RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
TaggedGene model (rodent): PBANKA_0204500; Gene model (P.falciparum): PF3D7_0109100; Gene product: LCCL domain-containing protein (LAP3; LCCL/lectin adhesive-like protein 2; CCp5)
Name tag: EGFP
Phenotype Gametocyte/Gamete; Fertilization and ookinete;
Last modified: 3 May 2015, 08:48
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-356
Principal namePbLAP3/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 LAP3 protein in gametocytes. The protein was observed distributed throughout the parasite cytoplasm in a somewhat punctate pattern
Fertilization and ookineteExpression of the GFP-tagged LAP3 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 CCp5 (LAP3; LCCL/lectin adhesive-like protein 3). The endogenous lap3 gene is tagged with gfp using a construct that integrates via a double cross-over recombination event.

Protein (function)
The lap3 (ccp5) 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 LAP3 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 different members of the LCCL domain-containing protein family (for example see LAP1/CCp3 RMgm-113, RMgm-114; LAP2/CCp1 RMgm-118, RMgm-121; LAP4/CCp2 RMgm-119; LAP6; CCp4 RMgm-120) indicate a role of several these proteins during oocyst development and sporozoite formation/production.
An unsuccessful attempt to disrupt LAP3/CCp5 has been described (RMgm-94)

The expression in gametocytes and ookinetes and the association of LAP3/CCp5 with the crystalloids is comparable to the expression and localisation pattern of two other LCCL domain-containing proteins, LAP1/CCp3/PbSR (RMgm-116) and LAP2/CCp1 (RMgm-355).

Additional information

Other mutants
RMgm-1249: The mutant expresses a GFP-tagged version of  an mutated form of LAP3. The entire LCCL domain of the endogenous lap3 gene is removed.
RMgm-1250: A mutant lacking expression of LAP3

  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0204500
Gene Model P. falciparum ortholog PF3D7_0109100
Gene productLCCL domain-containing protein
Gene product: Alternative nameLAP3; LCCL/lectin adhesive-like protein 2; CCp5
Details of the genetic modification
Name of the tagEGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/construct(Linear) plasmid double cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid KpnI, SacII
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 PbLAP3 a strategy was used of double crossover homologous recombination. The entire pblap3 coding sequence plus 0.6kb of upstream sequence was PCR amplified from genomic DNA with primers P3 (ACGAAGTTATCAGTCGAGGTACCTAGCGGAAACAACAATGTTC) and P4 (ATGAGGGCCCCTAAGCTATTTTTAATAATTTGTATCGAAAGTATAGTTG) and cloned into SalI/HindIII-digested pDNR-EGFP to give plasmid pDNR-PbLAP3/EGFP. The 3’UTR of pblap3 was amplified with primers P5 (CCTTCAATTTCGACATATAATGGATTAAAATTTTAGTTCGGT) and P6 (GCGGCCGCTCTAGCATAGGATTAGAAATACAGTAATAGCAATTTTG) and the resulting 0.7kb fragment cloned into NdeI-digested pLP-hDHFR by in-fusion cloning to give Plasmid pLP-hDHFR/PbLAP3. The pblap3/egfp-specific sequence from pDNRPbLAP3/EGFP was transferred to pLP-hDHFR/PbLAP3 by cre/loxp recombination to give the final construct pLP-PbLAP3/EGFP. pLP-PbLAP3/EGFP was doubly digested with KpnI and SacII 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 1P3; coding sequence + 0.6kb upstream F
Additional information primer 2P4; coding sequence + 0.6kb upstream R
Additional information primer 3P5; 3'UTR F
Additional information primer 4P6; 3'UTR R
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6