Summary

RMgm-117
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_1035200; Gene model (P.falciparum): PF3D7_1407000; Gene product: LCCL domain-containing protein | scavenger receptor-like protein (PbSR; P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; CCp3)
Name tag: EGFP
Phenotype Gametocyte/Gamete;
Last modified: 18 February 2009, 12:57
  *RMgm-117
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) : 18452513
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/ResearcherV. Carter; J.T. Dessens
Name Group/DepartmentDepartment of Infectious and Tropical Diseases
Name InstituteLondon School of Hygiene and Tropical Medicine
CityLondon
CountryUnited Kingdom
Name of the mutant parasite
RMgm numberRMgm-117
Principal namePbSR/EGFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteExpression of the PbSR/EGFP protein in female gametocytes (no expression in asexual blood stages and in male gametocytes). Analysis of purified gametocytes by Western blot using an anti-GFP antibody failed to detect the full-length PbSR-EGFP (predicted to be 175 kDa in size), but gave rise to a band of 27 kDa corresponding to monomeric GFP. This indicated that the C-terminal GFP tag was efficiently cleaved off the full-length PbSR protein. For this reason an additional mutant (RMgm 116; mCherry/PbSR/EGFP) has been generated which, in addition to the C-terminal GFP tag, contained an N-terminal red fluorescent protein (mCherry) tag.
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a GFP-tagged version of  PbSR (PbSR (P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; LCCL domain containing protein CCp3). The endogenous pbsr gene is replaced with a c-terminal gfp-tagged version of pbsr (see also the additional remarks under 'Phenotype').

Protein (function)
The pbsr gene is a member of a small conserved gene family, encoding proteins with multiple adhesive domains, for example a Lgl1 (LCCL)-lectin adhesive domain. In P. falciparum the LCCL domain-containing proteins are termed PfCCp's. PbSR contains four LCCL domains; a LH2 (lipoxygenase homology 2) domain; two SR (scavenger receptor cysteine-rich) domains and a PTX/LamG (pentraxin/laminin-G) domain related to concanavalin A-like module.
Phenotype analyses of mutants lacking expression of PbSR (RMgm-113, RMgm-114) indicate a role of PbSR in the formation of sporozoites in the oocyst.

Phenotype
Expression of the PbSR/EGFP protein in female gametocytes (no expression in asexual blood stages and in male gametocytes). Analysis of purified  gametocytes by Western blot using an anti-GFP antibody failed to detect the full-length PbSR-EGFP (predicted to be 175 kDa in size), but gave rise to a band of 27 kDa corresponding to monomeric GFP. This indicated that the C-terminal GFP tag was efficiently cleaved off the full-length PbSR protein. For this reason an additional mutant (RMgm-116; mCherry/PbSR/EGFP) has been generated which, in addition to the C-terminal GFP tag, contained an N-terminal red fluorescent protein (mCherry) tag.

Additional information

Other mutants
RMgm-113: A mutant lacking PbSR
RMgm-114: A mutant lacking PbSR
RMgm-115: A mutant containing a mutated form of PbSR
RMgm-116: A mutant expressing a GFP- and mCherry-tagged version of PbSR


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1035200
Gene Model P. falciparum ortholog PF3D7_1407000
Gene productLCCL domain-containing protein | scavenger receptor-like protein
Gene product: Alternative namePbSR; P. berghei Scavenger Receptor-like protein; PSLAP; LAP1; CCp3
Details of the genetic modification
Name of the tagEGFP
Details of taggingC-terminal
Additional remarks: taggingFull length expression of PbSR containing a c-terminal green fluorescent protein (GFP) tag
Commercial source of tag-antibodiesAnti GFP (Invitrogen 33-2600)
Type of plasmid/constructPlasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedureThe endogeneous pbsr gene was replaced with a c-terminal gfp-tagged version of pbsr.
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