RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-976

Malaria parasite	P. yoelii
Genotype
Tagged	Gene model (rodent): PY17X_0934900; Gene model (P.falciparum): PF3D7_1114800; Gene product: glycerol-3-phosphate dehydrogenase, putative (G3PDH; apiG3PDH) Name tag: 4xMyc
Phenotype	Liver stage;

Last modified: 24 December 2013, 21:51

*RMgm-976

Successful modification	The 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 Parasite	P. yoelii
Parent strain/line	P. 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/Researcher	S.E. Lindner; S.H.I. Kappe; A.M. Vaughan
Name Group/Department	Seattle Biomedical Research Institute
Name Institute	Seattle Biomedical Research Institute
City	Seattle
Country	USA
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Name of the mutant parasite
RMgm number	RMgm-976
Principal name	Py apiG3PDHmyc
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not tested
Sporozoite	Not different from wild type
Liver stage	apiG3PDHmyc expression in apicoplast of liver stages
Additional remarks phenotype	Mutant/mutation The mutant expresses a C-terminal 4xMyc-tagged version 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. Analysis of a mutant lacking expression of G3PDH (RMgm-975) provides evidence for an essential role of G3PDH for the maturation of liver stage schizonts and formation of viable liver-stage merozoites Phenotype Analysis of a mutant lacking expression of G3PDH (RMgm-975) provides evidence for an essential role of G3PDH for the maturation of liver stage schizonts and formation of viable liver-stage merozoites Analysis of the mutant expressing the tagged version of G3PDH showed apiG3PDHmyc expression in apicoplast of liver stages. No expression was detected in blood stages and sporozoites Additional information Other mutants RMgm-975: A mutant lacking expression of glycerol-3-phosphate dehydrogenase, putative (G3PDH).

Tagged: Mutant parasite with a tagged 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 product

glycerol-3-phosphate dehydrogenase, putative

Gene product: Alternative name

G3PDH; apiG3PDH

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Details of the genetic modification

Name of the tag

4xMyc

Details of tagging

C-terminal

Additional remarks: tagging

Commercial source of tag-antibodies

Type of plasmid/construct

Plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Selectable marker used to select the mutant parasite

tgdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

Additional remarks selection procedure

The 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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