Summary

RMgm-391
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0302500; Gene model (P.falciparum): PF3D7_0204700; Gene product: hexose transporter (hexose transporter 1; PfHT1, PfHT, PbHT)
Name tag: GFP
Phenotype Asexual bloodstage; Gametocyte/Gamete; Fertilization and ookinete; Oocyst; Sporozoite;
Last modified: 23 January 2011, 12:38
  *RMgm-391
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) : 20132450
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA 2.34
Other information parent lineP. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
The mutant parasite was generated by
Name PI/ResearcherK. Slavic; R. Tewari; S. Krishna
Name Group/DepartmentCentre for Infection, Cellular and Molecular Medicine
Name InstituteSt. George's University of London
CityLondon
CountryUK
Name of the mutant parasite
RMgm numberRMgm-391
Principal namepbht-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageGFP-expression in all blood stages. Localization of PbHT-GFP in the plasma membrane of early and late blood stages (see also 'Additional information')
Gametocyte/GameteGFP expression in female gametes and ookinetes. Colocalization of PbHT-GFP with protein P28 (PB000865.00.0), a surface marker of the ookinete, female gamete and zygote
Fertilization and ookineteGFP expression in female gametes and ookinetes. Colocalization of PbHT-GFP with protein P28 (PB000865.00.0), a surface marker of the ookinete, female gamete and zygote
OocystAnalysis of mosquito midgut oocysts, 11 days post infection, showed internal foci of GFP-fluorescence of developing sporoblasts and lower intensity membrane fluorescence signal. Twenty-one days post infection, strong fluorescence signal was detectable from sporulating oocysts and oocyst-derived sporozoites. Similarly, sporozoites released from dissected salivary glands 21 days post infection showed expression of PbHT-GFP.
SporozoiteTwenty-one days post infection, strong GFP-fluorescence signal was detectable from sporulating oocysts and oocyst-derived sporozoites. Similarly, sporozoites released from dissected salivary glands 21 days post infection showed expression of PbHT-GFP.
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal GFP-tagged version of the hexose transporter (HT)

Protein (function)
HT is a facilitative hexose transporter in the Major Facilitator Superfamily of integral membrane proteins that mediates the uptake of glucose and fructose by the parasite. HTis a single copy gene with no close paralogues. In P. falciparum three other proteins, PFI0955w, PFI0785c and PFE1455w have been annotated with putative sugar transport function, although they have diverged considerably from typical sugar transporters (21%, 13% and 7% amino acid sequence identity compared with PfHT, respectively). Unlike PfHT, PFI0955w and PFI0785c are expressed only late in the asexual cycle.
Unsuccessful attempts to disrupt the ht gene in P. berghei and P. falciparum indicate an essential function during asexual blood stage growth (see RMgm-390).
See also RMgm-603 for reports of unsuccessful attempts to disrupt pbht (pbht1) and analyses of a mutant that expresses a HA-tagged form of PbHT (PbHT1).
See also mutant RMgm-604, in which the endogenous hexose transporter 1 gene (pbht1) is replaced with the P. falciparum ortholog, pfht1.

Phenotype
All blood stages, female gametes, ookinetes, oocysts and sporozoites express PbHT-GFP as determined by GFP-fluorescence analysis.
Unsuccessful attempts to disrupt Pbht indicate an essential function during asexual blood stage growth (see RMgm-390). In the same paper, unsuccessful attempts are reported to disrupt the hexose transporter (Pfht) in P. falciparum.
See also RMgm-603 for analyses of a mutant that expresses a HA-tagged form of PbHT (PbHT1).

Additional information
Some of the observed fluorescence signal in blood stage parasites is internal and may be associated with perinuclear localization as well as the food vacuole. Observed internal PbHT-GFP signal could be associated with developing PbHT in its trafficking pathway or may come from a small proportion of PbHT-GFP protein that has been mislocalized.
Expression of PbHT-GFP in parasites from all analysed developmental stages was confirmed by Western blotting using an anti-GFP monoclonal antibody

Other mutants
RMgm-390: Unsuccessful attempts to disrupt the Pbht gene
RMgm-603: A mutant in which the endogenous hexose transporter 1 gene (pbht1) is replaced with a HA-tagged copy of pbht1.
RMgm-604: a mutant in which the endogenous hexose transporter 1 gene (pbht1) is replaced with the P. falciparum ortholog, pfht1


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0302500
Gene Model P. falciparum ortholog PF3D7_0204700
Gene producthexose transporter
Gene product: Alternative namehexose transporter 1; PfHT1, PfHT, PbHT
Details of the genetic modification
Name of the tagGFP
Details of taggingC-terminal
Additional remarks: taggingThe chosen integration strategy for the construct results in integration at the target locus by a single cross-over homologous recombination event.
Commercial source of tag-antibodiesRoche
Type of plasmid/constructPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid BstXI
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
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 1catGGTACCTAACATTTGGAATATTTGTTGCAGTTTTATTGGG
Additional information primer 1Pb10 (KpnI); PbHT-GFP fragment
Sequence Primer 2caaGGGCCCAACTCTTGATTTGCTTATATGTTTTTGTCTTTCTTC
Additional information primer 2Pb11 (ApaI); PbHT-GFP fragment
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