SummaryRMgm-5456
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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) : 37910326 |
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 | Not applicable |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | Mishra A, Mishra S |
Name Group/Department | Division of Molecular Microbiology and Immunology |
Name Institute | CSIR-Central Drug Research Institute |
City | Lucknow |
Country | India |
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Name of the mutant parasite | |
RMgm number | RMgm-5456 |
Principal name | Atg8-3XHA-mCherry |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | To check if Plasmodium Atg4 retains Atg8 processing activity despite its exposed glycine, we investigated the importance of cysteine protease for Atg8 conjugation. Atg8 was endogenously tagged with 3XHA–mCherry at the C-terminus to mask its exposed glycine. Atg8-3XHA-mCherry schizont cultures were treated with the cysteine protease inhibitor E64 to evaluate cysteine protease activity. The treated and untreated schizonts were fixed and analyzed by IFA using anti-Atg8 and anti-mCherry antibodies. Without an inhibitor, mCherry was cleaved, as confirmed by independent signals of Atg8 and mCherry that did not colocalize. However, E64-treated Atg8-3XHA-mCherry schizonts showed overlapping staining patterns. The treated Atg8-3XHAmCherry and WT schizonts also failed to properly segregate their nuclei and were found to be arrested during schizogony, which was due to the general inhibition of parasites by E64. Atg8 conjugation was not affected in the arrested WT parasites, suggesting that parasites could conjugate Atg8 with exposed glycine. |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0504100 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1019900 | ||||||||||||||||||||||||||
Gene product | autophagy-related protein 8 | ||||||||||||||||||||||||||
Gene product: Alternative name | ATG8 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | 3XHA–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 | hdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | For the endogenous tagging of Atg8 (PBANKA_0504100) to generate Atg8-3XHAmCherry transgenic parasites, two fragments, F1 (630 bp) and F2 (547 bp), were amplified using primers 1560/1561 and 1562/1563, respectively, and cloned sequentially into the pBC–3XHA–mCherry–hDHFR vector at XhoI/BglII and NotI/AscI, respectively. The plasmid was linearized using XhoI/AscI and transfected into P. berghei ANKA schizonts. Additional information on mutants generated in this study: To generate Atg4 (PBANKA_1025400) knockout parasites, double-crossover homologous recombination was used. For this, two fragments, F1 (967 bp) and F2 (556 bp), were amplified using primer sets 1655/1656 and 1333/1355. from the 5′ and 3′ UTRs of the gene, respectively, and were sequentially cloned into the pBC–GFP–hDHFR vector at XhoI/ClaI and NotI/AscI, respectively. The plasmid was linearized using XhoI/AscI and transfected into P. berghei ANKA schizonts. For the generation of Otu (PBANKA_0515350) knockout parasites, two fragments, F1 (591 bp) and F2 (546 bp), were amplified using primer sets 1964/1965 and 1966/1967 from the 5′ and 3′ UTRs, respectively, and sequentially cloned into the pBC–mCherry–TgDHFR vector at KpnI/ClaI and NotI/AscI, respectively. The plasmid was linearized using KpnI/AscI and transfected into P. berghei ANKA schizonts. For the generation of Atg4/Otu double knockout parasites, the linearized targeting cassette of Atg4 with GFP reporter and hDHFR selection marker was transfected into the purified schizonts of Otu KO parasites with mCherry reporter and TgDHFR selection marker. The transfected parasites were selected by the subcutaneous injection of WR drug (Sigma-Aldrich, SML2976) for 3 consecutive days. In the second approach, Otu–mCherry–TgDHFR and Atg4–GFP–hDHFR linearized cassettes were cotransfected into P. berghei ANKA schizonts. The transfected parasites were selected by the oral administration of pyrimethamine. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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