Summary

RMgm-171
Malaria parasiteP. yoelii
Genotype
DisruptedGene model (rodent): PY17X_0502200; Gene model (P.falciparum): PF3D7_1016900; Gene product: early transcribed membrane protein 10.3 | protein of early gametocyte 4 (UIS4, up-regulated in infective sporozoites, ETRAMP10.3)
Transgene
Transgene not Plasmodium: RFP
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Replacement locus: Gene model: PY00204; Gene product: Not available (UIS4, up-regulated in infective sporozoites gene 4)
PhenotypeNo phenotype has been described
Last modified: 16 September 2015, 18:45
  *RMgm-171
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene disruption, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 18242728
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/ResearcherS.A. Mikolajczak; S.H.I. Kappe
Name Group/DepartmentNot applicable
Name InstituteSeattle Biomedical Research Institute
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-171
Principal nameuis4ko
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 tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant lack expression of UIS4 (up-regulated in infective sporozoites gene 4) and expresses RFP under the control of the constitutive eef1a promoter.
A two step PCR technique was adapted for the efficient generation of the targeting construct. See figure 1: Mikolajczak et.al., Mol Biochem Parasitol 158 (2007) 213-216. In this paper a cloning procedure is described for gene replacement by double homologous recombination in P. yoelii, which requires only one digestion and ligation step. This significantly shortens the time required to complete the production of the targeting vector. Furthermore, for more efficient phenotypic evaluation of the mutant parasites, a fluorescent protein (RFP) cassette is introduced into the targeting vector in which RFP is under the control of the constitutive eef1a promoter. This allows for a more rapid assessment of parasite growth in all of its developmental stages. In addition, the introduction of the fluorescent marker via the replacement strategy confers the stable integration of the marker.

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 membrane.

Phenotype
See the mutants RMgm-35, RMgm-38, RMgm-39 for a description of the phenotype of mutants lacking expression of UIS4. This paper does not describe the phenotype of the parasites, only the method of generation of the parasites (see 'Mutant/mutation').

Additional information

Other mutants
Other mutants lacking expression of UIS4: RMgm-35, RMgm-38, RMgm-39


  Disrupted: Mutant parasite with a disrupted gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0502200
Gene Model P. falciparum ortholog PF3D7_1016900
Gene productearly transcribed membrane protein 10.3 | protein of early gametocyte 4
Gene product: Alternative nameUIS4, up-regulated in infective sporozoites, ETRAMP10.3
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 SbfI
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 modificationA two step PCR technique was adapted for the efficient generation of the targeting construct. See figure 1: Mikolajczak et.al., Mol Biochem Parasitol 158 (2007) 213-216. In this paper a cloning procedure is described for gene replacement by double homologous recombination in P. yoelii, which requires only one digestion and ligation step. This significantly shortens the time required to complete the production of the targeting vector. Furthermore, for more efficient phenotypic evaluation of the mutant parasites, a fluorescent protein (RFP) cassette is introduced into the targeting vector in which RFP is under the control of the constitutive eef1a promoter. This allows for a more rapid assessment of parasite growth in all of its developmental stages. In addition, the introduction of the fluorescent marker via the replacement strategy confers the stable integration of the marker.
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 1GCgatatcTTACAGATGATCAGAAGGAA
Additional information primer 1Pr.1 (EcoRV); 3'UTR forward + amplification of the final PCR product
Sequence Primer 2TAAATAAATGcctgcaggCAAAAATAATACACACTCTCAC
Additional information primer 2Pr.2 (SbfI); 3'UTR reverse + annealing of 5'UTR and 3'UTR
Sequence Primer 3TTATTTTTGcctgcaggCATTTATTTATTTTCCCTATT
Additional information primer 3Pr.3 (SbfI); 5'UTR forward + annealing of 5'UTR and 3'UTR
Sequence Primer 4GCgcggccgcATAAAAGGAAAGGATCAACC
Additional information primer 4Pr.4 (NotI); 5'UTR reverse + amplification of the final PCR product
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameRFP
Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/construct(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 SbfI
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationA two step PCR technique was adapted for the efficient generation of the targeting construct. See figure 1: Mikolajczak et.al., Mol Biochem Parasitol 158 (2007) 213-216. In this paper a cloning procedure is described for gene replacement by double homologous recombination in P. yoelii, which requires only one digestion and ligation step. This significantly shortens the time required to complete the production of the targeting vector. Furthermore, for more efficient phenotypic evaluation of the mutant parasites, a fluorescent protein (RFP) cassette is introduced into the targeting vector in which RFP is under the control of the constitutive eef1a promoter. This allows for a more rapid assessment of parasite growth in all of its developmental stages. In addition, the introduction of the fluorescent marker via the replacement strategy confers the stable integration of the marker.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
Primer information details of the primers used for amplification of the promoter sequence  Click to view information
Primer information details of the primers used for amplification of the promoter sequence  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0719300
Gene productbifunctional dihydrofolate reductase-thymidylate synthase, putative
Gene product: Alternative namedhfr/ts
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to view information
Primer information details of the primers used for amplification the 3'-UTR sequences  Click to hide information
Sequence Primer 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Insertion/Replacement locus
Replacement / InsertionReplacement locus
Gene Model of Parasite PY00204
Gene productNot available
Gene product: Alternative nameUIS4, up-regulated in infective sporozoites gene 4
Primer information details of the primers used for amplification of the target sequences  Click to view information
Primer information details of the primers used for amplification of the target sequences  Click to hide information
Sequence Primer 1GCgatatcTTACAGATGATCAGAAGGAA
Additional information primer 1Pr.1 (EcoRV); 3'UTR forward + amplification of the final PCR product
Sequence Primer 2TAAATAAATGcctgcaggCAAAAATAATACACACTCTCAC
Additional information primer 2Pr.2 (SbfI); 3'UTR reverse + annealing of 5'UTR and 3'UTR
Sequence Primer 3TTATTTTTGcctgcaggCATTTATTTATTTTCCCTATT
Additional information primer 3Pr.3 (SbfI); 5'UTR forward + annealing of 5'UTR and 3'UTR
Sequence Primer 4GCgcggccgcATAAAAGGAAAGGATCAACC
Additional information primer 4Pr.4 (NotI); 5'UTR reverse + amplification of the final PCR product