SummaryRMgm-1027
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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 |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 24755823 |
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 | P. berghei ANKA cl15cy1 |
Other information parent line | A reference wild type clone from the ANKA strain of P. berghei (PubMed: PMID: 17406255). |
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The mutant parasite was generated by | |
Name PI/Researcher | Manzoni G; Silvie O |
Name Group/Department | INSERM |
Name Institute | INSERM |
City | Paris |
Country | France |
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Name of the mutant parasite | |
RMgm number | RMgm-1027 |
Principal name | Δp230p-GFP-LUC |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | GFP-luciferase expression in blood stages |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation The construct (see Fig. B below) used contains i) GFP-Luciferase under control of the constitutive promoter of PbHSP70 and ii) a hDHFR-yFCU fusion gene, for positive negative selection, coupled to a second fluorescent cassette, encoding the red fluorescent protein mCherry. Both hDHFR-yFCU and mCherry are placed under control of a single bidirectional promoter of PbeEF1a (PBANKA_113330 and PBANKA_113340). The GFP-Luciferase and mCherry reporter genes are followed by an identical 1 kb fragment corresponding to the 3' untranslated region (UTR) of P. berghei DHFR-TS, placed in the same orientation, which serves both as a transcription terminator, for proper expression of the reporters, and for recycling of the drug resistance cassette. Spontaneous recombination between the two homologous PbDHFR-TS 3' UTR fragments results in excision of both the hDHFR-yFCU and the mCherry expression cassettes Therefore, with this strategy, parasites that have excised the drug-selectable marker become GFP-LUC+mCherry-, and can be easily distinguished from GFP-LUC+ mCherry+ parasites still harbouring the hDHFR-yFCU marker. Selection procedure (see Fig. C below): This new selection method eliminates the need for in vivo cloning of the parasites, and allows the rapid generation of drug-selectable marker-free P. berghei and P. yoelii mutants expressing a GFP or GFP-LUC cassette, using as few as three mice. From: A rapid and robust selection procedure for generating drug-selectable marker-free recombinant malaria parasites. Manzoni, et al. Sci Rep. 2014 Apr 23;4:4760. Other mutants |
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | GFP-Luciferase | ||||||||||||||||||
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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 | hdhfr/yfcu | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
Additional remarks genetic modification | |||||||||||||||||||
Additional remarks selection procedure | The parasites are selected by a combination of positive selection (pyrimethamine), negative selection (5-FC) and FACS sorting. 1) Transfected parasites are first selected in a mouse by pyrimethamine treatment 2) GFP-LUC+mCherry+ parasites are selected by FACS sorting and used to infect a mice 3) This mouse is treated with 5-FC to select for parasites that have the selectable marker removed 4) GFP-LUC+mCherry- and marker free parasites are selected by FAC sorting and used to infect a mouse | ||||||||||||||||||
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Other details transgene | |||||||||||||||||||
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Promoter | |||||||||||||||||||
Gene Model of Parasite | PBANKA_1133400 | ||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1357100 | ||||||||||||||||||
Gene product | elongation factor 1-alpha | ||||||||||||||||||
Gene product: Alternative name | EF-1alpha | ||||||||||||||||||
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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 | P230p | ||||||||||||||||||
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