SummaryRMgm-5316
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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) : 36869034 |
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 | Ferreira JL, Grünewald K |
Name Group/Department | Centre for Structural Systems Biology |
Name Institute | Centre for Structural Systems Biology |
City | Hamburg |
Country | Germany |
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Name of the mutant parasite | |
RMgm number | RMgm-5316 |
Principal name | PbSPM1-GFP |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | No |
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Phenotype | |
Asexual blood stage | Not tested |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | High expression in P. berghei sporozoites of PbSPM1-GFP |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Sporozoites and ookinetes are motile, elongated forms with a dense SPMT scaffold underneath the IMC. In the paper it is shown that SPMT's of these stages consist of 13- protofilament microtubules with twice-interrupted luminal helices. Similar structures, named Interrupted Luminal Helices (ILH), were first observed in flagellar ends of human spermatozoa, and more recently in equine and porcine spermatozoa, and tachyzoites of Toxoplasma gondii. The T. gondii ILH consists of thioredoxin-like proteins 1 and 2 (TrxL1, TrxL2) and subpellicular microtubule protein 1 (SPM1). Plasmodium spp. have homologues of TrxL1 (PfTrxL1; PBANKA_0820200; PF3D7_0919300) and SPM1 (PfSPM1; PBANKA_0810700; PF3D7_0909500) but lack TrxL2, which in the Toxoplasma ILH is present in both. Compatible with the ILH being composed of SPM1 and TrxL1 in Plasmodium, high expression in Plasmodium berghei (Pb) of PbSPM1-GFP and PbTrxL1-GFP was seen in the endogenously-tagged sporozoite lines, PbSPM1-GFP and PbTrxL1-GFP (see phenotype below).
Phenotype High expression in P. berghei sporozoites of PbSPM1-GFP (and PbTrxL1-GFP; see mutant RMgm-5317) Additional information Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0810700 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0909500 | ||||||||||||||||||||||||||
Gene product | subpellicular microtubule protein 1 | ||||||||||||||||||||||||||
Gene product: Alternative name | SPM1 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | GFP | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid single 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 | unknown | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | Endogenous tagging was performed essentially as described before using single-crossover integration. As both SPM1 and TrxL1 show an overall gene length of less than 1 kilobase, the entire open-reading frame was amplified from PbANKA wildtype genomic DNA. The resulting DNA fragment was cloned into the pL18 vector using EcoRI and BamHI restriction sites. The reverse primer encoded for six alanines that were used as a linker. The pL18 vector harbours the hDHFR gene for positive selection using the drug pyrimethamine. Prior to transfection, the vector was linearized using SwaI (SPM1) and BsmI (TrxL1), respectively, followed by ethanol precipitation. The linearized pL18-SPM1-GFP/pL18-TrxL1-GFP vectors were each transfected into an unmodified P. berghei ANKA strain using standard protocols | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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