SummaryRMgm-5321
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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) : 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-5321 |
Principal name | ARK2-GFP |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | ARK2 is expressed in the nucleus throughout the P. berghei life cycle. ARK2-GFP showed a punctate nuclear pattern with one or two focal points during blood schizogony and sporogony. ARK2-GFP was not detected in mature asexual (merozoites and sporozoites) and sexual (male gametes and ookinetes) stages of development |
Gametocyte/Gamete | It was present at a single focal point with an additional more diffuse nuclear location during early stages of male gamete formation and in the early zygote, but in later zygote stages it had a more dynamic location on the spindle and spindle pole. ARK2-GFP was not detected in mature asexual (merozoites and sporozoites) and sexual (male gametes and ookinetes) stages of development |
Fertilization and ookinete | ARK2-GFP was present at a single focal point with an additional more diffuse nuclear location during early stages of male gamete formation and in the early zygote, but in later zygote stages it had a more dynamic location on the spindle and spindle pole. ARK2-GFP was not detected in mature asexual (merozoites and sporozoites) and sexual (male gametes and ookinetes) stages of development. |
Oocyst | Not tested |
Sporozoite | ARK2-GFP was not detected in mature asexual (merozoites and sporozoites) stages |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation 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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