Back to search resultsSummaryRMgm-1467
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene disruption, Introduction of a transgene, Introduction of a transgene |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 27225796 |
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 | RMgm-1320 |
Other information parent line | This transgenic reporter line expresses luciferase under the control of the eef1α (PBANKA_113330) promoter and, in addition, mCherry under the control of the hsp70 (PBANKA_071190) promoter. Both reporter cassettes are introduced into the silent 230p locus, using a single DNA construct. This transgenic line does not contain a drug-selectable marker |
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The mutant parasite was generated by | |
Name PI/Researcher | De Niz M; Heussler V; Spielmann T |
Name Group/Department | Institute of Zoology |
Name Institute | Universtity of Hamburg |
City | Hamburg |
Country | Germany |
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Name of the mutant parasite | |
RMgm number | RMgm-1467 |
Principal name | PbΔmahrp1a |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | While no phenotypic differences were observed between the gene-deletion mutants and wild-type (wt) parasites during development in the mosquito or in the liver, blood stages of the mutants exhibited an attenuated phenotype in mice. While wt-infected C57B/6 mice died 7–9 days post infection as a result of complications typical for experimental cerebral malaria, PbΔmahrp1a-infected mice did not develop experimental cerebral malaria but died from hyperparasitemia 32–45 days post infection, respectively. A similar attenuation was also observed in infections in Balb/c mice. The parasitemia in the PbΔmahrp1a-infected mice was comparable to that of wt during the initial 2 days of infection, but was reduced compared with wt thereafter. Mutant parasites were predominantly found in reticulocytes throughout the course of infection, whereas virulent wt parasites were found in erythrocytes in the later course of infection. In addition, mice infected with thePbΔmahrp1a mutant showed a 3.6-fold increase in schizonts in the peripheral blood circulation compared to wt infections, respectively , indicating reduced sequestration of RBCs infected with mutant schizonts. PbΔmahrp1a parasites showed strongly reduced accumulation in the adipose tissue and lungs compared with wt, which is typical for a loss of CD36-mediated sequestration. Conversely, significantly increased accumulation of parasites was detected in the spleens of mice infected with PbΔmahrp1a mutants. Higher accumulation in the spleen of non-sequestered schizonts has been attributed to increased removal of non-sequestering schizonts by the spleen. Binding to mouse CD36 was significantly lower in PbΔmahrp1a schizonts compared with wt schizonts with both plate-coated CD36 or CD36 expressed on CHO-745 cells. |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Not different from wild type |
Additional remarks phenotype | Mutant/mutation While the overall amino-acid similarity between MAHRP1a of P. berghei and MAHRP1 of P. falciparum was rather low, the architecture of the protein features was similar and both proteins lack a PEXEL motif. In addition, three other findings indicated that these proteins are indeed orthologues. Firstly, the phylogenetic trees of these proteins are topologically concordant with the species tree of malaria parasites; secondly, a jackhmmer search arrived at the same proteins originally detected by our similarity searches; and finally, a re-examination of the genomic location revealed that the genes encoding MAHRP1 orthologues are in fact syntenic. Immunofluorescence assays with these sera showed that Pbmahrp1a was exported into P. berghei iRBCs where it was found in multiple discrete foci. By introducing the P. falciparum mahrp1a gene into PbΔmahrp1a evidence is presented that P. falciparum MAHRP1 complements the function of P. berghei mahrp1a: the growth-, sequestration- and virulence phenotype were (partly) restored in the complemented mutant)
Other mutants |
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Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1145800 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1370300 | ||||||||||||||||||||||||
Gene product | membrane associated histidine-rich protein | ||||||||||||||||||||||||
Gene product: Alternative name | MAHRP1; membrane associated histidine-rich protein 1a; MAHRP1a | ||||||||||||||||||||||||
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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 | pbdhfr | ||||||||||||||||||||||||
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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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | mCherry | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
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 | hdhfr | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | No | ||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||
Additional remarks selection procedure | This reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable marker. The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice. | ||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PBANKA_0711900 | ||||||||||||||||||
Gene product | PBANKA_0711900 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0306000 | ||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | 230p | ||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | Luciferase | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
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 | hdhfr | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||
Additional remarks selection procedure | This reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable marker. The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice. | ||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1133300 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1357100 | ||||||||||||||||||
Gene product | elongation factor 1-alpha | ||||||||||||||||||
Gene product: Alternative name | eef1a | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0306000 | ||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | 230p | ||||||||||||||||||
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