RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-975

Malaria parasite	P. yoelii
Genotype
Disrupted	Gene 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 modification	The 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 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-975
Principal name	Py apig3pdh(-)
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	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.
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 product

glycerol-3-phosphate dehydrogenase, putative

Gene product: Alternative name

G3PDH; apiG3PDH

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

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

Partial or complete disruption of the gene

Complete

Additional remarks partial/complete disruption

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

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	cggtaccGTGCGGAGCATTAAAAAATGTTGTTGCTTTAGGAGTAGG
Additional information primer 1	PY00789KO-3-Fwd: KO Construct, 3'UTR Fwd
Sequence Primer 2	tatgtgtgcgggcccagctagcCACGATTCTCCAGAATTTTATATATCTCCTTTGATGTG
Additional information primer 2	PY00789KO-3-Rev: KO Construct, 3'UTR Rev
Sequence Primer 3	atcgtggctagctgggcccGCACACATAATGGCAATTGAGGATAATTTTCACAAAAATAC
Additional information primer 3	PY00789KO-5-Fwd: KO Construct, 5'UTR Fwd
Sequence Primer 4	ggcggccgcTTTCAATGGTCCATTGTGCTTAACAGTTGGGGATATAAC
Additional information primer 4	PY00789KO-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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