RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-620

Malaria parasite	P. berghei
Genotype
Tagged	Gene model (rodent): PBANKA_0715500; Gene model (P.falciparum): PF3D7_0413500; Gene product: phosphoglucomutase-2 (PGM2) Name tag: GFP
Phenotype	Asexual bloodstage; Fertilization and ookinete;

Last modified: 24 July 2011, 12:11

*RMgm-620

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) : 21689687
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	P. berghei ANKA cl15cy1
Other information parent line	A reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255)
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The mutant parasite was generated by
Name PI/Researcher	T. Hills; A. Srivastava; AP Waters; J.C. Pizarro
Name Group/Department	The SGC (Structural Genomics Consortium)
Name Institute	University of Toronto
City	Toronto
Country	Canada
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Name of the mutant parasite
RMgm number	RMgm-620
Principal name	PbPGM2::GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Asexual blood stages express GFP-tagged PbPGM2
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Zygotes and ookinetes express GFP-tagged PbPGM2
Oocyst	Not different from wild type
Sporozoite	Not different from wild type
Liver stage	Not different from wild type
Additional remarks phenotype	Mutant/mutation The mutant expresses a GFP-tagged (C-terminal) version of PbPGM2 (phosphoglucomutase-2) Protein (function) Two P. falciparum genes code for putative phosphoglycerate mutases (PGMases; PfPGM1 - PF11_0208 and PfPGM2 - PFD0660w), a widespread protein group characterized by the involvement of histidine residues in their catalytic mechanism. Phosphoglycerate mutases (EC 5.4.2.1; PGMase) catalyze the reversible conversion of 2-phosphoglycerate (2-PG) to 3-phosphoglycerate (3-PG).This is an essential component of the glycolysis pathway providing 2-PG to the enzyme enolase and also of the gluconeogenesis pathway where it supplies 3-PG to the phosphoglycerate kinase. Members of the PGM family function as phosphotransferases or phosphohydrolases acting upon a variety of substrates. Aside from metabolic functions, a proportion of PGM family members are involved in cell signaling and/or regulation. In this study the P. berghei homologue of PGM2 (PBANKA_071550) was studied. The P. berghei homolog of PGM1 is PBANKA_092810 Phenotype GFP-tagged PbPGM2 is expressed in asexual blood stages, zygotes and ookinetes as shown by Western and IFA asnalysis using anti-GFP antibodies. IFA-analysis shows a cytoplasmic location. Additional information Repeated failures to disrupt the gene (see 619) indicates that PbPGM2 is essential for P. berghei asexual blood stage development by the. No information is provided on the expression in gametocytes Other mutants RMgm-619: Unsuccessful attempt to disrupt the pbpgm2 gene

Tagged: Mutant parasite with a tagged gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0715500

Gene Model P. falciparum ortholog

PF3D7_0413500

Gene product

phosphoglucomutase-2

Gene product: Alternative name

PGM2

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Details of the genetic modification

Name of the tag

GFP

Details of tagging

C-terminal

Additional remarks: tagging

Commercial source of tag-antibodies

anti-GFP mouse monoclonal (Roche)

Type of plasmid/construct

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

XbaI

Selectable marker used to select the mutant parasite

tgdhfr

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

C-terminal GFP-tagging of PGM2 was performed using a construct that integrates through single crossover homologous recombination into the PbPGM2-locus and contains the tgdhfr/ts selectable marker. Primers GU0626-SacII and GU0629-BamHI were used to amplify the 724bps targeting region of PbPGM2 (whilst incorporating an internal XbaI site using primers GU0627 and GU0628 by overlap extension PCR)

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	GGccgcggGGGTTTAACTTATGCATATGAAAATTATG
Additional information primer 1	GU0626 (SacII); targeting region forward
Sequence Primer 2	GGggatccAAAATATGTAACACTATTGAAAGGAAGATG
Additional information primer 2	GU0629 (BamHI); targeting region reverse
Sequence Primer 3	TAATTTAtctagaGCATTTGGAAAATATTTACTTATGATTTC
Additional information primer 3	GU0627 (XbaI); incorporation XbaI site
Sequence Primer 4	CCAAATGCtctagaTAAATTAATAAATGATCCGAATTTAAATGAAG
Additional information primer 4	GU0628 (XbaI); incorporation XbaI site
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

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