SummaryRMgm-853
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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) : 23500072 |
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 | Nagel A; Heussler V |
Name Group/Department | Institute of Cell Biology |
Name Institute | University of Bern |
City | Bern |
Country | Switzerland |
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Name of the mutant parasite | |
RMgm number | RMgm-853 |
Principal name | PbPFO(ls) |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Strongly reduced infectivity of sporozoites. Only a low number of mice developed blood stage parasitemia after infection with (high numbers of) sporozoites. FPO expression in (the cytosol of) liver stages (evidence is presented for partial co-localisation with the PVM). Evidence is presented for premature rupture/damage of the PVM in maturing liver stages. |
Additional remarks phenotype | Mutant/mutation Other mutants |
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | CDC perfringolysin O (PFO) | ||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||
Inducable system used | No | ||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||
Type of plasmid/construct | 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 | tgdhfr | ||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | For generation of a transgenic parasite line expressing PFO with a C-terminal V5-tag (Southern et al., 1991) in the liver stage and constitutively expressing cytosolic GFP (referred to as PbPFOLS), initially the coding sequence of PFO (NCBI reference sequence: NP_561079.1; http://ncbi.nlm.nih.gov) was PCR-amplified from C. perfringens genomic DNA (gDNA) using the oligonucleotide primers 5-GCGGATCCATGATAAGATTTAAGAAAACAAAAT-3'and 5'-GCGCGGCCGCATTGTAAGTAATACTAGATCCAGG-3'. The PCR product was cloned via BamHI and NotI restriction sites into the pGFP103464 plasmid(Helm et al., 2010), (resulting plasmid: pL0017.1.4), a modified pL0017 plasmid version (pL0017; MR4 MRA-786) cloned in our laboratory. Additionally the original gfp sequence of the pL0017 plasmid was excised using BamHI and XbaI digestion and was replaced by a double-stranded (ds)DNA oligonucleotide (obtained by annealing the single-stranded (ss)DNA oligonucleotides 5'-GATCCGCGGCCGCCCTAGGAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGTAGT-3' and 5'-CTAGACTACGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACCTCCTAGGGCGGCCGCG-3'), bearing the coding sequence for the V5-tag (bold letters) as well as a NotI restriction site, needed for the cloning of the pfo sequence. The full expression cassette for liver stage-specific expression of PFO::V5 was amplified from the pL0017.1.4 plasmid using the primers 5'-CGGAATTCGTTGCATTATCGTCAAAAGTG-3' and 5'-GCGAATTCCGAAATTGAAGGAAAAAACATCATTTG-3' and ligated via EcoRI restriction sites into a modified form of the pOB90 plasmid (obtained from Dr. Oliver Billker (Sanger Institute, UK)). This plasmid had been modified to allow stable double-cross-over integration into the non-essential gene locus of Pbp230p PBANKA_030600), by ligating into the plasmid, regions of the 50 (amplified by PCR using the primers 5'-ATGCGGTACCGTATATGGTAAAGAACCTACTAAC-3' and 5'-CTTAGGGCCCGATGTGTTTTATTTGGATGTGC-3'and 3' (amplified by PCR using the primers 5'-CTAGGCGGCCGCCTTGAGCCCGTTAATGAAATAG-3' and 5'-TGACCCGCGGGTATGGAACTACATCTATATAGG-3') of this gene locus. Additionally, the full expression cassette for constitutive expression of GFP (amplified from pL0017 plasmid DNA using the primers 5'-GCATCGATAGCTTAATTCTTTTCGAGCTCTTT-3'and 5'-GCAAGCTTCGAAATTGAAGGAAAAAACATCATTTG-3') was ligated via ClaI and HindIII restriction sites into this plasmid (final plasmid: pSI230-PFOLS). | ||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1003000 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0405300 | ||||||||||||||||||
Gene product | liver specific protein 2, putative | sequestrin | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | LISP2 | ||||||||||||||||||
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3'-UTR | |||||||||||||||||||
Gene Model of Parasite | Not available | ||||||||||||||||||
Gene product | Not available | ||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0306000 | ||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | 230p | ||||||||||||||||||
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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 | 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 | tgdhfr | ||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | For generation of a transgenic parasite line expressing PFO with a C-terminal V5-tag (Southern et al., 1991) in the liver stage and constitutively expressing cytosolic GFP (referred to as PbPFOLS), initially the coding sequence of PFO (NCBI reference sequence: NP_561079.1; http://ncbi.nlm.nih.gov) was PCR-amplified from C. perfringens genomic DNA (gDNA) using the oligonucleotide primers 5-GCGGATCCATGATAAGATTTAAGAAAACAAAAT-3'and 5'-GCGCGGCCGCATTGTAAGTAATACTAGATCCAGG-3'. The PCR product was cloned via BamHI and NotI restriction sites into the pGFP103464 plasmid(Helm et al., 2010), (resulting plasmid: pL0017.1.4), a modified pL0017 plasmid version (pL0017; MR4 MRA-786) cloned in our laboratory. Additionally the original gfp sequence of the pL0017 plasmid was excised using BamHI and XbaI digestion and was replaced by a double-stranded (ds)DNA oligonucleotide (obtained by annealing the single-stranded (ss)DNA oligonucleotides 5'-GATCCGCGGCCGCCCTAGGAGGTAAGCCTATCCCTAACCCTCTCCTCGGTCTCGATTCTACGTAGT-3' and 5'-CTAGACTACGTAGAATCGAGACCGAGGAGAGGGTTAGGGATAGGCTTACCTCCTAGGGCGGCCGCG-3'), bearing the coding sequence for the V5-tag (bold letters) as well as a NotI restriction site, needed for the cloning of the pfo sequence. The full expression cassette for liver stage-specific expression of PFO::V5 was amplified from the pL0017.1.4 plasmid using the primers 5'-CGGAATTCGTTGCATTATCGTCAAAAGTG-3' and 5'-GCGAATTCCGAAATTGAAGGAAAAAACATCATTTG-3' and ligated via EcoRI restriction sites into a modified form of the pOB90 plasmid (obtained from Dr. Oliver Billker (Sanger Institute, UK)). This plasmid had been modified to allow stable double-cross-over integration into the non-essential gene locus of Pbp230p PBANKA_030600), by ligating into the plasmid, regions of the 50 (amplified by PCR using the primers 5'-ATGCGGTACCGTATATGGTAAAGAACCTACTAAC-3' and 5'-CTTAGGGCCCGATGTGTTTTATTTGGATGTGC-3'and 3' (amplified by PCR using the primers 5'-CTAGGCGGCCGCCTTGAGCCCGTTAATGAAATAG-3' and 5'-TGACCCGCGGGTATGGAACTACATCTATATAGG-3') of this gene locus. Additionally, the full expression cassette for constitutive expression of GFP (amplified from pL0017 plasmid DNA using the primers 5'-GCATCGATAGCTTAATTCTTTTCGAGCTCTTT-3'and 5'-GCAAGCTTCGAAATTGAAGGAAAAAACATCATTTG-3') was ligated via ClaI and HindIII restriction sites into this plasmid (final plasmid: pSI230-PFOLS). | ||||||||||||||||||
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_0719300 | ||||||||||||||||||
Gene product | bifunctional dihydrofolate reductase-thymidylate synthase, putative | ||||||||||||||||||
Gene product: Alternative name | dhfr/ts | ||||||||||||||||||
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Insertion/Replacement locus | |||||||||||||||||||
Replacement / Insertion | Replacement locus | ||||||||||||||||||
Gene Model of Parasite | PBANKA_0306000 | ||||||||||||||||||
Gene product | 6-cysteine protein | ||||||||||||||||||
Gene product: Alternative name | 230p | ||||||||||||||||||
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