SummaryRMgm-5185
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Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene tagging, Introduction of a transgene |
Reference (PubMed-PMID number) | Not published (yet) |
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 676m1cl1 (RMgm-29) |
Other information parent line | 676m1cl1 (RMgm-29) is a reference ANKA mutant line which expresses GFP-luciferase 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 | Costa DM, Tavares J |
Name Group/Department | IBMC – Instituto de Biologia Molecular e Celular |
Name Institute | Universidade do Porto |
City | Porto |
Country | Portugal |
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Name of the mutant parasite | |
RMgm number | RMgm-5185 |
Principal name | SPATR-cmyc |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
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Phenotype | |
Asexual blood stage | Not tested |
Gametocyte/Gamete | Not tested |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Expression of cmyc-tagged SPATR in sporozoites (as shown by Western analysis) |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Published in bioRxiv preprint doi: https://doi.org/10.1101/2022.03.06.483110 Additional information Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0309500 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0212600 | ||||||||||||||||||||||||||
Gene product | secreted protein with altered thrombospondin repeat domain | ||||||||||||||||||||||||||
Gene product: Alternative name | SPATR | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | c-myc | ||||||||||||||||||||||||||
Details of tagging | C-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
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 | tgdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | To generate a parasite line expressing a c-Myc-tagged version of SPATR, the WT spatr open reading frame was replaced by a copy of spatr fused with a sequence encoding a 2x c-Myc tag at the C-terminus, by single cross-over homologous recombination. Since the linearization of the transfection vector was restricted to the AdhI restriction site within the spatr gene, modifications were made to pL0033 (BEI Resources) in in order to remove the AdhI restriction site originally present in the plasmid. The pL0033 fragment containing the ampicillin resistance cassette and the AdhI site that resulted from SapI and NcoI digestion was swapped by the fragment containing the kanamycin marker obtained by digestion of pET-28a(+) (Novagen) with the same restriction enzymes, generating pL0033_Kan. The genomic region encompassing 439 bp of the 5’ intergenic region and the open reading frame of spatr excluding the termination codon was then amplified by PCR using primers P24 + P25 and cloned into pL0033_Kan using the NotI and NcoI restriction sites. The 3’ regulatory sequence of spatr (358 bp) amplified with P26 + P27 was inserted downstream of the sequence encoding the 2x c-Myc tag using the FseI/NdeI restriction sites. A point mutation was inserted in the start codon to prevent the translation of the full-length untagged SPATR due to the duplication of part of the locus resulting from the genetic recombination event. To do this, the region containing the first 13 nucleotides of the coding sequence and the entire 5’ intergenic region previously inserted in the construct was swapped for the sequence amplified using primers P24 + P28 and using the NotI/AhdI restriction sites. Parasites were transfected using the AhdI-linearized construct. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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Type and details of transgene | |||||||||||||||||||
Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
Transgene name | A fusion of GFP (gfp-mu3) and Luciferase Firefly (LucIAV) | ||||||||||||||||||
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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 | gfp (FACS) | ||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||
Selection (positive) procedure | FACS (flowsorting) | ||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||
Additional remarks genetic modification | The GFP-Luciferase gene (1 copy) has been inserted into the 230p locus (PB000214.00.0) by double cross-over integration. | ||||||||||||||||||
Additional remarks selection procedure | This reporter mutant expressing GFP-Luciferase 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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