SummaryRMgm-4746
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene mutation |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 32034083 |
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 | Moreau CA, Frischknecht F |
Name Group/Department | Integrative Parasitology, Center for Infectious Diseases |
Name Institute | Heidelberg University Medical School |
City | Heidelberg |
Country | Germany |
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Name of the mutant parasite | |
RMgm number | RMgm-4746 |
Principal name | profilin chimeras |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | See below |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | See below |
Liver stage | See below |
Additional remarks phenotype | Mutant/mutation We first compared the blood stage growth rates of the chimeric parasite lines to those of wild type P. berghei and the previously reported P. berghei line expressing P. falciparum profilin. This showed that the parasites expressing the P. berghei profilin with the P. falciparum loop grew as fast as wild type P. berghei. Those lines expressing P. falciparum profilin and P. falciparum profilin with the P. berghei profilin loop grew somewhat faster but at comparable rates (Table 1). Intriguingly, expression of the P. falciparum chimera featuring the T. gondii loop slowed blood stage growth indicating that this chimeric profilin might not perform as efficiently in vivo. Infection of Anopheles stephensi mosquitoes however revealed similar infection rates and numbers of sporozoites. Infections of mice by mosquito bite showed a mild reduction in infectivity of the parasite lines expressing P. falciparum profilins containing the loops of P. berghei or T. gondii. P. berghei sporozoites expressing P. falciparum profilin migrated faster than wild type P. berghei sporozoites although fewer parasites were gliding. Examination of the parasite lines expressing loop chimeras showed that sporozoites of all chimeras moved in the typical circular fashion of wild type parasites. About the same percentage of sporozoites were gliding, but curiously, those expressing P. falciparum profilin containing the loop of T. gondii showed a higher percentage of persistently moving sporozoites than those just expressing the P. falciparum profilin. The quantification of their average and instantaneous speeds showed however, that all sporozoites expressing chimeras were over 50% slower than their respective control parasite lines. |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0833000 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0932200 | ||||||||||||||||||||||||||
Gene product | profilin | ||||||||||||||||||||||||||
Gene product: Alternative name | PFN, PRF | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | P. berghei profilin replaced by different chimeric forms of profilin | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||||
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 | tgdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | Vectors used for in vivo work are based on the b3D+ vector. We modified the vector for homologous recombination in the profilin (PBANKA_0833000) locus on chromosome 8 as follows. The P. berghei profilin 5’ upstream region (871 bp) was amplified from P. berghei ANKA WT genomic DNA using primer combination 5 (see Table S1) and subsequently inserted into b3D+ via SacII and NotI digestion and ligation. The profilin 3’ downstream region (805 bp) was amplified with primer combination 6 and inserted using ClaI and KpnI. P. berghei wild type profilin was amplified with primer combination 3 and cloned with NotI and XbaI. P. falciparum wild type was amplified with primer combination 4 and cloned into b3D+ using NotI and XbaI. Profilin chimeras were generated by overlap extension PCR where the respective loop regions were encoded by the interior primers generating two fragments (A and B). The loop regions present in both fragments were then used to anneal and fuse the fragments together. The Pb Pfn loop was encoded in primers 7a and 7b, the Pf Pfn loop in primers 8a and 8b and the Tg Pfn loop by primers 9a and 9b. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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