RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-980
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_1312000; Gene model (P.falciparum): PF3D7_1444300; Gene product: 1-acyl-sn-glycerol-3-phosphate acyltransferase, putative (LPAAT; AGPAT)
Name tag: 4xMyc
Phenotype Asexual bloodstage; Liver stage;
Last modified: 27 December 2013, 22:09
  *RMgm-980
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
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-980
Principal namePy 01678myc
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageStrong expression of LPAATmyc in blood stages. Evidence is presented for a location in the ER and NOT in the apicoplast.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageStrong expression of LPAATmyc in 24 and 48 hour liver stages. No apicoplast location
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal 4xMyc-tagged version of 1-acyl-sn-glycerol-3-phosphate acyltransferase, putative (LPAAT).

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.

Multiple unsuccessful attempts to disrupt the lpaat gene (RMgm-979, RMgm-485) indicates an essential function for growth/multiplication of asexual blood stages.

Phenotype
Multiple unsuccessful attempts to disrupt the lpaat gene (RMgm-979, RMgm-485) indicates an essential function for growth/multiplication of asexual blood stages.


Analysis of the mutant expressing the tagged version of LPAAT showed strong expression of LPAATmyc in blood stages. Evidence is presented for a location in the ER and NOT in the apicoplast. Strong expression of LPAATmyc in 24 and 48 hour liver stages. No apicoplast location

Additional information

Other mutants
RMgm-979: A mutant lacking expression of 1-acyl-sn-glycerol-3-phosphate acyltransferase, putative (LPAAT).

  Tagged: Mutant parasite with a tagged gene
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Details of the target gene
Gene Model of Rodent Parasite PY17X_1312000
Gene Model P. falciparum ortholog PF3D7_1444300
Gene product1-acyl-sn-glycerol-3-phosphate acyltransferase, putative
Gene product: Alternative nameLPAAT; AGPAT
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Details of the genetic modification
Name of the tag4xMyc
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid 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 parasitetgdhfr
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedureThe transgenic myc-epitope expressing parasites were created by gene replacement and thus contained a single epitope-tagged copy of the gene under its endogenous promoter.
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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren