RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-1443

Malaria parasite	P. berghei
Genotype
Mutated	Gene model (rodent): PY17X_0405400; Gene model (P.falciparum): PF3D7_0304600; Gene product: circumsporozoite (CS) protein (CS; CSP) Details mutation: The P. berghei csp gene replaced with (part of) the csp gene (247 allele) of P. vivax PVP01_0835600)
Phenotype	No phenotype has been described

Last modified: 6 May 2016, 19:29

*RMgm-1443

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) : 27129682
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	Mizutani M; Yoshida S
Name Group/Department	Laboratory of Vaccinology and Applied Immunology
Name Institute	Kanazawa University School of Pharmacy
City	Kanazawa
Country	Japan
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Name of the mutant parasite
RMgm number	RMgm-1443
Principal name	PvCSP(VK247)/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 different from wild type
Fertilization and ookinete	Not different from wild type
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 P. berghei csp gene is replaced with (part of) the csp gene (247 allele) of P. vivax PVP01_0835600). See also 'Additional information genetic modification'. Protein (function) Phenotype The mutant produced normal numbers of infectious sporozoites Additional information Other mutants RMgm-1104: In the mutant the endogenous P. berghei csp gene is replaced with (part of) the csp gene (210 allele) of P. vivax PVP01_0835600).

Mutated: Mutant parasite with a mutated gene

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Details of the target gene

Gene Model of Rodent Parasite

PY17X_0405400

Gene Model P. falciparum ortholog

PF3D7_0304600

Gene product

circumsporozoite (CS) protein

Gene product: Alternative name

CS; CSP

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Details of the genetic modification

Short description of the mutation

The P. berghei csp gene replaced with (part of) the csp gene (247 allele) of P. vivax PVP01_0835600)

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

pbdhfr

Promoter of the selectable marker

pbdhfr

Selection (positive) procedure

pyrimethamine

Selection (negative) procedure

Additional remarks genetic modification

To construct the transfer plasmid for generating the PvCSP(VK247)/Pb transgenic parasite line in place of native PbCSP, the DNA sequence corresponding to amino acids His24-Asp370 of the entire PvCSP VK247 gene (GenBank accession number M69059) minus its signal peptide and glycosylphosphatidylinositol (GPI)-anchor sequence was amplified from pEU3-PvCSP(VK247) using pPvCSP-VK247-F4 and pPvCSP-VK247-R1 primers. The PCR product was then cloned into pENTR (Invitrogen life Technologies, Carlsbad, CA, USA) to construct pENTR-PvCSP-VK247-MunI. In a previous study confirmed that the GPI anchor of the PfCSP moieties derived from PvCSP(VK210) were able to function in the transgenic rodent parasites. The DNA sequence corresponding to amino acids Lys372-Asn395 of the GPI anchor of PfCSP (Accession number AAN87615) was amplified from pBS-5′UTRPfCSP-Tcell using pPvCSP-VK247-F1 and pPfCSPR7 primers, and then cloned into pENTR to construct the pENTR-PvCSP-VK247-XmaI/MunI plasmid. A 1.1-kb fragment of PvCSP(VK247) was excised from pENTR-PvCSP-VK247-MunI by digestion with XmaI and MunI and then inserted into the XmaI/MunI sites of pENTR-PvCSP-VK247-XmaI/MunI to construct pENTR-PvCSP-VK247-R1. A 1.7-kb fragment of the PvCSP(VK247) nucleotide sequence and GPI anchor nucleotide sequence of PfCSP was excised from pENTR-PvCSP-VK247-R1 by digestion with XmaI and SfuI and then inserted into the XmaI/SfuI sites of pBS-5′UTR-PvCSP(VK210)-dihydrofolate reductase (DHFR)-3′ to construct pBS-5′UTR-PvCSP(VK247)-DHFR-3′-R1.

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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