RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-395
Malaria parasiteP. yoelii
Genotype
TaggedGene 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)
Name tag: c-myc
PhenotypeNo phenotype has been described
Last modified: 16 September 2015, 18:47
  *RMgm-395
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene tagging
Reference (PubMed-PMID number) Reference 1 (PMID number) : 20228203
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/ResearcherD.C. MacKellar; S.H.I. Kappe
Name Group/DepartmentNot applicable
Name InstituteSeattle Biomedical Research Institute, University of Washington
CitySeattle
CountryUSA
Name of the mutant parasite
RMgm numberRMgm-395
Principal namePyuis4myc
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 stageNot different from wild type
Additional remarks phenotype

Mutant/mutation
In the mutant the endogenous uis4 gene (up-regulated in infective sporozoites 4) is replaced with a C-terminal myc-tagged copy of uis4.

Protein (function)
UIS4 has been identified by transcription-profiling of P. berghei sporozoites (Kaiser et al., (2001). Mol. Microbiol. 51, 1221-32). The protein is expressed in sporozoites and throughout liver stage development and localizes to the parasitophorous vacuole membrane. Mutant parasites lacking expression of UIS4 (RMgm-35, RMgm-38) produce normal numbers of sporozoites that are able to infect liver cells. These parasites are however unable to develop into mature liver stages

Phenotype
The mutant shows a normal development throughout the life cycle. It produces infective sporozoites and liver stage development is comparable to that of wild type parasites, indicating that the myc-tag does not affect the function of the protein. Mutant parasites lacking expression of UIS4 (RMgm-35, RMgm-38) produce normal numbers of sporozoites that are able to infect liver cells. These parasites are however unable to develop into mature liver stages.

Additional information

Other mutants
RMgm-35, RMgm-38: P. berghei/P. yoelii mutants lacking expression of UIS4


  Tagged: Mutant parasite with a tagged 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
Name of the tagc-myc
Details of taggingC-terminal
Additional remarks: tagging4x tandem myc epitope tag
Commercial source of tag-antibodiesanti-myc (Santa Cruz Biotechnology SC-789, Santa Cruz, CA)
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 SacII, KpnI
Selectable marker used to select the mutant parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationTo create the PyUIS4myc construct, the PyUIS4 promoter and ORF were amplified as one product using the primers SP/PyUIS4Prom-B3D and ASP/PyUIS4-B3D and inserted into the B3D-myc vector with the SacII and SpeI enzymes. The PyUIS4 3’ untranslated region was amplified from 17XNL genomic DNA using the primers SP/PyUIS4-B3D/3’UTR and ASP/PyUIS4-B3D/3’UTR, and this was inserted into the B3D-myc vector containing PyUIS4 with the HindIII and KpnI enzymes. This construct was digested with SacII and KpnI, and transfected into P.yoelii 17XNL schizonts.
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 1CCGCGGGCTTCTTTGAGCAAATACTG
Additional information primer 1SP/PyUIS4Prom-B3D (Promoter PyUIS4)
Sequence Primer 2ACTAGTTATGTATGGGTCAAATGGTT
Additional information primer 2ASP/PyUIS4-B3D (ORF PyUIS4)
Sequence Primer 3AAAAGCTTTTCATTATGAGGGTAATTCAGAAAG
Additional information primer 3SP/PyUIS4-B3D/3'UTR (PyUIS4 3'UTR)
Sequence Primer 4AAGGTACCGACTTTTAAAAATAATATATATGAAA
Additional information primer 4ASP/PyUIS4-B3D/3'UTR (PyUIS4 3'UTR)
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6