RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4542
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1315700; Gene model (P.falciparum): PF3D7_1452000; Gene product: rhoptry neck protein 2 (RON2)
Details mutation: 'promoter-swap' mutant; the promoter of RON2 replaced by the promoter of PBANKA_1443300 (msp9)
Transgene
 Transgene not Plasmodium: GFP (gfp-mu3)
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 (230p)
Phenotype Sporozoite; Liver stage;
Last modified: 7 September 2020, 17:00
  *RMgm-4542
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 30307699
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 P. berghei ANKA 507cl1 (RMgm-7)
Other information parent line
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The mutant parasite was generated by
Name PI/ResearcherIshino T, Torii M
Name Group/DepartmentDivision of Molecular Parasitology
Name InstituteProteo-Science Center, Ehime University
CityToon, Ehime
CountryJapan
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Name of the mutant parasite
RMgm numberRMgm-4542
Principal nameP(msp9)-RON2
Alternative name
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
SporozoiteMutant sporozoites developed and matured normally in oocysts. ron2 mRNA levels in mutant sporozoites were approximately 50-fold less, compared to wild-type sporozoites and RON2 protein amount is decreased approximately 50-fold. Mutant (hemolymph) sporozoites were strongly reduced (20-fold reduction) in invading salivary glands. Reduced motility of mutant sporozoites. Mutant sporozoites showed reduced capacity to invade hepatocytes (and reduced infectivity to mice).
Liver stageMutant (hemolymph) sporozoites were strongly reduced (20-fold reduction) in invading salivary glands. Reduced motility of mutant sporozoites. Mutant sporozoites showed reduced capacity to invade hepatocytes (and reduced infectivity to mice).
Additional remarks phenotype

Mutant/mutation
In the 'promoter-swap' mutant the promoter of RON2 is replaced by the promoter (1252bp) of PBANKA_1443300 (msp9). This promoter is active in asexual blood stages (in schizonts) but is silent in mosquito stages, such as oocysts and sporozoites.

Protein (function)
RON2 is a rhoptry neck protein expressed in rhoptries of merozoites and sporozoites. Unsuccessful attempts to knock-out the ron2 gene (RMgm-214) indicates an essential function in blood stages. A number of rhoptry neck proteins (RONs), including RON2, RON4, and RON5 are secreted and inserted into the target cellular membrane as a complex. RON2 interacts with the micronemal protein AMA1.

Phenotype
Unsuccessful attempts to knock-out the ron2 gene (RMgm-214) indicates an essential function in blood stages.
Growth and multiplication of blood stages of the 'promoter-swap' mutant was comparable to that of wild type parasites.
Mutant sporozoites developed and matured normally in oocysts. ron2 mRNA levels in mutant sporozoites were approximately 50-fold less, compared to wild-type sporozoites and RON2 protein amount is decreased  approximately 50-fold. Mutant (hemolymph) sporozoites were strongly reduced (20-fold reduction) in invading salivary glands. Reduced motility of mutant sporozoites. Mutant (salivary gland) sporozoites showed reduced capacity to invade hepatocytes (and reduced infectivity to mice).

Additional information
Evidence is presented for localisation of RON2 in the rhoptry neck of rhoptries of merozoites while in rhoptries of sporozoites RON2 is distributed throughout the rhoptry body. See also RMgm-4543 for a mutant expressing a Cmyc tagged version of RON2.

In the paper two other 'promoter-swap' mutants are described where the ron2 promoter regions has been replaced with promoter regions of rap1 (PBANKA_1032100) and msp1 (BANKA_0831000)

Other mutants

  Mutated: Mutant parasite with a mutated gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1315700
Gene Model P. falciparum ortholog PF3D7_1452000
Gene productrhoptry neck protein 2
Gene product: Alternative nameRON2
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Details of the genetic modification
Short description of the mutation'promoter-swap' mutant; the promoter of RON2 replaced by the promoter of PBANKA_1443300 (msp9)
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo generate sporozoite stage-specific ron2 silencing transgenic parasites, the 5′UTR region of ron2 was replaced with a merozoite-specific candidate promoter region by double crossover homologous recombination within the GFP background strain. Two homologous recombination cassettes, ron2 upstream (-1683 to -845 bp relative to RON2 start of translation) and RON2-N (-105 to +945 bp) respectively, were sub-cloned into a transgenic vector containing a human DHFR expression cassette (hDHFR) used for drug selection. The promoter region of msp9 (PBANKA_1443300) was also sub-cloned into the transgenic vector immediately before the RON2-N region.
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 nameGFP (gfp-mu3)
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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 parasitegfp (FACS)
Promoter of the selectable markereef1a
Selection (positive) procedureFACS (flowsorting)
Selection (negative) procedureNo
Additional remarks genetic modificationThe GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration.
Additional remarks selection procedureThis reporter mutant expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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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 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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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