RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4736
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1400800; Gene model (P.falciparum): PF3D7_1302200; Gene product: protein UIS3 (ETRAMP13)
Details mutation: the P. berghei uis3 gene is replaced with the uis3 gene of P. falciparum
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 Liver stage;
Last modified: 9 March 2020, 14:08
  *RMgm-4736
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) Not published (yet)
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone 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/ResearcherPul, FJA (Geurten, FJA), Janse CJ
Name Group/DepartmentLeiden Malaria Research Group, Parasitology
Name InstituteLeiden University Medical Center
CityLeiden
CountryThe Netherlands
Name of the mutant parasite
RMgm numberRMgm-4736
Principal name2551cl1, 2552cl1,2
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageIn a few experiments mice were injected with mutant sporozoites. Infectivity of the mutant sporozoites appeared to be (slightly) reduced to normal (whereas P. berghei mutants lacking uis3 show strongly reduced liver stage development)
Additional remarks phenotype

Mutant/mutation
In the mutant the uis3 gene is replaced with the uis3 gene of P. falciparum. The P. falciparum uis3 gene is under control of the 5'UTR of P. berghei uis3 and the 3'UTR of the P. berghei cam gene. In addition the mutant expresses the fusion protein GFP-Luciferase under control of the constitutive eef1a promoter

Protein (function)
UIS3 is a member of the ETRAMP family of proteins. these proteins are frequently located in the parasitophorous vacuole (of blood stages and liver stages). P. berghei UIS3 is expressed in both blood stages and in sporozoites (very high transcript levels) and liver stages

Phenotype
The phenotype has not been analysed in detail. Direct replacement of P. berghei uis3 with P. falciparum uis3 was successful, indicating that expression of P. falciparum uis3 is not 'toxic' for blood stages (see also mutant RMgm-4735). However, expression of P. falciparum uis3 has not been established in this mutant.

In a few experiments mice were injected with mutant sporozoites. Infectivity of the mutant sporozoites appeared to be (slightly) reduced to normal (whereas P. berghei mutants lacking uis3 show strongly reduced liver stage development)

Additional information


Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1400800
Gene Model P. falciparum ortholog PF3D7_1302200
Gene productprotein UIS3
Gene product: Alternative nameETRAMP13
Details of the genetic modification
Short description of the mutationthe P. berghei uis3 gene is replaced with the uis3 gene of P. falciparum
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
Click to view information
Click to hide information
Plasmid PL2107
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 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) procedurepyrimethamine
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