RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5003
Malaria parasiteP. berghei
Genotype
MutatedGene model (rodent): PBANKA_1028300; Gene model (P.falciparum): PF3D7_1414400; Gene product: serine/threonine protein phosphatase PP1 (PP1)
Details mutation: 'promoter swap mutant': the promoter of pp1 replaced with the ama1 promoter (PBANKA_0915000).
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; Gametocyte/Gamete; Fertilization and ookinete; Oocyst;
Last modified: 21 June 2021, 12:47
  *RMgm-5003
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) : 34145386
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/ResearcherZeeshan M, Tewari R
Name Group/DepartmentSchool of Life Sciences, Queens Medical Centre
Name InstituteUniversity of Nottingham
CityNottingham
CountryUK
Name of the mutant parasite
RMgm numberRMgm-5003
Principal namePP1PTD
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageThe PP1PTD parasites grew slower than the WT-GFP controls in the asexual blood stage but produced normal numbers of gametocytes in mice.
Gametocyte/GameteThe PP1PTD parasites grew slower than the WT-GFP controls in the asexual blood stage but produced normal numbers of gametocytes in mice.
Reduced numbers of exflagellation (male gamete formation).
Fertilization and ookineteReduced numbers of exflagellation (male gamete formation). Strongly reduced ookinete formation.
OocystStrongly reduced ookinete formation. No oocyst formation.
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
In the 'promoter swap mutant': the promoter of pp1 has been replaced with the asexual blood stage promoter ama1 (PBANKA_0915000). This promoter is active in asexual blood stages but not in gametocytes.

Protein (function)
PP1 is a serine/threonine protein phosphatase

Phenotype
To investigate the function of PP1 during cell division in male gametogenesis, we used a promoter trap double homologous recombination (PTD) approach to downregulate gene expression at this stage by placing the pp1 gene under the control of the AMA1 promoter. AMA1 is known to be highly expressed in asexual blood stages, but not during sexual differentiation.

PP1 is expressed both in asexual blood stages and (male) gametocytes. Knock-out of the pp1 gene is not possible since it has an essential role in asexual blood stages. In the promoter swap mutant PP1 is expressed in asexual blood stages but is down-regulated in (male) gametocytes.

The PP1PTD parasites grew slower than the WT-GFP controls in the asexual blood stage but produced normal numbers of gametocytes in mice.
Reduced numbers of exflagellation (male gamete formation). Strongly reduced ookinete formation. No oocyst formation.

Additional information
Evidence is presented that:
- Ultrastructure analysis of PP1PTD male gametocytes shows defects in nuclear pole and axoneme assembly after activation. The development of PP1PTD male gametocytes was severely retarded, although axoneme growth increased.

From the Abstract:
'Using real-time live-cell and ultrastructural imaging, conditional gene knockdown, RNA-seq and proteomic approaches, we show that Plasmodium PP1 is implicated in both mitotic exit and, potentially, establishing cell polarity during zygote development in the mosquito midgut'.

From the paper: 'we used an auxin-inducible degron (AID) system to try and study the effect of rapid PP1 degradation in gametocytes. We tagged the endogenous pp1 gene with an AID/HA epitope tag to degrade the fusion protein in presence of auxin (Philip and Waters, 2015), and successfully generated pp1- AID parasite lines as shown by integration PCR but could not deplete kinesin-13 protein by auxin treatment'.

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1028300
Gene Model P. falciparum ortholog PF3D7_1414400
Gene productserine/threonine protein phosphatase PP1
Gene product: Alternative namePP1
Details of the genetic modification
Short description of the mutation'promoter swap mutant': the promoter of pp1 replaced with the ama1 promoter (PBANKA_0915000).
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 modificationConditional gene knockdown construct was designed using construct Pama1 (pSS368) (Sebastian et al., 2012).
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 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.
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