RMgmDB - Rodent Malaria genetically modified Parasites

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Summary

RMgm-4142
Malaria parasiteP. berghei
Genotype
Transgene
Transgene not Plasmodium: FRB::mCherry::TM
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_0514900; Gene product: 28 kDa ookinete surface protein (Pbs21; P28)
Replacement locus: Gene model: PBANKA_0306000; Gene product: 6-cysteine protein (230p)
PhenotypeNo phenotype has been described
Last modified: 11 May 2017, 18:48
  *RMgm-4142
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) : 28428983
MR4 number
Parent parasite used to introduce the genetic modification
Rodent Malaria ParasiteP. berghei
Parent strain/lineP. berghei ANKA
Name parent line/clone P. berghei ANKA cl15cy1
Other information parent line
The mutant parasite was generated by
Name PI/ResearcherHughes KR, Waters AP
Name Group/DepartmentWellcome Centre for Molecular Parasitology, Institute of Infection, Immunity and Inflammation, Colle
Name InstituteUniversity of Glasgow
CityGlasgow
CountryUK
Name of the mutant parasite
RMgm numberRMgm-4142
Principal nameKnockSideways Parental 2 (KSP2)
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 tested
OocystNot tested
SporozoiteNot tested
Liver stageNot tested
Additional remarks phenotype

Mutant/mutation
The mutant expresses a transgene which is a fusion protein (FRB::mCherry::TM; see below for an explanation) introduced into the silent 230p locus. The transgenic parasite does not express a drug-selectable marker (removed by negative selection). The transgene is mainly located at the plasma membrane.

Additional information
This transgenic line is used for inducible protein displacement in Plasmodium in vivo and in vitro using knocksideways (KS) technology. This technology can potentially rapidly inactivate proteins of interest through relocalisation/redistribution.

The addition of rapamycin (rap) induces heterodimerisation of two rapamycin-binding protein domains, FKBP and FRB, which are used to tag the Protein of Interest (POI) and serve as a (membrane-localised) sink for displaced proteins, respectively.

The authors have developed KS in P. berghei to be applicable in principle to a wide variety of proteins, and which combines the relocalising domains with fluorescent markers (GFP and mCherry fused to the anchor FRB and the POI FKBP, respectively) to enable rapid proof of relocalisation through microscopy and visual phenotyping assays.

Two parental lines for KS were generated to express a high level of FRB anchor protein localised to a defined, but accessible, subcellular localisation in P. berghei. The FRB domain was fused to a transmembrane (TM) protein (PBANKA_110790), which is the P. berghei homologue of triose phosphate transporter protein PfoTPT, an apicoplast outer membrane protein predicted to have 10 TM domains with both N- and C-termini cytoplasmically exposed. mCherry was also included generating the fusion protein FRB::mCherry::TM.
High expression was achieved using 1.7 kbp of HSP70 promoter (PBANKA_0711190) and the construct was integrated into the 230p locus to generate the KS parental line. Two similar parental lines were generated, KnockSideways Parental 1 (KSP1), in which the FRB::mCherry::TM protein regulated with a P45/48 stable 3’ UTR (RMgm-4141), and KSP2 with the FRB::mCherry::TM regulated by a P28 3’ UTR (RMgm-4142). KSP2 gave approximately 50% reduced expression of mCherry signal, but resulted better conversion rate to ookinetes.

Preliminary experiments carried out in both backgrounds gave identical results. The fusion protein predominantly localised to the parasite plasma membrane in live parasites, including defining the central cavity. Closer examination revealed some expression on intracellular membranous structures, including a structure morphologically consistent with the apicoplast as predicted by the native localisation of the original PboTPT TM domain protein anchor.
The membrane localisation is conferred by the PboTPT domain, as a line expressing FRB::mCherry showed mCherry throughout the cytoplasm.

Expression of FRB::mCherry::TM appeared to have no detrimental effect on asexual growth and expression was stable over several passages and through mosquito transmission.

The authors demonstrate the characteristics of the system using transgenically expressed cytoplasmic GFP and provide proof of principle by inducibly redistributing a number of proteins with different native, subcellular locations.

Other mutants
Parental 1 (KSP1), in which the FRB::mCherry::TM protein regulated with a P45/48 stable 3’ UTR (RMgm-4141), and KSP2 with the FRB::mCherry::TM regulated by a P28 3’ UTR (RMgm-4142).


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameFRB::mCherry::TM
Details of the genetic modification
Inducable system usedNo
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
Selectable marker used to select the mutant parasitehdhfr/yfcu
Promoter of the selectable markereef1a
Selection (positive) procedurepyrimethamine
Selection (negative) procedure5-fluorocytosine (5-FC)
Additional remarks genetic modificationTwo parental lines for KS were generated to express a high level of FRB anchor protein localised to a defined, but accessible, subcellular localisation in P. berghei. The FRB domain was fused to a transmembrane (TM) protein (PBANKA_110790), which is the P. berghei homologue of triose phosphate transporter protein PfoTPT, an apicoplast outer membrane protein predicted to have 10 TM domains with both N- and C-termini cytoplasmically exposed. mCherry was also included generating the fusion protein FRB::mCherry::TM.
High expression was achieved using 1.7 kbp of HSP70 promoter (PBANKA_0711190) and the construct was integrated into the 230p locus to generate the KS parental line. Two similar parental lines were generated, KnockSideways Parental 1 (KSP1), in which the FRB::mCherry::TM protein regulated with a P45/48 stable 3’ UTR, and KSP2 with the FRB::mCherry::TM regulated by a P28 3’ UTR (RMgm-4142). KSP2 gave approximately 50% reduced expression of mCherry signal, but resulted better conversion rate to ookinetes.

Plasmids used to generate the KS parental lines were created by modification of plasmid pG073 (Sinha et al., 2014) by addition of the FRB rapamycin dependent heterodimerisation partner domain and MCherry (red fluorescence; Clontech) domains fused to PbOTPT (PBANKA_110790; transmembrane domain protein) downstream of 1.6 kbp of HSP70 (PBANKA_071190) 5’ promoter/intergenic region to generate plasmid pG0089, which was used to generate the Knock-Sideways Parental line 1 (KSP1). This parasite line was negatively selected to generate line KSP1-M0 and cloned using conventional single cell cloning to generate line KSP1-m0cl2, which was used as a parental line for further transfections (referred to as KSP1 in the text). The plasmid pG0089 was then modified by replacement of the 3’ UTR region. The first ~500 bp of the p45/48 3’ UTR was excised with SmaI/AfeI and replaced with 492 bp of the translationally repressive 3’ UTR from P28 (PBANKA_051490) to generate plasmid PG0089-P28. This plasmid was digested with SacII prior to transfection into a WT P. berghei line to generate the parental parasite line KSP2. KSP2 was cloned to generate KSP2cl2, negatively selected to generate KSP2Cl2M1 and cloned again to generate KSP2cl2m1cl3 which was used as a parental line for further transfections (referred to as KSP2).
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_0711900
Gene Model P. falciparum ortholog PF3D7_0818900
Gene productheat shock protein 70
Gene product: Alternative nameHSP70
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 1
Additional information primer 1
Sequence Primer 2
Additional information primer 2
3'-UTR
Gene Model of Parasite PBANKA_0514900
Gene product28 kDa ookinete surface protein
Gene product: Alternative namePbs21; P28
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 / InsertionReplacement locus
Gene Model of Parasite PBANKA_0306000
Gene product6-cysteine protein
Gene product: Alternative name230p
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