SummaryRMgm-4634
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene tagging, Introduction of a transgene |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 31138850 |
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-928 |
Other information parent line | A P. berghei reference line expressing mCherry under the control of the P. berghei hsp70 promoter. The mutant does not contain a drug-selectable marker. The mCherry expression cassette has been introduced into the silent p23p locus |
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The mutant parasite was generated by | |
Name PI/Researcher | Niklaus L, Heussler VT |
Name Group/Department | Institute of Cell Biology |
Name Institute | University of Bern |
City | Bern |
Country | Switzerland |
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Name of the mutant parasite | |
RMgm number | RMgm-4634 |
Principal name | PbMitoKillerRed |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not tested |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | To generate parasites that mitochondrially express KillerRed (PbMitoKillerRed), we fused KillerRed with the mitochondrial targeting sequence of P. berghei citrate synthase (CS). After verifying integration of the plasmid into the P. berghei parasites’ genome and confirming mitochondrial expression, quantifications revealed that PbMitoKillerRed liver-stages were indeed efficiently killed after illumination with green light. During the time imaged, 2% of PbmCherry liver stages spontaneously exhibited a strong smooth LAMP1 rim, which was associated with parasite elimination, whereas 90% of PbMitoKillerRed parasites showed this phenotype upon illumination. |
Additional remarks phenotype | Mutant/mutation |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0506700 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1022500 | ||||||||||||||||||||||||||
Gene product | citrate synthase, mitochondrial, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | CS | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | KillerRed | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | KillerRed targeted to the mitochondrion by fusion to the targeting sequence of citrate synthase (CS). Expression under control of the constitutive eef1αa promoter (PBANKA_113330). | ||||||||||||||||||||||||||
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 | tgdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | These transgenic parasites express the photosensitizer protein KillerRed, which generates toxic reactive oxygen species (ROS) upon illumination with green light. Eukaryotic cells are most efficiently killed if KillerRed is expressed in their mitochondria or plasma membrane. To generate parasites that mitochondrially express KillerRed (PbMitoKillerRed), we fused KillerRed with the mitochondrial targeting sequence of P. berghei citrate synthase (CS). After verifying integration of the plasmid into the P. berghei parasites’ genome and confirming mitochondrial expression, quantifications revealed that PbMitoKillerRed parasites were indeed efficiently killed after illumination with green light. During the time imaged, 2% of PbmCherry parasites spontaneously exhibited a strong smooth LAMP1 rim, which was associated with parasite elimination, whereas 90% of PbMitoKillerRed parasites showed this phenotype upon illumination. To generate pL0017-MitoKillerRed, we amplified the cDNA of KillerRed by PCR with the forward primer KillerRed-fw 5′-TGGATCCATGGGTTCAGAG-3′ and the reverse primer KillerRed-rev 5′-TTCTAGATTAATCCTCGTCGCTACC-3′ with the KillerRed plasmid from Prof. Markus Meissner as a template. The cDNA of KillerRed was subcloned via blunt ends into pJET1.2 and the sequence was verified by the sequencing service of Microsynth AG in Balgach, Switzerland. The KillerRed cDNA fragment was excised with BamHI and XbaI. The final vector derived from the linearized pcmCherryMITO26. The sequence of mCherry was replaced with the sequence of KillerRed, which resulted in the pL0017-MitoKillerRed plasmid. The plasmid was linearized with ApaI and SacII before transfecting DCs. The drinking water of infected mice was supplemented with pyrimethamine (Sigma) to select for transfected parasites. The plasmid allows integration into the c- and dssu-rrna locus by single crossover. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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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/yfcu | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | No | ||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
Additional remarks genetic modification | This reporter mutant expressing mCherry 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 | ||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0818900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
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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