RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_1300700; Gene model (P.falciparum): PF3D7_1475500; Gene product: LCCL domain-containing protein (LAP2; LCCL/lectin adhesive-like protein 2; CCp1)
Name tag: EGFP
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 9 December 2009, 21:59
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) : 19932717
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/ResearcherS. Saeed; V. Carter; J.T. Dessens
Name Group/DepartmentDepartment of Infectious and Tropical Diseases
Name InstituteLondon School of Hygiene & Tropical Medicine
Name of the mutant parasite
RMgm numberRMgm-355
Principal namePbLAP2/EGFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteExpression of the GFP-tagged LAP2 protein in gametocytes. The protein was observed distributed throughout the parasite cytoplasm in a somewhat punctate pattern
Fertilization and ookineteExpression of the GFP-tagged LAP2 protein in ookinetes. The protein was confined to two focal spots, often visibly associated with clusters of malaria pigment. Immunoelectronmicroscopy showed association of the protein with the crystalloids.
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot tested
Additional remarks phenotype

The mutant expresses a GFP-tagged version of  LCCL domain-containing protein CCp1 (LAP2; LCCL/lectin adhesive-like protein 2). The endogenous lap2 gene is tagged with gfp using a construct that integrates via a single cross-over recombination event, resulting in a gfp-tagged lap2 gene and a partial, non-functional lap2 copy.

Protein (function)
The lap2 (ccp1) 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 and in P. berghei PbLAP's.

Analysis of the location of the GFP-tagged LAP2 by fluorescence microscopy and immunoelectronmicroscopy shows that the protein is expressed in gametocytes and ookinetes and is associated with crystalloids in the ookinetes. Crystalloids are transient organelles that form in developing ookinetes and disappear after ookinete-to-oocyst transition. Plasmodium crystalloids are cytoplasmic aggregations of closely packed spherical particles 25–35 nm in diameter. In P. berghei, haemozoin-containing vacuoles accumulate around the edges of the crystalloid inclusion bodies.

Phenotype analyses of mutants lacking expression of LAP2/CCp1 (RMgm-118, RMgm-121, RMgm-124) indicate a role of this protein during oocyst development and sporozoite formation/production

The expression in gametocytes and ookinetes and the association of LAP2/CCp1 with the crystalloids is comparable to the expression and localisation pattern of two other LCCL domain-containing proteins, LAP1/CCp3/PbSR (RMgm-116) and LAP3/CCp5 (RMgm-356).

Additional information

Other mutants
RMgm-118: A mutant lacking expression of LAP2/CCp1
RMgm-121: A mutant lacking expression of LAP2/CCp1
RMgm-122: A mutant expressing GFP under the control of the promoter LAP2/CCp1
RMgm-124: A mutant lacking expression of both LAP2/CCP1  and PbSR/PSLAP/LAP1/CCp3

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_1300700
Gene Model P. falciparum ortholog PF3D7_1475500
Gene productLCCL domain-containing protein
Gene product: Alternative nameLAP2; LCCL/lectin adhesive-like protein 2; CCp1
Details of the genetic modification
Name of the tagEGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
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 PacI
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo achieve GFP-tagging of PbLAP2, a strategy of single crossover homologous recombination was adapted. A 2.3kb fragment of pblap2 corresponding to the 3’-part of the coding sequence was PCR amplified from genomic DNA with primers P1 (ACGAAGTTATCAGTCGACATGAGTCATTACTAGACATAATTACAAGTGAA) and P2 (ATGAGGGCCCCTAAGCTTTCAGTAATTCCATGAGTTACTTTGC) and introduced into SalI/HindIII-digested pDNR-EGFP, via in-fusion cloning (BD Bioscienses) to give plasmid pDNR-PbLAP2/EGFP. The pblap2/egfp specific sequence was then transferred to pLP-hDHFR via cre-loxp recombination to give plasmid pLP-PbLAP2/EGFP. pLP-PbLAP2/EGFP was linearized with PacI prior to transfection of purified schizonts.
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
Additional information primer 1P1 (SalI); 3' part coding sequence
Additional information primer 2P2 (HindIII); 3' part coding sequence
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