RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-975
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_0934900; Gene model (P.falciparum): PF3D7_1114800; Gene product: glycerol-3-phosphate dehydrogenase, putative (G3PDH; apiG3PDH)
Phenotype Liver stage;
Last modified: 4 February 2014, 11:20
  *RMgm-975
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 24330260
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/ResearcherS.E. Lindner; S.H.I. Kappe; A.M. Vaughan
Name Group/DepartmentSeattle Biomedical Research Institute
Name InstituteSeattle Biomedical Research Institute
CitySeattle
CountryUSA
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Name of the mutant parasite
RMgm numberRMgm-975
Principal namePy apig3pdh(-)
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot different from wild type
Liver stageNormal infection of liver cells by sporozoites. Normal development of liver stages up to 24 hours. Aberrant formation of liver-stage merozoites. No viable liver-stage merozoites are formed. No blood stage infection after infection of mice with mutant sporozoites.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of glycerol-3-phosphate dehydrogenase, putative (G3PDH).

Protein (function)
Fatty acids are a major component of the phospholipids that make up all cellular membranes and one hypothesis would be that the fatty acids are required for the enormous amount of membrane biosynthesis necessary late in liver stage development for the formation of tens of thousands of exoerythrocytic merozoites. The precursor of phospholipid biosynthesis is phosphatidic acid. Phosphatidic acid is formed in a three-step enzymatic reaction, the first step of which is the conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate by glycerol 3-phosphate dehydrogenase (G3PDH). Two fatty acid side chains are then transferred to glycerol 3-phosphate, firstly by glycerol 3-phosphate acyltransferase (G3PAT) to form lysophosphatidic acid and then lysophosphatidic acid acyltranferase (LPAAT) to form phosphatidic acid. Analysis of the Plasmodium genome has also uncovered two sets of genes for phosphatidic acid biosynthesis and one set is predicted to target to the apicoplast. In the rodent malaria parasite P. yoelii  G3PDH and G3PAT are indeed ocalized to the apicoplast only during liver stage development, where they are essential for production of viable merozoites. Unexpectedly, there appears to be no specific apicoplast-targeted LPAAT.

Phenotype
Normal production of sporozoites. Normal infection of liver cells by sporozoites. Normal development of liver stages up to 24 hours. Aberrant formation of liver-stage merozoites. No viable liver-stage merozoites are formed. No blood stage infection after infection of mice with mutant sporozoites. Aberrant formation of apicoplast and MSP1 expression in maturing liver-stage schizonts.

Additional information
Analysis of a mutant expressing a C-terminal 4x-Myc-tagged version of G3PDH (RMgm-976) shows expression in the apicoplast of liver stages. No expression was detected in blood stages and sporozoites.

Other mutants
RMgm-976: A mutant expressing a C-terminal 4x-Myc-tagged version of G3PDH

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PY17X_0934900
Gene Model P. falciparum ortholog PF3D7_1114800
Gene productglycerol-3-phosphate dehydrogenase, putative
Gene product: Alternative nameG3PDH; apiG3PDH
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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 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 1cggtaccGTGCGGAGCATTAAAAAATGTTGTTGCTTTAGGAGTAGG
Additional information primer 1PY00789KO-3-Fwd: KO Construct, 3'UTR Fwd
Sequence Primer 2tatgtgtgcgggcccagctagcCACGATTCTCCAGAATTTTATATATCTCCTTTGATGTG
Additional information primer 2PY00789KO-3-Rev: KO Construct, 3'UTR Rev
Sequence Primer 3atcgtggctagctgggcccGCACACATAATGGCAATTGAGGATAATTTTCACAAAAATAC
Additional information primer 3PY00789KO-5-Fwd: KO Construct, 5'UTR Fwd
Sequence Primer 4ggcggccgcTTTCAATGGTCCATTGTGCTTAACAGTTGGGGATATAAC
Additional information primer 4PY00789KO-5-Rev: KO Construct, 5'UTR Rev
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: StudioDrie StudioDrie; S. Mededovic and A. van Wigcheren