Summary

RMgm-272
Malaria parasiteP. berghei
Genotype
TaggedGene model (rodent): PBANKA_0304900; Gene model (P.falciparum): PF3D7_0207500; Gene product: serine repeat antigen 6 (PbSERA3)
Name tag: TAP
Phenotype Asexual bloodstage; Liver stage;
Last modified: 2 August 2011, 10:54
  *RMgm-272
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) : 18419771
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone Not applicable
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherA. Schmidt-Christensen, V.T. Heussler
Name Group/DepartmentBernhard Nocht Institute for Tropical Medicine
Name InstituteBernhard Nocht Institute for Tropical Medicine
CityHamburg
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-272
Principal namePbSERA3-TAP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
Phenotype
Asexual blood stageExpression (and processing) of PbSERA3-TAP in blood stages
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageExpression (and processing) of PbSERA3-TAP in late liver stages
Additional remarks phenotype

Mutant/mutation
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.

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 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 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 PbSERA3, -4 (PB000352.01.0) and -5 (PB000649.01.0) are orthologs of P. falciparum SERA6, -7, -8.

Phenotype
The mutants has been used to analyse processing of SERA3 in blood and liver stages by Western blotting and to analyse the location of SERA3 by IFA analysis. Processed PbSERA3 has been detected by Western blot analysis in cell extracts of schizont-infected cells and merozoite-infected detached hepatic cells. Immunofluorescence assays revealed that PbSERA3 leaks into the host cell cytoplasm during merozoite development, where it might contribute to host cell death or activate host cell proteases that execute cell death.
Expression of the additional tagged SERA protease had no effect on parasite development throughout the life cycle.

Additional information
In the paper RT-PCR data is presented on transcription of the five SERA genes of P. berghei. PbSERA1-4 are expressed both in blood and liver stages. PbSERA5 is expressed in oocysts and sporozoites.

Other 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-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-367: A knock-out mutant lacking expression of SERA1
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 PBANKA_0304900
Gene Model P. falciparum ortholog PF3D7_0207500
Gene productserine repeat antigen 6
Gene product: Alternative namePbSERA3
Details of the genetic modification
Name of the tagTAP
Details of taggingC-terminal
Additional remarks: taggingThe P. berghei TAP transfection plasmid pL0032, for homologous recombination into the genome, was obtained from MR4 (www.MR4.org). The 5' UTR and the complete PbSERA3 ORF targeting sequence were obtained by amplification of P. berghei gDNA using the primer set: SERA3-TAP/for (5'-GCTCTAGATTTAACAATAAACTTTGCAAAATAGTGAAT-3') and SERA3-TAP/rev (5'-CATGCCATGGACATAACAGAAGAGACATTTGTTTTTTCC-3'). The resulting fragment was cloned into the NcoI/XbaI cloning sites of pL0032 in frame with the TAP-tag coding sequence. The plasmid was linearized for transfection using the unique restriction site XcmI.
The TAP-tag was visualised by Western analysis and IFA using human gamma-globulins (anti-TAP, Sigma).
Commercial source of tag-antibodieshuman gamma-globulins (anti-TAP, Sigma).
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 parasitetgdhfr
Promoter of the selectable markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationThe P. berghei TAP transfection plasmid pL0032, for homologous recombination into the genome, was obtained from MR4 (www.MR4.org). The 5' UTR and the complete PbSERA3 ORF targeting sequence were obtained by amplification of P. berghei gDNA using the primer set: SERA3-TAP/for (5'-GCTCTAGATTTAACAATAAACTTTGCAAAATAGTGAAT-3') and SERA3-TAP/rev (5'-CATGCCATGGACATAACAGAAGAGACATTTGTTTTTTCC-3'). The resulting fragment was cloned into the NcoI/XbaI cloning sites of pL0032 in frame with the TAP-tag coding sequence. The plasmid was linearized for transfection using the unique restriction site XcmI.
Integration of the construct into the SERA3 locus results in the presence of a TAP-tagged copy of SERA3 in addition to the endogenous SERA3 gene. The TAP-tagged form of SERA3 is under control of its own 5'-UTR region and the dhfr/ts 3'-UTR.
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'-GCTCTAGATTTAACAATAAACTTTGCAAAATAGTGAAT-3'
Additional information primer 1SERA3-TAP/for
Sequence Primer 25'-CATGCCATGGACATAACAGAAGAGACATTTGTTTTTTCC-3'
Additional information primer 2SERA3-TAP/rev
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