Summary

RMgm-35
Malaria parasiteP. berghei
Genotype
DisruptedGene model (rodent): PBANKA_0501200; Gene model (P.falciparum): PF3D7_1016900; Gene product: early transcribed membrane protein up-regulated in infective sporozoites (UIS4; ETRAMP10.3)
Phenotype Liver stage;
Last modified: 23 November 2015, 19:17
  *RMgm-35
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) : 15699336
MR4 number
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
The mutant parasite was generated by
Name PI/ResearcherA.K. Mueller, K. Matuschewski, S.H.I. Kappe
Name Group/DepartmentDepartment of Pathobiology
Name InstituteUniversity of Washington
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-35
Principal nameuis4REP-
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. Liver stage numbers are reduced by ~50% twenty four hours after invasion of HepG2 cells. At later time points, mature liver schizonts are absent. Only developmentally arrested liver forms persist.
Infection of mice (C57BL/6) by bite of infected mosquitoes or subcutaneous inoculation of sporozoites did not result in blood stage infection. Only intraveneous inoculation of very high numbers of sporozoites (50.000) resulted in blood stage infection in ~50% of the mice. These mice showed a prolonged prepatent period.
Additional remarks phenotype

Mutant/mutation
The mutant lacks expression of the UIS4 protein ('up-regulated in infective sporozoites').

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

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

Additional information
A P. yoelli mutant has been generated that lacks the expression of this protein (RMgm-38). This mutant has the same phenotype as described above.
P. berghei and P. yoelii 'attenuated sporozoites' lacking expression of UIS4 can induce sterile (protective) immunity in mice against challenge with wild type sporozoites by immunization of mice (C57Bl/6) with the mutant sporozoites.

Other mutants
A P. yoelli mutant has been generated that lacks the expression of this protein (RMgm-38). 
A. P. berghei mutant has been generated that lacks the expression of not only UIS4 but also UIS3 (PB000892.03.0; RMgm-39).


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PBANKA_0501200
Gene Model P. falciparum ortholog PF3D7_1016900
Gene productearly transcribed membrane protein up-regulated in infective sporozoites
Gene product: Alternative nameUIS4; ETRAMP10.3
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct used(Linear) plasmid 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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe mutant described here, uis4REP-, is generated using a replacement vector resulting in disruption of uis4 through double cross-over recombination. In the same paper (Mueller et al., (2004) PNAS, 102: 3022-37) a mutant (uis4-) has been described that has been generated using a disruption vector, resulting in disruption of uis4 by single cross-over recombination.
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'-GAATTCTGGATTCATTTTTTGATGCATGA-3'
Additional information primer 1UIS4rep1 forward
Sequence Primer 25'- GGGGTACCTTTATTCAGACGTAATAATTATGTGC-3'
Additional information primer 2UIS4rep2 reverse
Sequence Primer 35'- AAAACTGCAGATAATTCATTATGAGTAGTGTAATTCAG-3'
Additional information primer 3UIS4rep3 forward
Sequence Primer 45'- CCCCAAGCTTAAGTTTGCATATACGGCTGCTTCC-3'
Additional information primer 4UIS4rep4 reverse
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6