Back to search resultsSummaryRMgm-5333
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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) : 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-5333 |
Principal name | SKP1-HA |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | While a faint cytoplasmic signal could be observed for RBX1-HA in blood-stage schizonts, no specific enrichment was observed. SKP1-HA, FBXO1-HA, and FBXL2-HA showed a pattern that was reminiscent of the dynamic localisation of the glideosome-associated protein 45 (GAP45). |
Gametocyte/Gamete | Immunoblotting confirmed the expression of fusion proteins in gametocytes with the expected mobility for RBX1-HA and SKP1-HA, but no signal above the background could be observed for CUL1-HA. Immunofluorescence assays (IFA) revealed a cell-wide distribution of both RBX1-HA and SKP1-HA in both activated macro- and microgametocytes, while no signal above the detection limit was detected for CUL1-HA gametocytes. |
Fertilization and ookinete | A signal above the detection limit was also observed for SKP1-HA, FBXO1-HA and FBXL2-HA in ookinetes |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Phenotype See further below. '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_1142900 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1367000 | ||||||||||||||||||||||||||
Gene product | suppressor of kinetochore protein 1, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | SKP1 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | triple-HA | ||||||||||||||||||||||||||
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 | Yes | ||||||||||||||||||||||||||
Name of PlasmoGEM construct/vector | - | ||||||||||||||||||||||||||
Modified PlasmoGEM construct/vector used | Yes See "Additional remarks genetic modification" | ||||||||||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||||||||||
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 | 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. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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