RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4361
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1321700; Gene model (P.falciparum): PF3D7_1458000; Gene product: cysteine proteinase falcipain 1 (FP1)
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 Asexual bloodstage; Liver stage;
Last modified: 17 October 2017, 18:08
  *RMgm-4361
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 28922424
MR4 number
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 lineP.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190).
The mutant parasite was generated by
Name PI/ResearcherHopp CS; Sinnis P
Name Group/DepartmentDepartment of Molecular Microbiology & Immunology
Name InstituteJohns Hopkins Bloomberg School of Public Health
CityBaltimore
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-4361
Principal nameBP1-KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stage(Strongly) reduced growth rate of blood stages
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageProlonged prepatent period (4 days delay) after infection of mice with sporozoites (see additional information). Normal hepatocyte invasion and development of liver stages. Normal merosome formation and rupture. Evidence is presented for reduced hepatic merozoite infectivity.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of BP1

Protein (function)
P. falciparum expresses four cysteine proteases namely, falcipain-1 (FP-1), falcipain-2 and -2' (FP-2A and FP-2B) and falcipain-3 (FP-3) at asexual blood stages of the parasite. These proteases perform multiple functions such as hemoglobin hydrolysis, erythrocyte rupture and erythrocyte invasion and differ in their timing of expression. Falcipain-1 is active at the invasive merozoite stage while falcipain-2/-2' and -3 are expressed mainly at early and late trophozoite stages respectively.

Both P. falciparum and P. berghei have an papain-like cysteine protease encoded by a syntenic gene (falcipain 1, FP1, PF3D7_1458000 and berghepain 1, BP1, PBANKA_132170). Evidence has been presented that this protease is located (and active) in the (apical end) of merozoites and not in the digestive vacuole, suggesting that it has no role in hemoglobin digestion.

P. berghe has an additional papain-like cysteine protease, Berghepain-2 (BP2), which is equivalent to the three P. falciparum digestive vacuole falcipains (papain-like cysteine endoproteases), FP-2 (FP2a, PF3D7_1115700; FP2b, PF3D7_1115300) and -3 (PF3D7_1115400). The gene encoding BP2 has a syntenic location to the three falcipain genes of P. falciparum and also the the single copy vivapain-2 of P. Vivax (PVX_086040).

Phenotype
From the Abstract: 'We found that this mutant parasite displays a pronounced delay in blood stage infection after inoculation of sporozoites. Experiments designed to pinpoint the defect of berghepain-1 knockout parasites found that it was not due to alterations in gliding motility, hepatocyte invasion or liver stage development and that injection of berghepain-1 knockout merosomes replicated the phenotype of delayed blood stage growth after sporozoite inoculation. We identified an additional role for berghepain-1 in preparing blood stage merozoites for infection of erythrocytes and observed that berghepain-1 knockout parasites exhibit a reticulocyte restriction, suggesting that berghepain-1 activity broadens the erythrocyte repertoire of the parasite. The lack of berghepain-1 expression resulted in a greater reduction in erythrocyte infectivity in hepatocyte-derived merozoites than it did in erythrocyte-derived merozoites.'

Additional information

Other mutants
See PF3D7_1458000
 


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1321700
Gene Model P. falciparum ortholog PF3D7_1458000
Gene productcysteine proteinase falcipain 1
Gene product: Alternative nameFP1
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 parasitehdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
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 nameGFP (gfp-mu3)
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 modification
Additional remarks selection procedure
Other details transgene
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
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