RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-271
Malaria parasiteP. berghei
Genotype
Transgene
 Transgene not Plasmodium: GFP (GFP-mut3)
Promoter: Gene model: PBANKA_0304900; Gene model (P.falciparum): PF3D7_0207500; Gene product: serine repeat antigen 6 (PbSERA3)
3'UTR: Gene model: PBANKA_0719300; Gene product: bifunctional dihydrofolate reductase-thymidylate synthase, putative (dhfr/ts)
Phenotype Liver stage;
Last modified: 7 January 2010, 19:55
  *RMgm-271
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 18419771
MR4 number
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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
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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
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Name of the mutant parasite
RMgm numberRMgm-271
Principal nameSERA3 5'-UTR-GFP
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationNo
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Phenotype
Asexual blood stageNot tested
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot tested
SporozoiteNot tested
Liver stageGFP expression in late liver stages at 50h and 64h after infection of HepG2 cells with sporozoites (no expression at 16h and 39h).
Additional remarks phenotype

Mutant/mutation
The mutant expresses GFP under the control of the 5'-UTR of SERA3 (the ortholog of P. falciparum SERA6)

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 phenotype analyses of the liverstages indicate that the promoter of PbSERA3 is active towards the end of the exoerythrocytic stage, in particular during merozoite formation, confirming RT-PCR analyses of transcription (see 'Additional Information').

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-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-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

  Transgene: Mutant parasite expressing a transgene
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Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP (GFP-mut3)
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Details of the genetic modification
Inducable system usedNo
Additional remarks inducable system
Type of plasmid/constructCircular plasmid
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 entire 5′ UTR of SERA3 gene was cloned in front of GFP into the plasmid pL0031 (www.MR4.org). No further information is provided on the size and sequence of the 5'UTR.
Additional remarks selection procedure
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Other details transgene
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Promoter
Gene Model of Parasite PBANKA_0304900
Gene Model P. falciparum ortholog PF3D7_0207500
Gene productserine repeat antigen 6
Gene product: Alternative namePbSERA3
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 15'-GCTCTAGATTTAACAATAAACTTTGCAAAATAGTGAAT-3'
Additional information primer 1SERA3-5'UTR/for
Sequence Primer 25'-CATGCCATGGTTTTTCTTTATATTTTCATTAAC-3'
Additional information primer 2SERA3-5'UTR/rev
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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 / InsertionNot available
Gene Model of Parasite Not available
Gene productNot available
Gene product: Alternative name
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 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
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Conceptual design of the database: Dr. C.J. Janse (Leiden Malaria Research Group, Leiden, The Netherlands).
Technical design and realization: S. Mededovic and A. van Wigcheren