RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-4363
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Last modified: 27 October 2017, 15:58
*RMgm-4363| 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) : 29034214 |
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| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei NK65 |
| Name parent line/clone | RMgm-1115 |
| Other information parent line | This line is named GIMO mother line (gene insertion/marker out): GIMOPbNK65 (line 1596cl1; 1596cl2). The GIMO mother line shows a normal development during blood stage development although blood stage development has not been characterized in great detail. This line can be used as a reference line for introduction of transgenes. Several transgenic lines have been generated in this GIMO mother line of the NK65 strain. Analysis of blood infections of these transgenic lines has provided some evidence that these parasites induce infections in mice with slightly increased virulence features (higher parasitemia; earlier death) compared to the original 'NK65 Edinburg' parent line (unpublished observations). The GIMO mother line is used for introduction of transgenes into the modified 230p locus through transfection with constructs that target the 230p locus. These constructs insert into the 230p locus (‘gene insertion’), thereby removing the hdhfr::yfcu selectable marker (‘marker out’) from the genome of the mother lines. Transgenic parasites that are marker-free are subsequently selected by applying negative drug selection using 5-FC. This selection procedure is performed in vivo in mice. |
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| The mutant parasite was generated by | |
| Name PI/Researcher | Pham T, Janse CJ, Van den Steen PE |
| Name Group/Department | Laboratory of Immunobiology, Department of Microbiology and Immunology |
| Name Institute | Rega Institute for Medical Research, KU Leuven—University of Leuven |
| City | Leuven |
| Country | Belgium |
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| Name of the mutant parasite | |
| RMgm number | RMgm-4363 |
| Principal name | 2168cl23 |
| 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 Protein (function) Other mutants |
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-Luciferase fusion protein | ||||||||||||||||||
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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 | hdhfr/yfcu | ||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||
| Selection (positive) procedure | No | ||||||||||||||||||
| Selection (negative) procedure | 5-fluorocytosine (5-FC) | ||||||||||||||||||
| Additional remarks genetic modification | Parasites of the PbNK65 GIMO 1995cl2 mother cloned line were transfected with construct pL1156 that contains a GFP-luciferase expression cassette and 230p targeting sequences. The gfp-luciferase gene is under control of the schizont-specific ama1 promoter. After transfection, negative selection was applied to select for parasites in which the hdhfr::yfcu selection cassette in the 230p locus is replaced by the expression cassette. The selected parasites (2168 cloned lines) were cloned by the method of limiting dilution and the 2168cl2 cloned line was genotyped by Southern blot analysis of pulsed field gel electrophoresis (PFGE) separated chromosomes | ||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||
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| Other details transgene | |||||||||||||||||||
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| Promoter | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_0915000 | ||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1133400 | ||||||||||||||||||
| Gene product | apical membrane antigen 1 | ||||||||||||||||||
| Gene product: Alternative name | ama1 | ||||||||||||||||||
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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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