RMgmDB - Rodent Malaria genetically modified Parasites

Summary

RMgm-5155
Malaria parasiteP. berghei
Genotype
Transgene
Transgene Plasmodium: Gene model: PBANKA_0931200; Gene model (P.falciparum): PF3D7_1116800; Gene product: heat shock protein 101 (HSP101)
Promoter: Gene model: PBANKA_1133300; Gene model (P.falciparum): PF3D7_1357100; Gene product: elongation factor 1-alpha (eef1a)
3'UTR: Gene model: PBANKA_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (PbDHFS-FPGS)
Replacement locus: Gene model: Not available; Gene product: Not available
Transgene
Transgene not Plasmodium: GFP
Promoter: Gene model: PBANKA_0711900; Gene model (P.falciparum): PF3D7_0818900; Gene product: heat shock protein 70 (HSP70)
3'UTR: Gene model: PBANKA_1340000; Gene product: dihydrofolate synthase/folylpolyglutamate synthase, putative (PbDHFS-FPGS)
Replacement locus: Gene model: Not available; Gene product: Not available
Phenotype Asexual bloodstage; Liver stage;
Last modified: 4 February 2022, 14:25
  *RMgm-5155
Successful modificationThe parasite was generated by the genetic modification
The mutant contains the following genetic modification(s) Introduction of a transgene, Introduction of a transgene
Reference (PubMed-PMID number) Reference 1 (PMID number) : 34956312
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/ResearcherKreutzfeld O, Matuschewski K
Name Group/DepartmentMolecular Parasitology, Institute of Biology/Faculty for Life Sciences
Name InstituteHumboldt Universität zu Berlin
CityBerlin
CountryGermany
Name of the mutant parasite
RMgm numberRMgm-5155
Principal nameef1α:HSP101-myc or ef1α:HSP101-mCherry
Alternative name
Standardized name
Is the mutant parasite cloned after genetic modificationYes
Phenotype
Asexual blood stageef1α:HSP101-mCherry blood stages developed normally and displayed an mCherry signal consistent with PV localization, indicative of proper localization of the HSP101-mCherry fusion protein.
Gametocyte/GameteNot tested
Fertilization and ookineteNot tested
OocystNot different from wild type
SporozoiteNot different from wild type
Liver stageAfter sporozoite injection a slight delay of approximately 1 day in pre-patency in mice infected with ef1α:HSP101-mCherry and ef1α:HSP101-myc parasites in comparison to WT.
The sizes of liver stages were similar for all transgenic lines and WT parasites. In ef1α:HSP101-mCherry parasites successful expression of HSP101 could be detected, using the red mCherry and myc tags as indicator. HSP101-mCherry localized to the liver stage PVM. This localization was independently confirmed in hepatoma cells infected with ef1α:HSP101-myc sporozoites.
Additional remarks phenotype

Mutant/mutation
Two mutants were generated that express an additional copy of PbHSP101 (PbANKA_0931200) under the constitutive eef1a promoter inserted into a silent locus on chromosome 6. The additional copy of HSP101 is C-terminally tagged with either 3xcmyc (mutant ef1α:HSP101-myc) or mCherry (mutant ef1α:HSP101-mCherry).
In addition the mutants express GFP under control of the hsp70 promoter.

Protein (function)
Plasmodium parasites remodel their vertebrate host cells by translocating hundreds of proteins across an encasing membrane into the host cell cytosol via a putative export machinery termed PTEX (Plasmodium Translocon of EXported protein). HSP101 (PbANKA_094120), PTEX150 (PbANKA_100850), EXP2 (PbANKA_133430), PTEX88 (PbANKA_094130) and TRX2 (PbANKA_135800) have been identified as members of the PTEX complex. In contrast to most PTEX proteins, HSP101 is only expressed in blood stages and not in liver stages

Phenotype
ef1α:HSP101-mCherry blood stages developed normally and displayed an mCherry signal consistent with PV localization, indicative of proper localization of the HSP101-mCherry fusion protein.

After sporozoite injection a slight delay of approximately 1 day in pre-patency in mice infected with ef1α:HSP101-mCherry and ef1α:HSP101-myc parasites in comparison to WT. 
The sizes of liver stages were similar for all transgenic lines and WT parasites. In ef1α:HSP101-mCherry parasites successful expression of HSP101 could be detected, using the red mCherry and myc tags as indicator. HSP101-mCherry localized to the liver stage PVM. This localization was independently confirmed in hepatoma cells infected with ef1α:HSP101-myc sporozoites.  

Additional information
Evidence is presented that the constitutive expression of HSP101 in liver stages did not result in export of PEXEL-containing proteins during liver stage development, indicating that HSP101 is not a limiting factor for PEXEL-dependent protein export during this life cycle stage.
In this paper an additional mutant is generated with the promoter of HSP101 replaced by the promoter of ptex150 (PBANKA_1008500; promoter-swap mutant; RMgm-5156). The ptex150 promoter is also active in liver stages. Also in this mutant export of PEXEL-proteins was absent in liver stages.

Other mutants


  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: Plasmodium
Gene Model of Parasite PBANKA_0931200
Gene Model P. falciparum ortholog PF3D7_1116800
Gene productheat shock protein 101
Gene product: Alternative nameHSP101
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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modificationWe generated two targeting plasmids, which express an additional copy P. berghei HSP101 PbANKA_0931200) under the control of the constitutive promoter of elongation factor 1α (ef1α, PBANKA_113330) either as a fusion protein with a triple Myc tag only (ef1α:HSP101-myc) or an additional mCherry tag (ef1α:HSP101-mCherry).

To express an additional copy of PbHSP101 (PbANKA_0931200) under a constitutive promoter the expression cassette was inserted into a silent locus on chromosome 6, using the pBART-SIL6 vector (Kooij et al., 2012). This plasmid harbors a drug selectable hDHFRyFcu cassette for positive/negative selection, a GFP under the PbHSP70 promoter for parasite life cycle analysis, an mCherry/triple c-Myc (3xMyc) tag sequence for protein tagging under the control of the 3′ region of PbPPPK-DHPS for mRNA stability, and two homologous sequences for stable integration into the silent locus on chromosome 6. The HSP101 open reading frame was amplified using primers HSP101for and HSP101rev. The fragment was inserted via XbaI andAgeI 5′ to the mCherry/3xMyc tag. For the plasmids EF1α:HSP101-mCherry and EF1α:
HSP101-myc the 5′ region of EF1α (PbANKA_113330) was amplified using primers EF1for and EF1rev. The fragment was inserted via BssHII and XbaI 5′ next to the HSP101 open reading frame. mCherry was excised in the plasmids EF1α:HSP101-mCherry, and replaced by a linker sequence, generated with Linkfor und Linkrev, fusing the HSP101 open reading frame and the 3xMyc tag.

The plasmid is integrated by double cross-over integration in a locus on P. berghei chromosome 6 between two hypothetical open reading frames (PBANKA_061210 and PBANKA_061220), both displaying only limited gene expression in the stages studied thus far. The integration of pBART-SIL6 and pBAT-SIL6 at this locus was designed to occur 1 kb 3' of PBANKA_061210 and 0.9 kb of PBANKA_260 061220.
Additional remarks selection procedure
Other details transgene
Promoter
Gene Model of Parasite PBANKA_1133300
Gene Model P. falciparum ortholog PF3D7_1357100
Gene productelongation factor 1-alpha
Gene product: Alternative nameeef1a
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_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative namePbDHFS-FPGS
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 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

  Transgene: Mutant parasite expressing a transgene
Type and details of transgene
Is the transgene Plasmodium derived Transgene: not Plasmodium
Transgene nameGFP
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 markerpbdhfr
Selection (positive) procedurepyrimethamine
Selection (negative) procedureNo
Additional remarks genetic modification
Additional remarks selection procedureWe generated two targeting plasmids, which express an additional copy P. berghei HSP101 PbANKA_0931200) under the control of the constitutive promoter of elongation factor 1α (ef1α, PBANKA_113330) either as a fusion protein with a triple Myc tag only (ef1α:HSP101-myc) or an additional mCherry tag (ef1α:HSP101-mCherry).

To express an additional copy of PbHSP101 (PbANKA_0931200) under a constitutive promoter the expression cassette was inserted into a silent locus on chromosome 6, using the pBART-SIL6 vector (Kooij et al., 2012). This plasmid harbors a drug selectable hDHFRyFcu cassette for positive/negative selection, a GFP under the PbHSP70 promoter for parasite life cycle analysis, an mCherry/triple c-Myc (3xMyc) tag sequence for protein tagging under the control of the 3′ region of PbPPPK-DHPS for mRNA stability, and two homologous sequences for stable integration into the silent locus on chromosome 6. The HSP101 open reading frame was amplified using primers HSP101for and HSP101rev. The fragment was inserted via XbaI andAgeI 5′ to the mCherry/3xMyc tag. For the plasmids EF1α:HSP101-mCherry and EF1α:
HSP101-myc the 5′ region of EF1α (PbANKA_113330) was amplified using primers EF1for and EF1rev. The fragment was inserted via BssHII and XbaI 5′ next to the HSP101 open reading frame. mCherry was excised in the plasmids EF1α:HSP101-mCherry, and replaced by a linker sequence, generated with Linkfor und Linkrev, fusing the HSP101 open reading frame and the 3xMyc tag.

The plasmids are integrated by double cross-over integration in a locus on P. berghei chromosome 6 between two hypothetical open reading frames (PBANKA_061210 and PBANKA_061220), both displaying only limited gene expression in the stages studied thus far. The integration of pBART-SIL6 and pBAT-SIL6 at this locus was designed to occur 1 kb 3' of PBANKA_061210 and 0.9 kb of PBANKA_260 061220.
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_1340000
Gene productdihydrofolate synthase/folylpolyglutamate synthase, putative
Gene product: Alternative namePbDHFS-FPGS
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 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