RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-53
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1354800; Gene model (P.falciparum): PF3D7_1335900; Gene product: thrombospondin-related anonymous protein | sporozoite surface protein 2 (sporozoite surface protein 2; SSP2; SSP-2; TRAP)
Phenotype Sporozoite; Liver stage;
Last modified: 24 May 2009, 23:38
  *RMgm-53
Successful modificationThe 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) : 11295181
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17X
Name parent line/clone Not applicable
Other information parent lineP. yoelii 17X was obtained as a cloned line from the WHO Registry of Standard Malaria Parasites, University of Edinburgh
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The mutant parasite was generated by
Name PI/ResearcherM.M. Mota, V. Nussenzweig
Name Group/DepartmentDepartment of Pathology, Kaplan Cancer Center
Name InstituteNew York University Medical Center
CityNew York
CountryUSA
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Name of the mutant parasite
RMgm numberRMgm-53
Principal namePyINT-GFP1; PyINT-GFP2
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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Phenotype
Asexual blood stageNot different from wild type
Gametocyte/GameteNot different from wild type
Fertilization and ookineteNot different from wild type
OocystNot different from wild type
SporozoiteNormal numbers of midgut sporozoites are formed. Sporozoites do not display gliding motility. Sporozoites are strongly affected in their capacity to invade salivary glands. In wild type infected mosquitoes the proportion of total salivary gland-associated sporozoites released from within the glands was ~80% and in mutant infected mosquitoes only ~4%. Sporozoites are also affected in their infectivity to BALB/c mice (mutant sporozoites were at least 100 times less infective than WT sporozoites).
Liver stageSporozoites are affected in their infectivity to BALB/c mice (mutant sporozoites were at least 100 times less infective than WT sporozoites).
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of TRAP (thrombospondin-related anonymous protein) and expresses GFP as a reporter protein (see 'Additional remarks genetic modification').

Protein (function)
TRAP 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). TRAP is located in the micronemes of sporozoites. The protein plays a role in the gliding motility of sporozoites and invasion of host cells. The role of the different domains of the protein in motility and invasion has been analysed in mutant parasites expressing mutated forms of TRAP (see below). 

Phenotype
The phenotype analyses show that the lack of expression of TRAP results in the loss of the gliding motility of the sprozoites. The mutant sprozoites show a strongly reduced capacity to invade salivary glands and infectivity to the mammalian host.

Other mutants
A. P. berghei mutant has been generated that lacks expression of TRAP (RMgm-47).
P. berghei mutants have been generated with mutated cytoplasmic tails of TRAP (RMgm-54; RMgm-55; RMgm-56; RMgm-57; RMgm-149; RMgm-150; RMgm-151).
Other P. berghei mutants have been generated with mutated extracellular (adhesive) domains (A-domain: RMgm-48; RMgm-49; TSR domain: RMgm-50; RMgm-51; A-domain and TSR domain: RMgm-52).
A P. berghei mutant has been generated in which the endogenous P. berghei trap was replaced with (mutated forms of) P. falciparum trap (RMgm-58)

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PY17X_1354800
Gene Model P. falciparum ortholog PF3D7_1335900
Gene productthrombospondin-related anonymous protein | sporozoite surface protein 2
Gene product: Alternative namesporozoite surface protein 2; SSP2; SSP-2; TRAP
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Partial or complete disruption of the geneComplete
Additional remarks partial/complete disruption
Selectable marker used to select the mutant parasitepbdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe mutant has been generated using a construct that disrupt the trap gene by single cross-over integration (insertion vector). The disadvantage of using an insertion construct is that the construct can be removed from the genome, thereby restoring the wild type genotype.
The construct contains a pyrimethamine-resistant form of P. berghei dihydrofolate reductase-thymidylate synthase (dhfr-ts) gene fused to the gfpmut2 gene.
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 15'ggcggatccgcgtgttccttaatggtcaggaaactctt-3'
Additional information primer 1PyTRAP-F sense
Sequence Primer 25'-ggc gcggccgc atatccattattagatttagactggtttt-3'
Additional information primer 2PyTRAP-R antisense
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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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren