RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-37
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_1402400; Gene model (P.falciparum): PF3D7_1302200; Gene product: early transcribed membrane protein 13 (UIS3; up-regulated in infective sporozoites; ETRAMP13)
Phenotype Liver stage;
Last modified: 19 February 2009, 20:38
  *RMgm-37
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) : 17624848
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii 17XNL
Name parent line/clone Not applicable
Other information parent line17XNL is a non-lethal strain of P. yoelii
The mutant parasite was generated by
Name PI/ResearcherA.S. Tarun, S.H.I. Kappe
Name Group/DepartmentDepartment of Pathobiology
Name InstituteUniversity of Washington
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-37
Principal namePyuis3[-]
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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. In vivo (BALB/c mice), the number of liver stages at 2 hours after intravenous injection of sporozoites is reduced by 75%. The number of liver stages at 24h were reduced by >95%. liver stages could not be detected at 40h after infection. Liver stages were morphologically similar to wt liver stages at 6 and 12h after infection, a time when the intrahepatocytic transformation from elongate sporozoite to spherical trophozoites commences. Antibody staining for UIS4, a resident parasitophorous vacuole membrane (PVM)protein, showed a typical circumferential pattern, which indicates that the PVM is present. Liver stages did not develop into schizonts but appeared to be arrested at the trophozoite stage. Arrested liver stages did not persist in the liver after 40h.
Infection of BALB/c mice by intravenous inoculation of high numbers of sporozoites (50.000) did not result in blood stage infection.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the UIS3 protein ('up-regulated in infective sporozoites'; early transcribed membrane protein 13, ETRAMP13).

Protein (function)
UIS3 has been identified by transcription-profiling of sporozoites (Kaiser et al., (2001). Mol. Microbiol. 51, 1221-32).

Phenotype
The phenotype analysis demonstates a role of this protein in liver stage development.

Additional information
A P. berghei mutant has been generated that lacks the expression of this protein (RMgm-36). This mutant has the same phenotype as described above.
P. berghei and P. yoelii  'attenuated sporozoites' lacking expression of UIS3 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
A P. berghei mutant has been generated that lacks the expression of this protein (RMgm-36).
A. P. berghei mutant has been generated that lacks the expression of not only UIS3 but also UIS4 (RMgm-39).


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_1402400
Gene Model P. falciparum ortholog PF3D7_1302200
Gene productearly transcribed membrane protein 13
Gene product: Alternative nameUIS3; up-regulated in infective sporozoites; ETRAMP13
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct usedPlasmid 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 1ATATGGTACCATTTAAATGTGTTAAGTGACTATGTTAGCA
Additional information primer 1PY3REP1F
Sequence Primer 2ATATAAGCTTATCTTTGAGTCACAATGATAAATGTG
Additional information primer 2PY3REP2R
Sequence Primer 3ATATGAATTCATTTTCTTAAAGAACAATATCAACCAAA
Additional information primer 3PY3REP3F
Sequence Primer 4ATATGGATCCATGAATCATAAACACTTTTCCAGCC
Additional information primer 4PY3REP4R
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6