RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4634
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0506700; Gene model (P.falciparum): PF3D7_1022500; Gene product: citrate synthase, mitochondrial, putative (CS)
Name tag: KillerRed
Transgene
Transgene not Plasmodium: mCherry
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0711900; Gene product: heat shock protein 70 (HSP70)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
Phenotype Liver stage;
Last modified: 4 June 2019, 17:33
  *RMgm-4634
Successful modificationThe 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
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-928
Other information parent lineA 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
The mutant parasite was generated by
Name PI/ResearcherNiklaus L, Heussler VT
Name Group/DepartmentInstitute of Cell Biology
Name InstituteUniversity of Bern
CityBern
CountrySwitzerland
Name of the mutant parasite
RMgm numberRMgm-4634
Principal namePbMitoKillerRed
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageTo 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
These transgenic parasites express the photosensitizer protein KillerRed, which generates toxic reactive oxygen species (ROS) upon illumination with green light. The KillerRed was fused with the mitochondrial targeting sequence of P. berghei citrate synthase (CS). The KillerRed-CS fusion gene is under control of the constitutive eef1a promoter and introduced into the c/d-ssu-rRNA gene locus. In addition the parasites express mCherry (under control of the hsp70 promoter and introduced into the silent 230p gene).

Protein (function)

Phenotype
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 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.

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 information

Other mutants


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0506700
Gene Model P. falciparum ortholog PF3D7_1022500
Gene productcitrate synthase, mitochondrial, putative
Gene product: Alternative nameCS
Details of the genetic modification
Name of the tagKillerRed
Details of taggingC-terminal
Additional remarks: taggingKillerRed 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 usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThese 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
Primer information: Primers used for amplification of the target sequences  Click to view information
Primer information: Primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemCherry
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedureNo
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationThis 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
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0711900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4