RMgmDB - Rodent Malaria genetically modified Parasites


Malaria parasiteP. yoelii
MutatedGene 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)
Details mutation: Replacement of P. yoelii uis4 with P. falciparum PF10_0164, C-terminally myc-tagged
Phenotype Sporozoite; Liver stage;
Last modified: 16 September 2015, 18:46
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Gene mutation
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
Name of the mutant parasite
RMgm numberRMgm-393
Principal namePyuis4(-)PF10_0164myc
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
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
SporozoitePF10_0164myc expression in sporozoites. Mutant sporozoites are able to infect liver cells in vitro. Infection of mice by intravenous injection of mutant sporozoites did not result in blood infections.
Liver stageMutant sporozoites are able to infect liver cells in vitro. PF10_0164myc is expressed in young liver stages. Evidence is presented for a localisation in the parasitophorous vacuole membrane.
Infection of mice by intravenous injection of mutant sporozoites did not result in blood infections. A strong reduction in number of liver stages is observed between 12 and 24 hours after infection of liver cells.
Additional remarks phenotype

In the mutant the uis4 gene (up-regulated in infective sporozoites 4) is replaced with the P. falciparum gene PF10_0164 (ETRAMP10.3; early transcribed membrane protein 10.3), which is C-terminally myc-tagged. PF10_0164 is under the control of the endogenous uis4 promoter

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

P. falciparum gene PF10_0164 (ETRAMP10.3; early transcribed membrane protein 10.3) is a member of the 'early transcribed membrane protein (ETRAMP)' family. This is a Plasmodium conserved family with greater than ten members in P. falciparum. ETRAMPs are abundantly expressed early in the intraerythrocytic cycle and are small (frequently less than 200 aa) integral membrane proteins that are localized within the parasitophorous vacuolar membrane (PVM). All members have signal peptides plus a transmembrane domain. PF10_0164 is expressed in sporozoites and blood stages, where it localizes to the parasitophorous vacuole, and is also exported to the host erythrocyte. In the paper evidence is presented that PF10_0164 is refractory to disruption in asexual blood stages. PF10_0164 is syntenic with UIS4 and it has been proposed that this P. falciparum gene is the functional ortholog of P.berghei/P.yoelii UIS4

In the P. yoelii mutant PF10_0164myc localized to the parasitophorous vacuole membrane of liver stages, but the mutant parasites are unable to complete liver stage development. These data indicate that PF10_0164 is a parasitophorous vacuole protein that  does not complement P. yoelii UIS4, and is thus likely not a functional ortholog of UIS4. Mutant sporozoites are able to invade liver cells but liver stage development is strongly impaired. This phenotype is comparable to the phenotype of mutant parasites lacking expression of UIS4 (see RMgm-35, RMgm-38).

Additional information
The amino acid sequence identity between the PF10_0164 and UIS4 is 9% (17% conserved residues), and the predicted PF10_0164 protein is considerably shorter. Topology prediction tools suggest that the transmembrane domain is located at a position in the protein that makes the length of the N-terminus comparable between the proteins, whereas the C-terminus of PF10_0164 is considerably shorter. Both, however, possess a high density of charged residues in their C-termini (51% and 64% of residues in PF10_0164 and PyUIS4, respectively), with a net negative charge for each.

As controls, two additional mutant P. yoelii lines have been generated in which the endogenous uis4 has been replaced with either PF10_0164 without myc-tag or with the P. yoelii uis4 gene with a myc tag (PyUIS4myc). Liver stages of the first line were not able to develop into mature liver schizonts whereas liver stages of the second line showed normal liver stage development (RMgm-395).
Since the C-terminal domain of PF10_0164 is significantly shorter than that of UIS4, it can be argued that the introduction of a myc epitope tag to the C-terminus of either protein may affect their functions differently, thus compromising the use of PyUIS4myc parasites (RMgm-395) as a control. Therefore, as an additional control to ensure that the failure of PF10_0164 to complement the function of UIS4was not due to the presence of the epitope tag, the mutant line was generated in which uis4  was replaced with PF10_0164 without a tag. These parasites formed liver stages in vitro that expressed PF10_0164 without a myc tag, and the protein localized in a similar manner as seen in the Pyuis4(-)PF10_0164myc parasites. However, infecting mice by intravenous injection of sporozoites of this line did not result in blood stage infections  indicating that that PF10_0164 does not complement UIS4 function in the liver, regardless of the presence or absence of an epitope tag.

Other mutants
RMgm-35, RMgm-38: P. berghei/P. yoelii mutants lacking expression of UIS4
RMgm-395: A P. yoelii mutant expressing a c-terminal myc tagged version of UIS4

  Mutated: Mutant parasite with a mutated 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
Short description of the mutationReplacement of P. yoelii uis4 with P. falciparum PF10_0164, C-terminally myc-tagged
Inducable system usedNo
Short description of the conditional mutagenesisNot available
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 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 PF10_0164myc construct, the PF10_0164 ORF was PCR amplified from 3D7 strain genomic DNA template with the primers SP/PfUIS4-B3D and ASP/PfUIS4-B3D. The promoter from PyUIS4 was amplified from 17XNL strain genomic DNA with the primers SP/PyUIS4Prom-B3D and ASP/PyUIS4Prom-B3D. The amplified PF10_0164 ORF and the PyUIS4 promoter were combined and PCR was performed to fuse the products into a new amplicon using the primers SP/PyUIS4Prom-B3D
and ASP/PfUIS4-B3D. The resulting product was ligated into the B3D vector that encodes a 4x tandem myc epitope tag with the SacII and SpeI restriction endonucleases.
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 PF10_0164 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
Additional information primer 1SP/PfUIS4-B3D (PF10_0164 ORF)
Additional information primer 2ASP/PfUIS4-B3D (PF10_0164 ORF and PCR fusion of PyUIS4 promoter with PF10_0164 ORF)
Additional information primer 3SP/PyUIS4Prom-B3D (Promoter PyUIS4 and PCR fusion of PyUIS4 promoter with PF10_0164 ORF)
Additional information primer 4ASP/PyUIS4Prom-B3D (Promoter PyUIS4)
Additional information primer 5SP/PyUIS4-B3D/3'UTR (PyUIS4 3'UTR)
Additional information primer 6ASP/PyUIS4-B3D/3'UTR (PyUIS4 3'UTR)