RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-1226
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Last modified: 25 March 2015, 17:07
*RMgm-1226| 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) : 25786000 |
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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 | RMgm-687 |
| Other information parent line | GIMO-PbANKA (RMgm-687) contains as a selectable marker (SM) the fusion gene of hdhfr (human dihydrofolate reductase; positive SM) and yfcu (yeast cytosine deaminase and uridyl phosphoribosyl transferase; negative SM) stably integrated into the 230p locus (PBANKA_030600) through double cross-over recombination. The SM is under control of the P. berghei eef1α promoter. This reference line of P. berghei ANKA is used for rapid introduction of transgenes free of drug-resistance genes (RMgm-687; PubMed: PMID: 22216235). |
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| The mutant parasite was generated by | |
| Name PI/Researcher | Burda PC; Janse CJ; Heussler VT |
| Name Group/Department | Institute of Cell Biology |
| Name Institute | University of Bern |
| City | Bern |
| Country | Switzerland |
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| Name of the mutant parasite | |
| RMgm number | RMgm-1226 |
| Principal name | PbPL-GFP |
| 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 | Not tested |
| Liver stage | Localisation of PbPL::GFP at the parasitophorous vacuole membrane (PVM) |
| Additional remarks phenotype | Mutant/mutation Protein (function) See also mutant RMgm-1225 with a disrupted PL gene. Analyses of the phenotype of this mutant provide evidence for a role of PbPL in PVM disruption and egress of liver merozoites Other mutants See RMgm-202, RMgm-203 and RMgm-1225 for mutants with a disrupted/mutated PL gene |
Transgene: Mutant parasite expressing a transgene| top of page | |||||||||||||||||||
| Type and details of transgene | |||||||||||||||||||
| Is the transgene Plasmodium derived | Transgene: Plasmodium | ||||||||||||||||||
| Gene Model of Parasite | PBANKA_1128100 | ||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0629300 | ||||||||||||||||||
| Gene product | phosphatidylcholine-sterol acyltransferase, putative | phospholipase, putative | ||||||||||||||||||
| Gene product: Alternative name | PL, UIS10 | ||||||||||||||||||
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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 | The PbPL-GFP expression vector pL0043(LS)PbPL-GFP(C)mCherry was generated by first amplifying the PbPL coding sequence from blood stage cDNA using primer pair PbPL-GFP-fw/PbPL-GFP-rev, which was subsequently digested with BglII and ligated into the BamHI digested liver stage-specific expression vector pGFP(103464) in frame with GFP. From there the (LS)PbPL-GFP expression cassette was cloned via EcoRV and KpnI into the pL0043 vector, which targets the P. berghei 230p locus by double crossover homologous recombination. Finally, a constitutive mCherry expression cassette was integrated via KpnI, which had been amplified before from the p(C)mCherry plasmid using primers mCherry-fw and mCherry-rev. The linearized plasmid pL0043(LS)PbPL-GFP(C)mCherry was transfected into blood stage schizonts of the GIMO(ANKA) parasite line | ||||||||||||||||||
| Additional remarks selection procedure | This mutant expressing PbPL::GFP does not contain a drug-selectable marker. The mutant has been generated in the reference line GIMOPbANKA (RMgm-687). 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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| Other details transgene | |||||||||||||||||||
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| Promoter | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_1003000 | ||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0405300 | ||||||||||||||||||
| Gene product | liver specific protein 2, putative | sequestrin | 6-cysteine protein | ||||||||||||||||||
| Gene product: Alternative name | 6-cysteine protein; sequestrin; LISP2 | ||||||||||||||||||
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| 3'-UTR | |||||||||||||||||||
| Gene Model of Parasite | Not available | ||||||||||||||||||
| Gene product | Not available | ||||||||||||||||||
| Gene product: Alternative name | |||||||||||||||||||
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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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