SummaryRMgm-1503
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene tagging, Gene tagging |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 27427910 |
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 | Kehrer J; Mair GR |
Name Group/Department | Integrative Parasitology, Center for Infectious Diseases |
Name Institute | University of Heidelberg Medical School |
City | Heidelberg |
Country | Germany |
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Name of the mutant parasite | |
RMgm number | RMgm-1503 |
Principal name | pat::gfp;g377:mcherry |
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 | In female gametocytes co-localisation of PAT with the osmiophilic body (OB) protein G377. The presence of additional PAT-positive but G377-negative vesicles suggests that additional vesicles defined by PAT exist that are distinct from OBs containing G377 |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation A mutant lacking expression of PAT (Δpat; RMgm-1500); A mutant in which the endogeneous pat gene is replaced with the pat gene of P. falciparum 3D7: Δpat;pat(PF3D7)::mcherry; RMgm-1501) A mutant expressing GFP-tagged PAT and mCherry-tagged G377 (pat::gfp;g377:mcherry; RMgm-1503): A mutant lacking expression of PAT and expressing mCherry-tagged G377 (PBANKA_1463000) and a mutant lacking expression of PAT and expressing mCherry-tagged PPLP2 (PBANKA_1463000) A mutant expressing a GFP-tagged version of TRAP (ss::gfp::trap; RMgm-1505) A 'promoter-swap' mutant expressing PAT under the promoter of the ccp1/lap2 promoter (PBANKA_1300700)(patccp.PP; RMgm-1506) |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0303900 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0206200 | ||||||||||||||||||||||||||
Gene product | pantothenate transporter | ||||||||||||||||||||||||||
Gene product: Alternative name | PAT | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | GFP | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid single 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 | A 2417 bp product encompassing the entire ORF of pat (1626 bp) as well as 771 bp of the 5’UTR region was amplified from P. berghei genomic DNA with primers g1025 and g1027. The amplicon was cut with HincII and BamHI to release 1539 bp of the 3’ end of the ORF; this fragment was ligated into pLIS0010 (ΔNheI) containing the Toxoplasma dhfr/ts selection cassette to yield a C-terminal GFP fusion. The final plasmid pLIS0301 was digested with NheI, precipitated with 2.5 volumes ethanol and transfected into wildtype schizonts. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1463000 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1250100 | ||||||||||||||||||||||||||
Gene product | osmiophilic body protein G377 | ||||||||||||||||||||||||||
Gene product: Alternative name | Pfg377; G377 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | mCherry | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | (Linear) plasmid single 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 | ||||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | A 1493 bp fragment encompassing the 3’ end of the ORF of P. berghei G377 (PBANKA_146300) ORF (starting from amino acid 4) was PCR-amplified from P. berghei genomic DNA with primers g3092 (PacI) and 3093 (BamHI) and cloned into pLIS0201 to create a C-terminal mCherry fusion. The final plasmid pLIS0244 was digested with BstZ17I, precipitated with 2.5 volumes ethanol and transfected into pat::gfp schizonts. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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