SummaryRMgm-5216
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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) |
Reference 1 (PMID number) : 35628522 |
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 that 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 | Sá M, Tavares J |
Name Group/Department | Host-Parasite Interactions Group, Instituto de Investigação e Inovação em Saúde |
Name Institute | Universidade do Porto |
City | Porto |
Country | Portugal |
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Name of the mutant parasite | |
RMgm number | RMgm-5216 |
Principal name | maebl:myc |
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 | Analyses of the mutant expressing a C-terminal cmyc tagged version of MAEBL showed expression in micronemes of sporozoites (with higher expression in hemolymph sporozoites compared to salivary gland sporozoites). Immunoelectron microscopy analyses provided evidence that myc-tagged MAEBL was detected not only associated with micronemes but also to the surface of sporozoites, i.e., near the plasma membrane and/or inner membrane complex |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Additional information |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0901300 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1147800 | ||||||||||||||||||||||||||
Gene product | membrane associated erythrocyte binding-like protein | ||||||||||||||||||||||||||
Gene product: Alternative name | MAEBL | ||||||||||||||||||||||||||
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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 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 | The transfection vector containing the tagged maebl ORF was obtained through modification of the pL0007_MAEBLcomp plasmid by inserting a sequence encoding 2 copies of the myc tag epitope (2 EQKLISEEDL) right before the stop codon. The maebl 3'UTR (497 bp) and the last 669 bp of the maebl ORF (excluding the stop codon) were amplified using the primer pairs P5/P9 and P10/P11, respectively; the latter primer including the coding sequence of the tag. Both fragments were subcloned into the XhoI/EcoRV and EcoRV/EcoRI sites of a pL0007 vector, originating the plasmid pL0007_MAEBLmyc_3'UTR. The pL0007_MAEBLcomp plasmid was digested with the BstBI restriction enzyme to remove a 6689 bp fragment corresponding to the last 13 bp of the PBANKA_0901400 gene sequence, the entire maebl 5'UTR and the first 5475 bp of the maebl ORF, henceforth named 5'UTR_ORF fragment. After religation, the resultant plasmid was digested with BstBI/HincII to replace the maebl 3'UTR and the final portion of the maebl ORF by a myc-tagged version, obtained through the digestion of pL0007_MAEBLmyc_3'UTR with the same restriction enzymes. Finally, the plasmid was digested with BstBI to allow the insertion of the 5'UTR_ORF fragment, originating the final transfection vector. The correct orientation of the insert and the presence of the myc tag epitope coding sequence in the transfection vector were confirmed by DNA sequencing. The plasmid was linearized with PmeI before transfection. | ||||||||||||||||||||||||||
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) PCR construct 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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