RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
DisruptedGene model (rodent): PBANKA_0204500; Gene model (P.falciparum): PF3D7_0109100; Gene product: LCCL domain-containing protein (LAP3; LCCL/lectin adhesive-like protein 2; CCp5)
Phenotype Fertilization and ookinete; Sporozoite;
Last modified: 3 May 2015, 09:00
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 25900212
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-1250
Principal namePbLAP3/KO
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot tested
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNormal ookinete production. However, ookinetes lack crystalloid bodies
OocystNot different from wild type
SporozoiteStrongly reduced sporozoite formation inside oocysts (the large majority of oocysts (~98%) failed to produce sporozoites). No infection of mice through the bite of infected mosquitoes
Liver stageNot different from wild type
Additional remarks phenotype

The mutant lacks expression of LAP3.

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 a mutant (RMgm-356) expressing a 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 indicate that LAP3 is essential for formation of crystalloid bodies in the ookinete. In addition, these analyses indicate that LAP3 plays a major role in the formation of sporozoites inside oocysts,

Additional information

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.

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-356: A mutant expressing a GFP-tagged LAP3

  Disrupted: Mutant parasite with a disrupted 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
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(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
Partial or complete disruption of the genePartial
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationPlasmid pLP-PbLAP3/EFGP (RMgm-356) served as a template for inverse PCR using primers LAP3-KO-F (50ATTCAAAAAGCTTAGGGGCCCTCAT30) and LAP3-KO-R (50CCTAAGCTTTTTGAATATATTAAAATGGTTGTAATAACCA30). The amplified plasmid DNA was circularised via In-Fusion cloning (Takara Bio, Japan), resulting in the transfection construct pLP-PbLAP3-KO, in which all but the first 21 codons of P. berghei LAP3 (pblap3) have been removed.
The plasmid were linearised with HindIII and SacII restriction enzymes to remove the vector backbone.
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
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