RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-984
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Last modified: 1 March 2014, 13:53
*RMgm-984| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Gene mutation, Introduction of a transgene |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 24446886 |
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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 507cl1 (RMgm-7) |
| Other information parent line | P.berghei ANKA 507cl1 (RMgm-7) is a reference ANKA mutant line which expresses GFP under control of a constitutive promoter. This reference line does not contain a drug-selectable marker (PubMed: PMID: 16242190). |
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| The mutant parasite was generated by | |
| Name PI/Researcher | Silvie O; Matuschewski K; Ingmundson A. |
| Name Group/Department | Centre d’Immunologie et des 8 Maladies Infectieuses |
| Name Institute | Sorbonne Universités, UPMC Univ Paris (CIMI) |
| City | Paris |
| Country | France |
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| Name of the mutant parasite | |
| RMgm number | RMgm-984 |
| Principal name | UIS4-mCherry(ssu): lines L1-L9 |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | No |
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| Phenotype | |
| Asexual blood stage | Not tested |
| Gametocyte/Gamete | Not tested |
| Fertilization and ookinete | Not tested |
| Oocyst | Not tested |
| Sporozoite | mCherry expression is analysed in sporozoites and liver stages of 9 different mutants that contain UIS4 tagged with mCherry using different combinations of the promoter, the 3'UTR and tagged uis4 open reading frame. Evidence is presented that transcripts encoding UIS4 are stored in a translationally repressed state in sporozoites, allowing UIS4 protein synthesis only after hepatocyte invasion. Using a series of reporter transgenic parasite lines (see also RMgm-985) evidence is presented that the open reading frame of UIS4 mRNA is critical for gene silencing, whereas the 5’ and 3´ untranslated regions are dispensable. |
| Liver stage | mCherry expression is analysed in sporozoites and liver stages of 9 different mutants that contain UIS4 tagged with mCherry using different combinations of the promoter, the 3'UTR and tagged uis4 open reading frame. Evidence is presented that transcripts encoding UIS4 are stored in a translationally repressed state in sporozoites, allowing UIS4 protein synthesis only after hepatocyte invasion. Using a series of reporter transgenic parasite lines (see also RMgm-985) evidence is presented that the open reading frame of UIS4 mRNA is critical for gene silencing, whereas the 5’ and 3´ untranslated regions are dispensable. |
| Additional remarks phenotype | Mutant/mutation Phenotype Additional information |
Mutated: Mutant parasite with a mutated gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_0501200 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | Not available | ||||||||||||||||||||||||||
| Gene product | early transcribed membrane protein up-regulated in infective sporozoites | ||||||||||||||||||||||||||
| Gene product: Alternative name | UIS4; ETRAMP10.3 | ||||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||||
| Short description of the mutation | different promoters, UTR's to drive mCherry-tagged UIS4 | ||||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||||
| Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||||
| Type of plasmid/construct | single cross-over and centromere-containing episomes | ||||||||||||||||||||||||||
| 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 | pbdhfr | ||||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||||
| Additional remarks genetic modification | See the paper (PMID: 24446886) for details of the different constructs to generate the L1-L9 mutants that contain UIS4 tagged with mCherry using different combinations of promoters, the 3'UTRs and the tagged uis4 open reading frame (and selection cassettes). The mCherry-tagged versions of uis4 are integrated either i) into the (silent) locus of the C-type ssu rRNA gene or ii) the tagged uis4 gene is introduced as a stable episome using centromere-containing plasmids. | ||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
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Transgene: Mutant parasite expressing a transgene| top of page | |||||||||||||||||||
| Type and details of transgene | |||||||||||||||||||
| Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
| Transgene name | GFP (gfp-mu3) | ||||||||||||||||||
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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 | KspI (SacII) | ||||||||||||||||||
| Selectable marker used to select the mutant parasite | gfp (FACS) | ||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||
| Selection (positive) procedure | FACS (flowsorting) | ||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||
| Additional remarks genetic modification | The GFP gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration. | ||||||||||||||||||
| Additional remarks selection procedure | The reporter parent line expressing GFP does not contain a drug-selectable marker. This mutant has been selected by FACS sorting after transfection based on GFP fluorescence. | ||||||||||||||||||
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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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