SummaryRMgm-5155
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Successful modification | The 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 | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone | Not applicable |
Other information parent line | |
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The mutant parasite was generated by | |
Name PI/Researcher | Kreutzfeld O, Matuschewski K |
Name Group/Department | Molecular Parasitology, Institute of Biology/Faculty for Life Sciences |
Name Institute | Humboldt Universität zu Berlin |
City | Berlin |
Country | Germany |
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Name of the mutant parasite | |
RMgm number | RMgm-5155 |
Principal name | ef1α:HSP101-myc or ef1α:HSP101-mCherry |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | 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. |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | 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 remarks phenotype | Mutant/mutation 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. Additional information |
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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 product | heat shock protein 101 | ||||||||||||||||||
Gene product: Alternative name | HSP101 | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | We 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 | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1133300 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1357100 | ||||||||||||||||||
Gene product | elongation factor 1-alpha | ||||||||||||||||||
Gene product: Alternative name | eef1a | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1340000 | ||||||||||||||||||
Gene product | dihydrofolate synthase/folylpolyglutamate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | PbDHFS-FPGS | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | Not available | ||||||||||||||||||
Gene product | Not available | ||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | GFP | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid double cross-over | ||||||||||||||||||
PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||
Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||
Plasmid/construct map | |||||||||||||||||||
Plasmid/construct sequence | |||||||||||||||||||
Restriction sites to linearize plasmid | |||||||||||||||||||
Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||
Additional remarks selection procedure | We 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. | ||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_0711900 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0818900 | ||||||||||||||||||
Gene product | heat shock protein 70 | ||||||||||||||||||
Gene product: Alternative name | HSP70 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1340000 | ||||||||||||||||||
Gene product | dihydrofolate synthase/folylpolyglutamate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | PbDHFS-FPGS | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | Not available | ||||||||||||||||||
Gene product | Not available | ||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||
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