RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-39
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_1400800; Gene model (P.falciparum): PF3D7_1302200; Gene product: early transcribed membrane protein 13 (UIS3; up-regulated in infective sporozoites; ETRAMP13)
DisruptedGene model (rodent): PBANKA_0501200; Gene model (P.falciparum): PF3D7_1016900; Gene product: early transcribed membrane protein 10.3 | protein of early gametocyte 4 (UIS4; ETRAMP10.3)
Phenotype Liver stage;
Last modified: 23 November 2015, 19:20
  *RMgm-39
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Gene disruption
Reference (PubMed-PMID number) Reference 1 (PMID number) : 17624847
MR4 number
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Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei NK65
Name parent line/clone Not applicable
Other information parent line
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The mutant parasite was generated by
Name PI/ResearcherO. Jobe, S.H.I. Kappe, U. Krzych
Name Group/DepartmentDivision of Malaria Vaccine Development
Name InstituteWalter Reed Army Institute of Research
CitySilver Spring, Maryland
CountryUSA
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Name of the mutant parasite
RMgm numberRMgm-39
Principal namePbuis3(-)4(-)
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
SporozoiteNot different from wild type
Liver stageIn vitro invasion of hepatocytes by the sporozoites is not affected. Liver stage development is strongly impaired. Sporozoites lack the capacity to develop into mature liver schizonts.
Infection of C57BL/6 mice by intravenous inoculation of high numbers of sporozoites (75.000) did not result in blood stage infection.
Additional remarks phenotype

Mutant/mutation
This 'double knock-out' mutant lacks expression of both the UIS3 and UIS4 protein (UIS3: 'up-regulated in infective sporozoites'; early transcribed membrane protein 13, ETRAMP13; UIS4 'up-regulated in infective sporozoites').

Protein (function)
These proteins have been identified by transcription-profiling of sporozoites (Kaiser et al., (2001). Mol. Microbiol. 51, 1221-32). UIS4 is expressed throughout liver stage development and localizes to the parasitophorous vacuole.

Phenotype
The phenotype analysis demonstrates a role of these proteins in liver stage development.

Additional information
'Single knock-out' P. berghei and P. yoelli mutants have been generated that lack the expression of UIS3 (RMgm-36; RMgm-37). These mutant shows a comparable phenotype as described above.
'Single knock-out' P. berghei and P. yoelli mutants have been generated that lack the expression of UIS4 (RMgm-35; RMgm-38). These mutant shows a comparable phenotype as described above. P. berghei and P. yoelii  'attenuated sporozoites' lacking expression of UIS3 and UIS4 can induce sterile (protective) immunity in mice against challenge with wild type sporozoites by immunization of mice (C57Bl/6; BALB/c) with the mutant sporozoites.

Other mutants
'Single knock-out' P. berghei and P. yoelli mutants have been generated that lack the expression of UIS3 (RMgm-36; RMgm-37).
'Single knock-out' P. berghei and P. yoelli mutants have been generated that lack the expression of UIS4 (RMgm-35; RMgm-38).

  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_1400800
Gene Model P. falciparum ortholog PF3D7_1302200
Gene productearly transcribed membrane protein 13
Gene product: Alternative nameUIS3; up-regulated in infective sporozoites; ETRAMP13
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid double 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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
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 15'-GGGTACCCGCATTAGCATAACATCTCATTGG-3'
Additional information primer 1UIS3rep1for
Sequence Primer 25' -
CAAGCTTGCTTTCATATATTTGTTATTTGTC-3'
Additional information primer 2UIS3rep2rev
Sequence Primer 35'-GGAATTCCCATATGTTTGTGTAACATC-3'
Additional information primer 3UIS3rep3
Sequence Primer 45' -CTCTAGAGTGTGCTTAAATGTTTCTTTAAAC-3'
Additional information primer 4UIS3rep4rev
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6
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  Disrupted: Mutant parasite with a disrupted gene
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Details of the target gene
Gene Model of Rodent Parasite PBANKA_0501200
Gene Model P. falciparum ortholog PF3D7_1016900
Gene productearly transcribed membrane protein 10.3 | protein of early gametocyte 4
Gene product: Alternative nameUIS4; ETRAMP10.3
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct useddouble or 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 parasitehdhfr
Promoter of the selectable markerunknown
Selection (positive) procedureWR99210
Selection (negative) procedureNo
Additional remarks genetic modificationThe construct for disruption of UIS4 has not been described in this paper. It is unknown whether the locus is disrupted by single or double cross-over recombination. The promoter of the selectable marker is unknown.
Additional remarks selection procedureThe selection of the double knock-out mutant has not been described in this paper.
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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P.berghei model of malaria
Technical design and realization: S. Mededovic and A. van Wigcheren