RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-1469

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_1416700; Gene model (P.falciparum): PF3D7_1318200; Gene product: glycerol-3-phosphate 1-O-acyltransferase (G3PAT; Pb apiG3PAT) Name tag: HA-GFP
Phenotype	Liver stage;

Last modified: 24 June 2016, 13:18

*RMgm-1469

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) : 27324409
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	Shears MJ; McFadden GI
Name Group/Department	University of Melbourne, BioSciences
Name Institute	University of Melbourne, BioSciences
City	Melbourne
Country	Australia
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Name of the mutant parasite
RMgm number	RMgm-1469
Principal name	Pb apiG3PAT ha gfp
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 different from wild type
Sporozoite	Not different from wild type
Liver stage	GFP fluorescence was observed in liver stage parasites in a structure resembling the apicoplast. Apicoplast targeting of Pb apiG3PAT was confirmed by immunofluorescence microscopy and colocalization of the HA and GFP tags with the FASII enzyme FabI.
Additional remarks phenotype	Mutant/mutation The mutant expresses a C-terminal HA-GFP tagged version of G3PAT (Pb apiG3PAT) 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 (RMgm-977, RMgm-978). Phenotype Phenotype analyses of mutants lacking expression of G3PAT (Pb apiG3PAT) show an important role of this protein for full maturation of liver schizonts (see RMgm-977 and RMgm-1468). Analyses of mutants expressing tagged versions of G3PAT (Pb apiG3PAT) show expression in liver stages and an apicoplast location (this mutant and RMgm-978) GFP fluorescence was observed in liver stage parasites in a structure resembling the apicoplast. Apicoplast targeting of Pb apiG3PAT was confirmed by immunofluorescence microscopy and colocalization of the HA and GFP tags with the FASII enzyme FabI. Additional information Other mutants RMgm-977, RMgm-978 P. yoelii mutants lacking expression of G3PAT or expressing a Myc-tagged version of G3PAT, respectively. RMgm-1468 a P. berghei mutant lacking expression of G3PAT

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_1416700

Gene Model P. falciparum ortholog

PF3D7_1318200

Gene product

glycerol-3-phosphate 1-O-acyltransferase

Gene product: Alternative name

G3PAT; Pb apiG3PAT

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Details of the genetic modification

Name of the tag

HA-GFP

Details of tagging

C-terminal

Additional remarks: tagging

Commercial source of tag-antibodies

Type of plasmid/construct

(Linear) plasmid single 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

hdhfr

Promoter of the selectable marker

eef1a

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

The Pb apiG3PAT coding sequence (minus stop codon) was amplified from P. berghei genomic DNA using primers P14 and P15 and introduced into the pREP3 transfection vector, which was derived from the pL0006 vector (Malaria Research and Reference Reagent Resource Center) by addition of the GFP coding sequence downstream of the multiple cloning site.
Following single crossover homologous recombination, sequences for the hemagglutinin (HA) tag, fluorescent protein (GFP) and calmodulin 3’ untranslated region (CAM 3’) are introduced downstream of the Pb apiG3PAT coding sequence, enabling expression of the tagged protein from the endogenous promotor.

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
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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