RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. berghei
MutatedGene model (rodent): PBANKA_0403200; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CSP)
Details mutation: The endogenous P. berghei csp gene replaced by the P. falciparum csp gene
PhenotypeNo phenotype has been described
Last modified: 22 August 2020, 20:47
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
Reference (PubMed-PMID number) Reference 1 (PMID number) : 23176559
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/ResearcherSumitani M, Yoshida S.
Name Group/DepartmentGenetically Modified Organism Research Center
Name InstituteNational Institute of Agrobiological Sciences
CityOwashi, Tsukuba, Ibaraki
Name of the mutant parasite
RMgm numberRMgm-4851
Principal namePfCSP/Pb
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Asexual blood stageNot different from wild type
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageNot different from wild type
Additional remarks phenotype

In the mutant the endogenous csp gene is replaced by the csp gene of P. falciparum.
The wild-type P. berghei (WT-Pb) CSP genomic locus, without the signal peptide, was replaced through homologous recombination with the PfCSP gene and selectable marker (dhfr), thus generating PfCSP/Pb

Protein (function)
The CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

From the paper: 'Successful replacement of the gene in the PfCSP/Pb line was confirmed by CSP gene-specific PCR (data not shown), and the presence of PfCSP protein in PfCSP/Pb sporozoites using indirect fluorescence assays (IFAs). Immunoblot analysis using the anti-PfCSP mAb 2A10 and the anti-PbCSP mAb 3D11 also confirmed successful replacement of the CSP genes. The PfCSP/Pb line had a normal life cycle, i.e. it proliferated normally during blood-stage infections in mice and had normal oocyst to sporozoite stage development in mosquitoes (data not shown). In addition, we confirmed that the PfCSP/Pb and WT-Pb lines could be transmitted to naïve mice via the natural biting activity of sporozoite-infected mosquitoes (data not shown).'

Additional information

Other mutants


  Mutated: Mutant parasite with a mutated gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0403200
Gene Model P. falciparum ortholog PF3D7_0304600
Gene productcircumsporozoite (CS) protein
Gene product: Alternative nameCSP
Details of the genetic modification
Short description of the mutationThe endogenous P. berghei csp gene replaced by the P. falciparum csp gene
Inducable system usedNo
Short description of the conditional mutagenesisNot available
Additional remarks inducable system
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
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerunknown
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationA 1.2-kb fragment that included the upstream region of the PbCSP open reading frame (ORF) and its signal sequence (1–19 aa) was PCR-amplified from P. berghei ANKA genomic DNA using pPbCSP-5′ UTR-F1 (5′-GTCGACGCTTTTACTTTGTCCA
GGTATTATGCTC-3′) and pPbCSP-5′UTR-R1 (5′-CCCGGGAAGTAGAGAATTAACTAATAAAAG-3′) primers. The PCR product was cloned into pCR2.1-TOPO (Invitrogen, Carlsbad, CA, USA) to generate pCR-5′UTR-PbCSP-SP. A 1.5-kb fragment that included the entire PfCSP gene without its signal sequence was PCR-amplified from P. falciparum (3D7 strain) genomic DNA using pPfCSP-F3 (5′-CCCGGGTATGGATTATTCCAGGAATACCAGTGCTATGGAAGT-3′) and pPfCSP-R3 (5′-CTCGAGGTCGACTGTTAAATGAACTTCGAAGTA-3′) primers. The PCR product was inserted into the XmaI/XhoI sites to generate pCR-5′UTRPbPfCSP. A 2.7-kb fragment that included the 5′UTR of the PbCSP gene and the PfCSP gene was excised from pCR-5′UTRPbPfCSP by digestion with SalI, then cloned into the SalI site of pBS-DHFR (Dessens et al., 2001) to generate pBS5′UTR-PbCSP-DHFR. A 1.1-kb fragment that included the downstream region of the PbCSP ORF was PCR-amplified from P. berghei ANKA genomic DNA using pPbCSP-3’UTR-F1 (5′-GGATCCATAAACATTACGCATGATTATAAA-3′) and pPbCSP3′UTR-R1 (5′-GCGGCCGCAGTACTCACGAATCCGAAATAAGTTAC-3′) primers. The PCR product was cloned into the BamHI/NotI sites of pBS-5′UTR-PbCSP-DHFR to generate pBS-5′UTRPfCSP-DHFR-3′.
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