RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-4851

Malaria parasite	P. berghei
Genotype
Mutated	Gene 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
Phenotype	No phenotype has been described

Last modified: 22 August 2020, 20:47

*RMgm-4851

Successful modification	The 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
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Parent parasite used to introduce the genetic modification
Rodent Malaria Parasite	P. berghei
Parent strain/line	P. berghei ANKA
Name parent line/clone	P. berghei ANKA 2.34
Other information parent line	P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943).
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The mutant parasite was generated by
Name PI/Researcher	Sumitani M, Yoshida S.
Name Group/Department	Genetically Modified Organism Research Center
Name Institute	National Institute of Agrobiological Sciences
City	Owashi, Tsukuba, Ibaraki
Country	Japan
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Name of the mutant parasite
RMgm number	RMgm-4851
Principal name	PfCSP/Pb
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modification	Yes
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Phenotype
Asexual blood stage	Not different from wild type
Gametocyte/Gamete	Not tested
Fertilization and ookinete	Not tested
Oocyst	Not different from wild type
Sporozoite	Not different from wild type
Liver stage	Not different from wild type
Additional remarks phenotype	Mutant/mutation 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. Phenotype 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

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0403200

Gene Model P. falciparum ortholog

PF3D7_0304600

Gene product

circumsporozoite (CS) protein

Gene product: Alternative name

CSP

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Details of the genetic modification

Short description of the mutation

The endogenous P. berghei csp gene replaced by the P. falciparum csp gene

Inducable system used

Short description of the conditional mutagenesis

Not available

Additional remarks inducable system

Type of plasmid/construct

(Linear) plasmid double cross-over

PlasmoGEM (Sanger) construct/vector used

Modified PlasmoGEM construct/vector used

Plasmid/construct map

Plasmid/construct sequence

Restriction sites to linearize plasmid

Selectable marker used to select the mutant parasite

tgdhfr

Promoter of the selectable marker

unknown

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

A 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

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