SummaryRMgm-5459
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Successful modification | The gene/parasite could not be changed/generated by the genetic modification. |
The following genetic modifications were attempted | Gene disruption, Gene disruption |
Number of attempts to introduce the genetic modification | 2 |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 37910326 |
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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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Attempts to generate the mutant parasite were performed 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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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0515350 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1031400 | ||||||||||||||||||||||||
Gene product | OTU-like cysteine protease | ||||||||||||||||||||||||
Gene product: Alternative name | Otu | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (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 | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | tgdhfr | ||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||
Additional remarks genetic modification | To generate/select mutants with both genes simultaneously disrupted indicate that asexual blood stages require that at least one of the genes is active for growth/multiplication of asexual blood stages. See alos below for the information of single KO mutants of Atg4 and Otu. 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 information on mutants generated in this study: 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 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. 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. From the paper: 'We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 (RMgm-5457) and Otu (RMgm-5458) showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling'. Attempts to generate and select Atg4/Otu double knockout parasites were unsuccessful. | ||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||
Primer information: Primers used for amplification of the target sequences
![]() Primer information: Primers used for amplification of the target sequences
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1025400 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1417300 | ||||||||||||||||||||||||
Gene product | cysteine protease ATG4, putative | ||||||||||||||||||||||||
Gene product: Alternative name | ATG4 | ||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (Linear) PCR construct 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 | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
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 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 information on mutants generated in this study: 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 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. 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. From the paper: 'We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 (RMgm-5457) and Otu (RMgm-5458) showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling'. Attempts to generate and select Atg4/Otu double knockout parasites were unsuccessful. | ||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||
Primer information: Primers used for amplification of the target sequences
![]() Primer information: Primers used for amplification of the target sequences
![]()
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