RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4175
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1140700; Gene model (P.falciparum): PF3D7_1364900; Gene product: ferrochelatase (FC; FECH)
Transgene
Transgene not Plasmodium: mCherry
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_1140700; Gene product: ferrochelatase (FC; FECH)
Phenotype Oocyst; Sporozoite; Liver stage;
Last modified: 26 June 2017, 18:37
  *RMgm-4175
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) : 28617870
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherRathnapala UL; McFadden GI
Name Group/DepartmentSchool of BioSciences
Name InstituteUniversity of Melbourne
CityParkville, Victoria
CountryAustralia
Name of the mutant parasite
RMgm numberRMgm-4175
Principal nameFC(KOmCh)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystOocysts were produced but no salivary gland sporozoites
SporozoiteOocysts were produced but no salivary gland sporozoites
Liver stageSee additional information on the generation and analysis of sporozoites lacking the FC gene (these FC(KO) sporozoites are motile and liver infective both in vitro and in vivo but have severe growth defects and fail to complete liver stage development)
Additional remarks phenotype

Mutant/mutation
The parasite lacks expression of FC and expresses mCherry under control of the constitutive eef1a promoter.

Protein (function)
The malaria parasite is capable of de novo heme biosynthesis despite its ability to acquire heme from red blood cell (RBC) hemoglobin. The first enzyme of the de novo heme-biosynthetic pathway, δ-aminolevulinate synthase (ALAS) and the last two enzymes, Protoporphyrinogen IX oxidase (PPO) and Ferrochelatase (FC) localize to the mitochondrion. The enzymes that catalyze the intermediate steps: ALA dehydratase (ALAD), Porphobilinogen deaminase (PBGD) and Uroporphyrinogen III decarboxylase (UROD) localize to the apicoplast whereas, the next enzyme Coproporphyrinogen III oxidase (CPO) is cytosolic. Earlier studies showed that host ALAD and FC are imported into the parasites in the intraerythrocytic stages, suggesting that the host machinery may augment parasite heme synthesis. See the phenotype of mutant RMgm-925 which  lacks expression of FC indicating an essential role for oocyt/sporozoite production

Phenotype
Oocysts were produced but no salivary gland sporozoites

Additional information
Evidence is presented that:
- Crossing FC deficient parasites with those carrying a wild type FC gene complements the defect in oocyst development and sporozoite production
- By crossing with wild type parasites, sporozoites were obtained that lack the FC gene (FC(KO) sporozoites)
- FC(KO) sporozoites are motile and liver infective both in vitro and in vivo but have severe growth defects and fail to complete liver stage development

Other mutants
See PF3D7_1364900


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1140700
Gene Model P. falciparum ortholog PF3D7_1364900
Gene productferrochelatase
Gene product: Alternative nameFC; FECH
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 markereef1a
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 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
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationmCherry is fused to the hdhfr selectable marker
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_1140700
Gene productferrochelatase
Gene product: Alternative nameFC; FECH
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