RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-140

Malaria parasite	P. berghei
Genotype
Disrupted	Gene model (rodent): PBANKA_0412900; Gene model (P.falciparum): PF3D7_0315200; Gene product: circumsporozoite- and TRAP-related protein (CTRP)
Phenotype	Fertilization and ookinete; Oocyst;

Last modified: 18 February 2009, 14:20

*RMgm-140

Successful modification	The 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) : 10587361
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	Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/Researcher	M. Yuda, Y. Chinzei
Name Group/Department	Department of Medical Zoology
Name Institute	School of Medicine, Mie University
City	Tsu
Country	Japan
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Name of the mutant parasite
RMgm number	RMgm-140
Principal name	CTRP(-)1; CTRP(-)2
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	Normal numbers of ookinetes are produced (in vitro and in vivo in A. stephensi). Ookinetes do not show any morphological differences from wild-type parasites with Giemsa staining under microscopic observation. Ookinetes do not develop into oocysts.
Oocyst	Normal numbers of ookinetes are produced. Ookinetes do not develop into oocysts.
Sporozoite	Not tested
Liver stage	Not tested
Additional remarks phenotype	Mutant/mutation The mutant lacks expression of CTRP (circumsporozoite protein [CSP] and thrombospondin-related adhesive protein [TRAP]-related protein; CSP and TRAP-related protein (CTRP). Protein (function) CTRP is a type 1 transmembrane protein, containing two adhesive domains in its extracellular portion, an A-domain of von Willebrand factor and a thrombospondin type I repeat (TSR, TSP). CTRP has six tandemly arrayed A domains followed by seven TSP type 1-like domains. CTRP is located in the micronemes of ookinetes. This structure is similar to that of TRAP (PF13_0201; PB000374.03.0), a malaria sporozoite protein critical for sporozoite motility and invasion into host cells. CTRP plays a role in the motility of ookinetes and invasion of midgut epithelial cells. Phenotype The phenotype analyses indicate a role in the transformation of the ookinete into the oocyst. This and other studies indicate a role in motility and invasion of midgut epithelial cells. Additional information Ookinetes lacking expression of CTRP that are directly injected into the hemocoel, thereby by-passing the midgut wall, develop into oocysts and these ookinetes can transform in vitro into oocysts, indicating that CTRP is not essential for transformation of ookinetes into oocysts (Nacer, A. et al., Malar J. 19;7:82). A P. falciparum mutant lacking CTRP (PFC0640w) has been generated which show a comparable defect in the formation of oocysts (Templeton, T.J. 2000. Mol. Microbiol. 36, 1-9). A mutant has been generated (RMgm-150) that expresses a mutated form of TRAP (thrombospondin-related anonymous protein; PB000374.03.0; PF13_0201), in which the cytoplasmic tail domain (CTD) of TRAP is replaced with the CTD domain of CTRP. The CTD of CTRP can complement the function of the CTD of TRAP, albeit not as well as TRAP as shown by the reduced invasion of salivary glands and hepatocytes. Other mutants RMgm-141: an independent mutant lacking expression of CTRP. RMgm-150: A mutant that expresses a mutated form of TRAP (thrombospondin-related anonymous protein; PB000374.03.0; PF13_0201), in which the cytoplasmic tail domain (CTD) of TRAP is replaced with the CTD domain of CTRP.

Disrupted: Mutant parasite with a disrupted gene

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Details of the target gene

Gene Model of Rodent Parasite

PBANKA_0412900

Gene Model P. falciparum ortholog

PF3D7_0315200

Gene product

circumsporozoite- and TRAP-related protein

Gene product: Alternative name

CTRP

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Details of the genetic modification

Inducable system used

Additional remarks inducable system

Type of plasmid/construct used

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

Partial or complete disruption of the gene

Partial

Additional remarks partial/complete disruption

The DNA fragment containing the 5' portion of ctrp (2.05 kb) was subcloned into pBluescript II. The selectable marker gene pbdhfr was inserted into the MunI site of this fragment after ligation of EcoRI linkers to both ends. Integration of the construct results in disruption of the 5'-UTR region of ctrp.

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

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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