RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1049
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1346500; Gene model (P.falciparum): PF3D7_1331600; Gene product: protein tyrosine phosphatase-like protein, putative (PTPLA; 3-hydroxyacyl-CoA dehydratase; DEH)
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 Oocyst; Sporozoite; Liver stage;
Last modified: 23 September 2020, 15:30
  *RMgm-1049
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) : 25011111
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/ResearcherDS Guttery, AA Holder, R Tewari
Name Group/DepartmentMalaria Research Group/School of Life Sciences
Name InstituteUniversity of Nottingham
CityNottingham
CountryUK
Name of the mutant parasite
RMgm numberRMgm-1049
Principal nameΔptpla
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
OocystNormal oocyst size and numbers. No sporozoites formed 14 or 21 days post-infection
SporozoiteNormal oocyst size and numbers. No sporozoites formed 14 or 21 days post-infection
Liver stageNot transmitted
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of PTPLA and expresses GFP under the constitutive eef1a promoter.

Protein (function)
The gene was targetted for deletion/tagging in a systematic functional analysis of the entire P. berghei protein phosphatome, which comprises 30 predicted protein phosphatases (PPs), that exhibit differential and distinct expression patterns during various stages of the life-cycle. Gene disruption analysis of all P. berghei PPs revealed that half of the genes are likely essential for asexual blood stage development; whereas six are essential for sexual development/sporogony in the mosquito.
The parasite utilises a number of signal transduction mechanisms, including reversible protein phosphorylation catalysed by protein kinases (PKs) and phosphatases (PPs). This mechanism of signalling is a conserved, ubiquitous regulatory process for many eukaryotic and prokaryotic cellular pathways.

Sequence analysis of the P. falciparum parasite has revealed approximately 85 putative PK and 27 putative PP catalytic subunits encoded in its genome (the Plasmodium protein phosphatome being one of the smallest of the eukaryotic phyla).
The Plasmodium phosphatome has been classified into 4 major groups: phosphoprotein phosphatases (PPPs), metallo-dependent protein phosphatases (PPMs), protein tyrosine phosphatases (PTPs) and NLI interacting factor-like phosphatases (NIFs), as well as a number of smaller classes.

To define the phosphatome, PPs encoded in the genomes of P. berghei and P. falciparum were identified by similarity to hidden Markov models of known PP catalytic domains. PFam domains were used to define protein sets with similarity to PPP, PTP, PPM, NIF-like and PTP-like A families. There are no predicted PPs with good similarity to the Low-Molecular Weight Phosphatase (LMWP) or CDC25 families. There are also no good matches to models of SSU72 RNA polymerase II CTD phosphatase or Eyes Absent (EYA) phosphatase. Other PFam domains specific to PP catalytic domains are subclasses of the above families. The 5 identified Plasmodium PP families were compared to 4969 PP-like proteins from 44 diverse eukaryotes, to classify them and eliminate PP-like proteins with confirmed non-protein phosphatase functions.

In this study 30 and 29 PPs were identified in the genomes of P. berghei and P. falciparum respectively, encompassing 28 direct orthologues across the 5 PP families described above.
As found with the kinome, the phosphatome is highly conserved with only three proteins without direct orthology between P. falciparum and P. berghei.
On the basis of catalytic domain phylogeny and domain architecture, the Plasmodium PPPtype phosphatases can be further classified into subfamilies, with PPP1 to PPP7 corresponding to the animal PP1-PP7 types. Plasmodium PPPs also include the BSU-like phosphatase PPKL, an EF-hand containing phosphatase (EFPP) and the two SHLPs, none of which is present in the host.

Phenotype
Normal oocyst size and numbers. No sporozoites formed 14 or 21 days post-infection

Additional information

Other mutants


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1346500
Gene Model P. falciparum ortholog PF3D7_1331600
Gene productprotein tyrosine phosphatase-like protein, putative
Gene product: Alternative namePTPLA; 3-hydroxyacyl-CoA dehydratase; DEH
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 parasitetgdhfr
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 1CCCCGGGCCCCCTGAATTGATCTTATCTAATTGTATTG
Additional information primer 1P0471
Sequence Primer 2GGGGAAGCTTGGATAACGATTGAGTTATAGCTATC
Additional information primer 2P0472
Sequence Primer 3CCCCGAATTCGACCTCATTATTTTATTACTTCCC
Additional information primer 3P0473
Sequence Primer 4GGGGTCTAGAGAAGAATGGGATGGTAATTTATCTG
Additional information primer 4P0474
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 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