RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-71

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 gene replaced with the ortholog of P. falciparum, lacking region II
Phenotype	Oocyst; Sporozoite; Liver stage;

Last modified: 26 January 2011, 14:37

*RMgm-71

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) : 12244064
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	R. Tewari, A. Crisanti
Name Group/Department	Imperial College of Science, Technology and Medicine
Name Institute	Imperial College of Science
City	London
Country	UK
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Name of the mutant parasite
RMgm number	RMgm-71
Principal name	CSP(RII-)-7
Alternative name	CSP(RII-)-7 (replacement with PfCS without region II)
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	Normal numbers of of mature oocysts at day 14 after infection of A. stephensi mosquitoes.
Sporozoite	Compared to wild type, the mutant showed a 290-fold reduction in the number of salivary gland sporozoites. Sporozoites do not show gliding motility.
Liver stage	Sporozoites failed to infective mice (C57Bl/6) after intravenous injection of sporozoites or after infection by mosquito bite.
Additional remarks phenotype	Mutant/mutation In the mutant the endogenous P. berghei CS is replaced by a mutated form of P. falciparum CS (circumsporozoite protein) . The mutated form lacks Region II (WSPCSVTCGNGIQVRIK). 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 Analyses of a P. berghei mutant in which the endogenous P. berghei CS gene is replaced with the 'wild type' CS gene of P. falciparum (RMgm-69) show that the CS protein of P. falciparum can complement the activity of the endogenous P. berghei CS protein efficiently (although the mutant sporozoites invade less well the salivary glands). The phenotype analysis of the mutant expressing the mutated PfCS protein lacking Region II, indicates that Region-II is involved in gliding motility. The lack of Region-II affects both salivary gland invasion and infectivity of sporozoites to the mammalian host. In comparison with the P. berghei mutant expressing 'wild type' PfCS, the mutants sporozoites expressing PfCS lacking Region II show a >95% reduction in salivary gland invasion. A. P. berghei mutant has been described expressing a mutated from of CS lacking Region II-plus (RMgm-68). These mutants show a defect in egress of the oocysts. These sporozoites are also not infective to the mammalian host but they show normal gliding behavior. A difference between the mutated CS genes is that the Region II-plus deletion in RMgm-68 does not affect the thrombospondin (TSR) domain whereas in the mutant described here two of the four cysteines of the TSR domain are deleted. Other mutants A P. berghei mutant has been generated that express the 'wild type' form of P. falciparum CS (RMgm-69). A P. berghei mutant has been generated that express another mutated form of P. falciparum CS (RMgm-70 with a mutated Region I). A P. berghei mutant has been generated that express a hybrid form of CS of P. berghei and P. falciparum (the P. berghei CS repeat region is exchanged with the P. falciparum CS repeat region)(RMgm-76). A P. berghei mutant has been generated that lacks expression of CS (RMgm-9) . A. P. berghei mutant has been generated that produces reduced levels of CS (RMgm-72). P. berghei mutant has been generated with a mutated Region II-plus (RMgm-68). P. berghei mutants have been generated with a mutated GPI-anchor addition sequence (RMgm-73). A P. berghei mutant has been generated in which the endogenous P. berghei CS was replaced with P. gallinaceum CS (RMgm-74). A P. berghei mutant has been generated in which the endogenous P. berghei CS was replaced with P. yoelii CS (RMgm-75).

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 gene replaced with the ortholog of P. falciparum, lacking region II

Inducable system used

Short description of the conditional mutagenesis

Not available

Additional remarks inducable system

Type of plasmid/construct

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

The CS coding sequence(1600bp) from P. falciparum (Wellcome strain)lacking region II was introduced into the P. berghei genome.
Deletion of coding region II (WSPCSVTCGNGIQVRIK) was introduced by site-directed mutagenesis.
The mutated PfCS coding sequence linked to 250 nucleotides of its 3'-UTR was introduced into the genome, thereby replacing the endogenous P. berghei CS gene. The pfCS gene is under control of the PbCS regulatory sequences: i) a 5'-UTR of the PbCS gene encompassing nucleotides 1-1130 immediately upstream of the start codon and ii) the 3'-UTR encompassing nucleotides 1-1150 downstream of the stop codon.

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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