SummaryRMgm-4569
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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) : 30547015 |
MR4 number | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. yoelii |
Parent strain/line | P. y. yoelii 17XNL |
Name parent line/clone | Not applicable |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | Arredondo SA, Kappe SHI |
Name Group/Department | Center for Global Infectious Disease Research |
Name Institute | Seattle Children’s Research Institute |
City | Seattle |
Country | USA |
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Name of the mutant parasite | |
RMgm number | RMgm-4569 |
Principal name | P36(mCherry) |
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 tested |
Fertilization and ookinete | Not tested |
Oocyst | P36mCherry was found to be expressed in both oocyst (day 10) and salivary gland-derived sporozoites by live fluorescence microscopy and confirmed by western blot analysis using α-mCherry. |
Sporozoite | P36mCherry parasites successfully progressed through development within the mosquito vector, with an in vivo pre-patent period comparable to wild-type parasites; in vitro, sporozoites displayed normal gliding motility ( and were capable of invading hepatoma cells, indicating no significant negative impact on protein function derived from the fusion with the mCherry tag. P36mCherry was found to be expressed in both oocyst (day 10) and salivary gland-derived sporozoites by live fluorescence microscopy and confirmed by western blot analysis using α-mCherry. P36mCherry is expressed at significantly higher levels in salivary gland sporozoites as compared to oocyst sporozoites. P52, however, was only detected in salivary gland sporozoites. Evidence is presented for a micronemal location of P36mCherry and for the co-localization of P36 with P52 and/or TRAP in the micronemal organelles. Analysis of P36mCherry salivary gland sporozoites by IFA in the context of Hepa 1–6 cell invasion revealed the presence of an accumulation of P36mCherry protein toward one end of the sporozoite, reminiscent of the previously reported secretion of TRAP. This concentrated protein appeared to be released from the sporozoite when contrasted with the MTIP-labeled inner membrane complex, and it consistently co-localized with unprocessed, full-length TRAP. The staining pattern for both P36mCherry and TRAP regularly overlapped at what appeared to be progressive stages of secretion where the concentration of the proteins at the apical end became more prominent as the internal micronemal signal diminished. |
Liver stage | P36mCherry parasites successfully progressed through development within the mosquito vector, with an in vivo pre-patent period comparable to wild-type parasites; in vitro, sporozoites displayed normal gliding motility ( and were capable of invading hepatoma cells, indicating no significant negative impact on protein function derived from the fusion with the mCherry tag. |
Additional remarks phenotype | Mutant/mutation Protein (function) |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_1003500 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0404400 | ||||||||||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||||||||||
Gene product: Alternative name | P36 | ||||||||||||||||||||||||||
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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 double 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 | tgdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | The creation of P36(mCherry) relied upon double crossover homologous recombination using a modified plasmid pDEF (van Dijk et al., 2005), which allowed for the addition of a fluorescent mCherry epitope tag to the carboxy terminus of P36. The resultant P. yoelii P36mCherry parasite expresses a single copy of P36, with a C-terminal mCherry tag, under the control of its endogenous promoter. Briefly, the two regions of the targeted locus were PCR amplified with locus-specific primers. The two PCR products were then fused by Sequence Overlap Extension PCR (“SOE PCR”). This PCR product was then digested at the 5′ and 3′ ends with restriction sites incorporated into the primers and inserted into the modified pDEF vector. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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