SummaryRMgm-5340
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene disruption |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 36898988 |
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 | Rashpa R, Brochet M |
Name Group/Department | Faculty of Medicine, Department of Microbiology and Molecular Medicine |
Name Institute | University of Geneva |
City | Geneva |
Country | Switzerland |
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Name of the mutant parasite | |
RMgm number | RMgm-5340 |
Principal name | PBANKA_1358700-KO |
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 | Not different from wild type |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Protein (function) Phenotype 'These results indicate that gametocytes express a conserved SKP1/RBX1/CUL1 complex with at least two possible adaptor proteins, FBXO1 and FBXL2. The enrichment of CDPK1 in FBXO1-HA and SKP1-HA immuno-precipitates suggests a possible interplay between phosphorylation and ubiquitination to regulate sexual development.' 'The endogenous skp1, cul1 and rbx1 genes were taged with an auxin-inducible degron (AID) coupled to an HA epitope tag that allows degradation of the fusion protein in the presence of auxin in a strain expressing the Tir1 protein (RMgm-1305; Philip and Waters, 2015). However, no significant degradation of the targeted proteins nor defects in exflagellation could be observed upon auxin addition in non-clonal populations This prevented to further use this system to interrogate the functions of these proteins across multiple stages.' 'To infer potential function in gametocytes, we then opted for stage-specific knockdowns by placing the endogenous cul1 or rbx1 genes under the control of the promoter of the blood-stage schizont-specific promoter ama1 (PBANKA_0915000), which is active in schizonts but silent in gametocytes. Cloned parasite lines expressing cul1 (Pama1CUL1; RMgm-5338) and rbx (Pama1RBX; RMgm-5339) under control of the ama-1 promoter were obtained, but none of these showed quantifiable defects in exflagellation (possibly due to sufficient expression levels?). However, the Pama1CUL1 clone mainly formed retort ookinetes, a phenotype that was partially rescued by fertilisation with competent Nek4-KO microgametes, suggestive of a role for cul1 in either sexual lineage.' We then interrogated the requirement for the putative adaptor proteins FBXO1, FBXL2 and the protein of unknown function PBANKA_1358700 that was enriched in SCFFBXO1 immuno-precipitates. We were able to obtain a KO clonal line for PBANKA_1358700 (RMgm-5340), but no defects in asexual blood stages nor in exflagellation were detected. Other mutants |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1358700 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1345800 | ||||||||||||||||||||||||
Gene product | conserved Plasmodium protein, unknown function | ||||||||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||||||||
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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 | 3xHA, KO (disruption) or AID/HA targeting vectors were generated using phage recombineering in Escherichia coli TSA strain with PlasmoGEM vectors (https://plasmogem.umu.se/pbgem/). For final targeting vectors not available in the PlasmoGEM repository, generation of knockout and tagging constructs were performed using sequential recombineering and gateway steps. For each gene of interest (goi), the Zeocin-resistance/Phe-sensitivity cassette was introduced using oligonucleotides goi HA-F x goi HA-R and goi KO-F x goi KO-R for 3xHA, AID/HA tagging and KO targeting vectors, respectively. Insertion of the GWcassette following the gateway reaction was confirmed using primer pairs GW1 x goiQCR1 and GW2 x goiQCR2.The modified library inserts were then released from the plasmid backbone using NotI. The AID/HA targeting vectors were transfected into the 615 parasite line, while the KO/GD and triple HA targeting vectors were transfected into the 2.34 line unless otherwise specified. | ||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||||||||
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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