RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4939
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1122300; Gene model (P.falciparum): PF3D7_0623400; Gene product: MEI2-like RNA-binding protein (Mei2)
DisruptedGene model (rodent): PBANKA_1024600; Gene model (P.falciparum): PF3D7_1418100; Gene product: liver specific protein 1, putative (LISP1)
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: 4 December 2020, 17:10
  *RMgm-4939
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-4937
Other information parent lineThis mutant (2834cl2m1cl1; RMgm-4937) lacks the mei2 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 cassetes 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-4939
Principal name2901cl1
Alternative nameΔmei2Δlisp1
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 stage∆mei2∆lisp1 have lower parasite liver loads (determined by in vivo imaging) compared to mice infected with either WT or the single gene-deletion mutant ∆mei2. Some mice infected with 5 × 10(4) ∆mei2∆lisp1 sporozoites developed blood infections. These mice that established a blood-stage infection had a greatly prolonged prepatent period (5-9 days)
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of MEI2 and LISP1. The lisp1 gene has been deleted in mutant (2834cl2m1cl1; RMgm-4937) that lacks the mei2 gene and does not contain a drug selectable marker. This mutant also expresses mCherry and luciferase under constitutive promoters. 

Protein (function)
Mei2 is a member of the largest family of RNA binding proteins (RBPs) – those that contain a RNA recognition motif (RRM), a stretch of 70-90 amino acids that contain two consensus RNA-interacting motifs, RNP1 and RNP2. RRM-containing proteins are subdivided into ten separate families (RRM_1 thru RRM_10) based on shared amino acid identities between members of each family and Mei2 contains a C-terminal RRM_2, thought to be unique to fungi and plants. Plasmodium contains a single Mei2-like gene

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

Phenotype
Normal blood stage and mosquito stage development.
Mice intravenously infected with ∆mei2∆lisp1 sporozoites have lower parasite liver loads (determined by in vivo imaging) compared to mice infected with either WT or the single gene-deletion mutant ∆mei2. Some mice infected with 5 × 10(4) ∆mei2∆lisp1 sporozoites developed blood infections. These mice that established a blood-stage infection had a greatly prolonged prepatent period (5-9 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_1122300
Gene Model P. falciparum ortholog PF3D7_0623400
Gene productMEI2-like RNA-binding protein
Gene product: Alternative nameMei2
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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-300555
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 mei2, construct PbGEM-300555 (pL2206) was used (http://plasmogem.sanger.ac.uk/). This 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 pL2206 (exp. 2834) using standard transfection technologies and transformed parasites selected by positive selection with pyrimethamine. Selected parasites were cloned by limiting dilution and mutant 2834cl2 was used for genotype analysis and to generate the SM-free gene deletion mutant. To remove the hdhfr::yfcu SM cassette from the genomes of 2834cl2, 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 constructs pL2206, flanking the hdhfr::yfcu cassette and thereby removing the SM. Selection and cloning of the parasites resulted in the SM-free single gene-deletion mutant ∆mei2 (2834cl2m1cl1)
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_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 2834cl2m1cl1; (RMgm-4937) were transfected with construct pL2204 (exp. 2901) using standard transfection technologies and transformed parasites selected by positive selection with pyrimethamine. Selected parasites were cloned by limiting dilution and mutant 2901cl1 were used for genotype and phenotype analysis.
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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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren