SummaryRMgm-5102
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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) : 34715088 |
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 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 | Brandsma AM, Montagna GN |
Name Group/Department | Parasitology Unit |
Name Institute | Max Planck Institute for Infection Biology |
City | Berlin |
Country | Germany |
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Name of the mutant parasite | |
RMgm number | RMgm-5102 |
Principal name | uis4::Pfetramp8 |
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 different from wild type |
Fertilization and ookinete | Not different from wild type |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Strongly reduced liver stage development (comparable to mutants lacking expression of P. berghei uis4). No blood-stage infection after infection of mice by mosquito bite or intravenous injection of sporozoites. |
Additional remarks phenotype | Mutant/mutation Protein (function) Phenotype Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0501200 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1016900 | ||||||||||||||||||||||||||
Gene product | early transcribed membrane protein 10.3 | protein of early gametocyte 4 | ||||||||||||||||||||||||||
Gene product: Alternative name | UIS4, up-regulated in infective sporozoites, ETRAMP10.3 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Short description of the mutation | Pbuis4 gene replaced with P. falciparum etramp8 gene (PF3D7_0829600) | ||||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||||
Short description of the conditional mutagenesis | Not available | ||||||||||||||||||||||||||
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 | tgdhfr | ||||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | In order to generate the trans-species complementation parasites, we amplified the 5’UTR of PbUIS4 gene and fused it to the ETRAMP8 or ETRAMP10.3 ORF, respectively, by PCR. We used a first step PCR cycle of 95°C, 5’ and 60°C 5' for the fragment annealing, followed by addition of the appropriate primers and 34 cycles of PCR under standard conditions (primers are listed in Supplementary Table S1). The fragments containing the PbUIS4 5’UTR fused to the corresponding PfETRAMP ORF were cloned into a B3D+ vector (Silvie et al., 2008). Next, the PbUIS4 3’UTR was added to facilitate the integration of the constructs in the PbUIS4 locus by a double recombination event. The resulting B3D+ uis4::etramp8 and B3D+ uis4::etramp10.3 plasmids contained the 3’ UTR of GAPDH following the PfETRAMP ORF. For replacement of the 3’ UTR of GAPDH with the 3’ UTR of UIS3, a 750 bp fragment of the 3’ UTR of UIS3 was amplified from P. berghei WT ANKA-GFP gDNA by PCR using primers 3’UTR PbUIS3fd-BamHI and 3’UTRPbUIS3 rv-EcoRI. After restriction digestion with BamHI and EcoRI, the 3’ UTR of UIS3 was cloned into the B3D+ uis4::etramp8 and B3D+ uis4::etramp10.3 vectors, generating the B3D uis4::etramp8 and B3D uis4::etramp10.3 vectors. The correct replacements were checked by sequencing. The two vectors were linearized with SacII and KpnI. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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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 | (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 gene (1 copy) has been inserted into the 230p locus (PBANKA_030600) by double cross-over integration. | ||||||||||||||||||
Additional remarks selection procedure | This reporter mutant 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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