Summary

RMgm-864
Malaria parasiteP. yoelii
Genotype
TaggedGene model (rodent): PY17X_0305700; Gene model (P.falciparum): Not available; Gene product: Not available (SERA1)
Name tag: eGFP
Phenotype Asexual bloodstage;
Last modified: 1 December 2013, 10:53
  *RMgm-864
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) : 23634205
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. yoelii
Parent strain/lineP. y. yoelii YM
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherHuang, X; Preiser, PR
Name Group/DepartmentDivision of Molecular Genetics & Cell Biology
Name InstituteSchool of Biological Sciences, Nanyang Technological University
CitySingapore
CountrySingapore
Name of the mutant parasite
RMgm numberRMgm-864
Principal nameeGFP-tagged SERA1
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageeGFP-SERA1 expression in blood stages (see further 'Additional remarks phenotype').
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a C-terminal eGFP-tagged version of serine repeat antigen 1 (SERA1).
(gene model sera 1 in P. yoelii YM: PYYM_0306000)

Protein (function)
Plasmodium species possesses SERAs of two major groups, specified as 'cysteine-type SERAs' and 'serine-type SERAs'. Serine-type SERAs contain an active site serine residue instead of the canonical cysteine residue. The genomes of different Plasmodium species contain different numbers of SERA genes. P. falciparum possesses nine SERA protease genes whereas the P. berghei and P. yoelii genome contains five. P. falciparum possesses six “serine-type” (SERA1 to SERA5 and SERA9) and three “cysteine-type” (SERA6 to SERA8) SERAs. In P. falciparum mutants have been generated lacking expression of SERA1, 2, 3, 4 , 7, 8 and 9 without a distinct phenotype in the blood stages (MvCoubrie J.E. et al,  2007, Infection and Immunity, 75:5565-74).

SERA genes of P. berghei and P. yoelii are arranged in a tandem cluster that contains two serine-type SERAs (PbSERA1, -2) and three cysteine-type SERAs (PbSERA3, -4 and -5). 

Sequence homology and syntenic organisation indicate that PySERA3 (PY000293), -4 (PY02062) and -5 (PY02063) are orthologs of P. falciparum SERA6, (PF3D7_0207500) -7 (PF3D7_0207400), -8 (PF3D7_0207300)

P. yoelii encodes two members of the SERAser subfamily, PySERA1 and PySERA2.
SERA1 (PY00291) and SERA2 (PY00292) previously recognized as SERA3 and a putative cysteine protease respectively.

Phenotype analyses of P. yoelii YM mutants lacling expression of SERA1 indicate that SERA1 does not have an essential role during blood stage development. Growth,multiplication and virulence of blood stages comparable to wild type (WT) parasites although evidence is presented for subtle differences in growth between mutant and WT parasites.

Phenotype
Analyses of mutants expressing eGFP-tagged SERA1 and SERA2 (RMgm-865) it appears that both PySERA1 and 2 are expressed in the later stage of parasite maturation and that the proteases are located insight the parasitophorous vacuole (PV). However, it cannot be ruled out that the observed location in the PV is either due to the GFP tag preventing proper export or due to the fact that SERA are processed at the C-terminal end prior to activation which could result in the GFP tag remaining within the PV while the protease is exported further. However, no obvious free GFP was detected in the parasite schizont extract using anti-GFP antibody.

Additional information
See also below the P. berghei mutants lacking expression of SERA1, SERA2 and lacking both SERA1 and SERA2. These mutants showed a normal growth of blood stages under the conditions tested.

Evidence has been presented that transcription of sera1 and sera2 are upregulated in the lethal YM strain compared to the non-lethal YA strain of P. yoelii.

Other mutants
RMgm-862: A P. yoelii mutant lacking expression of SERA1
RMgm-863: A P. yoelii mutant lacking expression of SERA2
RMgm-864, RMgm-865: Mutants expressing C-terminal GFP-tagged versions of SERA1 and SERA2 respectively

P. berghei mutants:
RMgm-102: A mutant lacking expression of SERA8 (PB000649.01.0; PFB0325c), a cysteine-type SERA homologue (serine repeat antigen 8, SERA-8; ECP1, egress cysteine protease 1; PbSERA5)
RMgm-271: A mutant expressing GFP under the control of the 5'-UTR of PbSERA3 (an ortholog of P. falciparum SERA6)
RMgm-272: The mutant expresses in addition to the endogenous SERA3  (the ortholog of P. falciparum SERA6) a TAP-tagged form of SERA3 under control of its own 5'-UTR and the dhfr/ts 3'-UTR
RMgm-365: A mutant expressing the endogenous SERA1 protein fused to the mCherry red fluorescent protein
RMgm-366: A mutant expressing the endogenous SERA2 protein fused to the mCherry red fluorescent protein
RMgm-368: A knock-out mutant lacking expression of SERA2
RMgm-369: A double knock-out mutant lacking expression of SERA1 and SERA2


  Tagged: Mutant parasite with a tagged gene
Details of the target gene
Gene Model of Rodent Parasite PY17X_0305700
Gene Model P. falciparum ortholog Not available
Gene productNot available
Gene product: Alternative nameSERA1
Details of the genetic modification
Name of the tageGFP
Details of taggingC-terminal
Additional remarks: tagging
Commercial source of tag-antibodies
Type of plasmid/constructPlasmid single cross-over
PlasmoGEM (Sanger) construct/vector usedNo
Modified PlasmoGEM construct/vector usedNo
Plasmid/construct map
Plasmid/construct sequence
Restriction sites to linearize plasmid
Selectable marker used to select the mutant parasitehdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationA single cross-over gene targeting construct strategy was used. C-terminal fragment of SERA1 gene was cloned into B3DhRap2/3-eGFP vector with the eGFP in frame. Homologous recombination with the linearized plasmid containing the selectable marker hDHFR results in eGFP fused towards the C-terminal of SERA1 such that the expression of eGFP is controlled by the SERA1 endogenous promoter.

No information is provided on the primer sequences used to PCR-amplify the target sequence
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 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
Sequence Primer 3
Additional information primer 3
Sequence Primer 4
Additional information primer 4
Sequence Primer 5
Additional information primer 5
Sequence Primer 6
Additional information primer 6