Summary

RMgm-4481
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1401000; Gene model (P.falciparum): PF3D7_1302500; Gene product: conserved Plasmodium protein, unknown function (PbmaLS_05)
Transgene
Transgene not Plasmodium: A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV
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; Sporozoite; Liver stage;
Last modified: 3 September 2018, 15:09
  *RMgm-4481
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) : 30154793
Reference 2 (PMID number) : 29515528
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 676m1cl1 (RMgm-29)
Other information parent line676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase 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/ResearcherFernandes P, Mueller AK
Name Group/DepartmentCentre for Infectious Diseases, Parasitology Unit
Name InstituteUniversity Hospital Heidelberg
CityHeidelberg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-4481
Principal namePbmaLS_05 (-)
Alternative namePbmaLS_05 (−) GFP Luc(con)
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageParasites lacking PbmaLS_05 showed a decreased ability to infect RBC (and immature reticulocytes), resulting in reduced peripheral parasite loads. Reduced mortality due to experimental cerebral malaria (ECM). None of the mice infected with mutant sporozoites developed ECM; in contrast, mice infected with WT sporozoites, displayed signs of ECM between day 7 and 9 p.i. Mice die from high parasitemia and anemia.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNo data provided. Assuming normal sporozoite production.
Liver stageQuantification of both numbers and development through size measurements of in vitro exo-erythrocytic forms, suggested that deletion of PbmaLS_05 had no effect on the number or developmental size of intra-hepatic parasites, in vitro. A minor developmental delay was observed for KO parasites at 24 h post-invasion but not after 48 and 63 h.p.i.. Moreover, the branching of the apicoplast and segregation between daughter nuclei was also comparable between WT and KO intra-hepatic parasites, thus excluding any defect in apicoplast inheritance. Wild type parasite liver loads in vivo in mice. all KO-infected mice became patent with blood-stage infection on the same day as those infected with WT sporozoites, regardless of the route of infection used, indicating that deletion of PbmaLS_05 has no effect on both infectivity and intra-hepatic development.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PbmaLS_05 and expresses the fusion gene GFP-Luciferase under control of the constitutive eef1a promoter (in this study the gene PbmaLS_05 was also deleted in wild type P. berghei ANKA parasites).

Protein (function)
PbmaLS_05 has a predicted protein size of 306 kDa that contains two predicted transmembrane domains and one predicted P loop containing nucleoside triphosphate hydrolase domain.
The ortholog of PBANKA_140100 (PbmaLS_05), i.e., PF3D7_1302500, was initially identified as a putative antigen that is differentially expressed in intra-hepatic stages of P. falciparum radiation-attenuated sporozoites in comparison to Pf wild-type.
Evidence is presented for an apicoplast location.

Phenotype
Parasites lacking PbmaLS_05 showed a decreased ability to infect RBC (and immature reticulocytes), resulting in reduced peripheral parasite load. Reduced mortality due to experimental cerebral malaria (ECM). None of the mice infected with mutant sporozoites developed ECM; in contrast, mice infected with WT sporozoites, displayed signs of ECM between day 7 and 9 p.i. Mice die from high parasitemia and anemia.
No information is provided on mosquito stages. Assuming that wild type numbers of sporozoites are produced.
Quantification of both numbers and development through size measurements of in vitro exo-erythrocytic forms, suggested that deletion of PbmaLS_05 had no effect on the number or developmental size of intra-hepatic parasites, in vitro. A minor developmental delay was observed for KO parasites at 24 h post-invasion but not after 48 and 63 h.p.i.. Moreover, the branching of the apicoplast and segregation between daughter nuclei was also comparable between WT and KO intra-hepatic parasites, thus excluding any defect in apicoplast inheritance. Wild type parasite liver loads in vivo in mice. all KO-infected mice became patent with blood-stage infection on the same day as those infected with WT sporozoites, regardless of the route of infection used, indicating that deletion of PbmaLS_05 has no effect on both infectivity and intra-hepatic development.

Additional information
Expression throughout the complete life cycle (in all stages). Evidence presented for an apicoplast location. See also RMgm-4482 for a mutant expressing a C-terminal GFP-tagged version of PbmaLS_05.

Other mutants 
See also RMgm-4482 for a mutant expressing a C-terminal GFP-tagged version of PbmaLS_05.


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1401000
Gene Model P. falciparum ortholog PF3D7_1302500
Gene productconserved Plasmodium protein, unknown function
Gene product: Alternative namePbmaLS_05
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/yfcu
Promoter of the selectable markerpbdhfr
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 nameA fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV
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 procedureThis reporter mutant expressing GFP-Luciferase does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence.
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