF‐GFP was restricted to the apical end, whereas in oocysts, it was more evenly distributed throughout the developing sporozoites. In contrast, MyoJ‐GFP was only observed in mature oocysts, located at the junction between the differentiating sporozoites and the oocyst bodies. Following oocyst rupture, MyoJ‐GFP remained associated with the oocyst body and was not present in sporozoites. Finally, MyoK‐GFP was found exclusively associated with the nucleus of gametocytes, appearing as an arc across the cell, and in zygotes/early ookinetes, as two distinctive dots. These dots were either absent or only remnants were seen in mature ookinetes.
Other mutantsLive cell imaging reveals distinct spatio-temporal profiles for each myosin during ookinete development We showed previously that MyoA-GFP is associated with the pellicle of this protuberance during zygote to ookinete development. MyoA-GFP was only detectable from stage II of ookinete development, initially located throughout the cytoplasm until, after stage V, when it was associated with the pellicle of the growing protuberance and the mature ookinete. MyoB-GFP was not detected until the very last stage of ookinete development, appearing as a dot at the apical end of the parasite. Since PfMLC-B was also expressed at the apical end of merozoites, we next checked its location in zygotes and ookinetes. Interestingly, MLC-B-GFP was detected much earlier in ookinete development, visible as an apical dot several hours before MyoB-GFP was observed. This suggests that MLC-B-GFP is made and positioned, well before its partner, MyoB. MyoE-GFP was detectable as very faint fluorescence in the cytosol until the final stage of differentiation when it was apparent at the basal end of the ookinete. As with MyoB, the localised appearance of MyoE was only seen after the body of the retort was finally reduced to the tapered base of the mature ookinete. MyoF-GFP expression appeared to translocate from the boundary of the stage II ookinetes - where the newly formed ookinete protrudes from the retort - to the apical end of the mature ookinete. As described above, MyoK-GFP was initially detected as a single dot in zygotes, then as two dots in the developing ookinete associated with the nucleus, but the signal almost completely disappeared from mature ookinetes. To resolve the structure of the apical and basal ‘dots’ of MyoB and MyoE (respectively) in mature ookinetes, we used 3D-SIM super resolution microscopy. This showed MyoB-GFP as an apical ring with an aperture in the centre, whereas MyoE-GFP formed a continuous basal ‘cap’ . Co-localisation of myosins with MyoA and the apical marker, ISP1 To further study the location of the GFP-tagged myosins, we expressed them in parasite lines that were also expressing either MyoA-mCherry or ISP1-mCherry. ISP1-mCherry is a marker for early ookinete development located and maintained at the protuberance that becomes the apical end of ookinetes. The MyoA-mCherry line was generated in this study and validated by integration PCR. Expression was identical to that seen previously for MyoA-GFP. MyoA-mCherry and MyoB-GFP co-localise at the apical end of ookinetes whereas MyoA-mCherry and MyoE-GFP co-localise at the basal end. These locations were confirmed by MyoB-GFP co-localised with ISP1-mCherry at the apical end, and MyoE-GFP located at the opposite end of the ookinete from ISP1-mCherry. There was no co-localisation between MyoA-mCherry and MyoJ-GFP, expressed in the sporozoite bodies of oocysts and oocyst body, respectively. However, the two myosins did overlap at the point where the sporozoites detach from the oocyst body. In contrast, MyoA-mCherry partially co-localised with MyoF-GFP in the sporozoite bodies of oocysts. Finally, and as seen with ookinetes, MyoA-mCherry also partially co-localises with MyoE-GFP in liver stage schizonts.
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1344100 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1329100 | ||||||||||||||||||||||||||
Gene product | myosin F | ||||||||||||||||||||||||||
Gene product: Alternative name | MyoF | ||||||||||||||||||||||||||
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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 | For GFP-tagging of each myosin/MLC-B by single homologous recombination, a region of the 3’ end of the gene, but omitting the stop codon, was PCR amplified. This fragment was inserted into p277 vector, upstream of the gfp sequence, using KpnI and ApaI restriction sites. The p277 vector contains the human dhfr cassette, conveying resistance to pyrimethamine. Before transfection, the vector was linearised. The GFP tagging constructs and cell lines for MyoA and MyoB have been reported previously. MyoA-mCherry was constructed using the same primers as described previously. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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