RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4943
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1024600; Gene model (P.falciparum): PF3D7_1418100; Gene product: liver specific protein 1, putative (LISP1)
DisruptedGene model (rodent): PBANKA_1003000; Gene model (P.falciparum): PF3D7_0405300; Gene product: liver specific protein 2, putative (LISP2)
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)
Transgene
 Transgene not Plasmodium: Luciferase
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (P230p)
Phenotype Liver stage;
Last modified: 8 December 2020, 13:02
  *RMgm-4943
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene disruption, Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Not published (yet)
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone RMgm-4938
Other information parent lineThis mutant (2832cl3m0cl1; RMgm-4938) lacks the lisp1 gene and expresses luciferase under the control of the eef1α (PBANKA_1133300) promoter and, in addition, mCherry under the control of the hsp70 (PBANKA_0711900) promoter. Both reporter cassettes are introduced into the silent 230p locus, using a single DNA construct. This mutant does not contain a drug-selectable marker
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The mutant parasite was generated by
Name PI/ResearcherOthman AS, Franke-Fayard B, Janse CJ
Name Group/DepartmentLeiden Malaria Group, Department of Parasitology
Name InstituteLeiden University Medical Center, LUMC
CityLeiden
CountryThe Netherlands
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Name of the mutant parasite
RMgm numberRMgm-4943
Principal name2961cl1
Alternative nameΔlisp1Δlisp2
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stage100% of the mice intravenously infected with 5 × 10(4) ∆lisp1∆lisp2 sporozoites developed blood infections. Mice with blood infections showed a prolonged prepatent period (2-3 days).
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of LISP1 and LISP2. It also expresses mCherry and luciferase under constitutive promoters. The mutant does not contain a drug-selectable marker.

Protein (function)
Evidence has been presented that LISP1 plays a role in egress of merozoites from liver cells.

Evidence has been presented that LISP2 plays a role in development of maturing liver schizonts. LISP2 is specifically expressed in (late) liver stages. Evidence has been presented for export of LISP2 into the cytoplasm of the hepatocyte. The protein has been named LISP2 and sequestrin. 

Phenotype
100% of the mice intravenously infected with 5 × 10(4) ∆lisp1∆lisp2 sporozoites developed blood infections. Mice with blood infections showed a  prolonged prepatent period (2-3 days).

Additional information

Other mutants

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1024600
Gene Model P. falciparum ortholog PF3D7_1418100
Gene productliver specific protein 1, putative
Gene product: Alternative nameLISP1
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedYes
Name of PlasmoGEM construct/vectorPbGEM-334115
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationFor deletion of lisp1, construct PbGEM-334115 (pL2204) was used (http://plasmogem.sanger.ac.uk/). The construct contains the hdhfr::yfcu selectable marker (SM) cassette with the P. berghei eef1α promoter region and 3’ terminal sequence of pbdhfr. Before transfection, the construct was linearized by digesting with NotI. Parasites of line 1868cl1 were transfected with construct pL2204 (exp. 2832) using standard transfection technologies and transformed parasites selected by positive selection with pyrimethamine. Selected parasites were cloned by limiting dilution and mutant 2832cl3 were used for genotype analysis and to generate the SM-free gene deletion mutants. To remove the hdhfr::yfcu SM cassette from the genome of 2832cl3, these parasites were selected (negative selection) by treatment of infected mice with 5-Fluorocytosine (5-FC) as described. This treatment selects for parasites that have undergone homologous recombination between the two 3’-UTR of pbdhfr untranslated regions present in the integrated construct pL2204, flanking the hdhfr::yfcu cassette and thereby removing the SM. Selection and cloning of the parasites resulted in the SM-free single gene-deletion mutants ∆lisp1 (2832cl3m0cl1)
Additional remarks selection procedureSee above for applying negative selection to remove the drug selectable marker cassette
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
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  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1003000
Gene Model P. falciparum ortholog PF3D7_0405300
Gene productliver specific protein 2, putative
Gene product: Alternative nameLISP2
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo delete the lisp2 gene plasmid pL1462 was used (see RMgm-930). This construct was used to delete lisp2 in mutant 2832cl3m0cl1 (RMgm-4938).
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
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene namemCherry
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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 and Luciferase does not contain a drug-selectable
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 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
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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
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  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameLuciferase
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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 modification
Additional remarks selection procedureThis reporter mutant expressing mCherry and Luciferase does not contain a drug-selectable
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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 productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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
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3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
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 nameP230p
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
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P.berghei model of malaria
Technical design and realization: S. Mededovic and A. van Wigcheren