RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-737
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*RMgm-737| 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) : 22355110 |
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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 | P. berghei NK65 TRAP/FlpL(-) |
| Other information parent line | TRAP/FlpL(-)(RMgm-268) is a P. berghei NK65 mutant that expresses the yeast Flpl recombinase under the control of the trap promoter. The mutant does not contain a drug-selectable marker (PubMed: PMID: 19380117). |
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| The mutant parasite was generated by | |
| Name PI/Researcher | M. Zhang; V. Nussenzweig |
| Name Group/Department | Department of Pathology |
| Name Institute | New York University School of Medicine |
| City | New York |
| Country | USA |
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| Name of the mutant parasite | |
| RMgm number | RMgm-737 |
| Principal name | PbPK4cKO |
| 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 | Mutant sporozoites lacking expression of PK4 showed normal development in vitro in HepG2 cells and developed into mature liver stages. However, the ability of mutant sporozoites to infect mice was dramatically reduced. When mice were injected i.v. with 10(4) sporozoites, fewer mice were infected with mutant sporozoites. Resulting blood infections consisted of only wild type and not mutant parasites. When the dose was reduced to 10(3) sporozoites, only the control mice injected with wild type sporozoites developed blood infection. |
| Liver stage | Mutant sporozoites lacking expression of PK4 showed normal development in vitro in HepG2 cells and developed into mature liver stages. However, the ability of mutant sporozoites to infect mice was dramatically reduced. When mice were injected i.v. with 10(4) sporozoites, fewer mice were infected with mutant sporozoites. Resulting blood infections consisted of only wild type and not mutant parasites. When the dose was reduced to 10(3) sporozoites, only the control mice injected with wild type sporozoites developed blood infection. |
| Additional remarks phenotype | Mutant/mutation Expression of PK4 is silenced by using the Flp/FRT site-specific recombination (SSR) system (see RMgm-268). Removal of the FRTed TRAP 3' regulatory sequences controlling expression of PK4 has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of TRAP 3' regulatory sequences and the hdhfr selectable marker which were flanked by FRT sequences. In the mutant described here the Flp/FRT site-specific recombination (SSR) system of yeast has been used to silence PK4 expression specifically in liver stages. Removal of the FRTed TRAP 3' regulatory sequences controlling expression of PK4 has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the Flp recombinase in sporozoites that resulted in the excision of TRAP 3' regulatory sequences and the hdhfr selectable marker which were flanked by FRT sequences. The phenotype analyses indicate that PK4 is essential for blood stages. In addition PK4 is not essential for development of liver stages in vitro. The absence of blood infections after infecting mice with mutant sporozoites may be due to either only a developmental block of blood stages or due to a failure to produce fully infectious liver merozoites in combination with a developmental block of blood stages. |
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| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1126900 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0628200 | ||||||||||||||||||||||||||
| Gene product | protein kinase PK4 | eukaryotic translation initiation factor 2-alpha kinase | ||||||||||||||||||||||||||
| Gene product: Alternative name | PK4 | ||||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||||
| Short description of the mutation | The mutant contains a mutated pk4 gene with FRT-flanked TRAP 3' regulatory sequences | ||||||||||||||||||||||||||
| Inducable system used | Flp/FRT | ||||||||||||||||||||||||||
| Short description of the conditional mutagenesis | The FRTed 3' regulatory sequence of pk4 is removed in the sporozoite stage by the Flp/FRT system | ||||||||||||||||||||||||||
| Additional remarks inducable system |
  A recently developed P. berghei conditional mutagenesis approach was utilized that uses stage-specific expression of a temperature-sensitive variant of the yeast recombinase, flippase (FlpL) to catalyze the excision of DNA placed between two Flp Recognition Target sequences (FRT) (see RMgm-268). FlpL catalyzes site-specific DNA recombination at a temperature range of 25°-30°C. The pk4 locus was modified by placing it under control of FRT-flanked 3’ TRAP regulatory sequences. The pUC-PbPK4cKO targeting vector was constructed by cloning two PCR products encompassing the extreme C terminus of the gene and its 3’UTR into plasmid p3′TRAP-hDHFRflirte′3 that carries the two Flp recognition target sequences (FRT sites) flanking 3′ UTR of TRAP gene and the hDHFR expression cassette. | ||||||||||||||||||||||||||
| Type of plasmid/construct | Plasmid double cross-over | ||||||||||||||||||||||||||
| PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||||
| Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||||
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| Plasmid/construct sequence | |||||||||||||||||||||||||||
| Restriction sites to linearize plasmid | SphI/KpnI | ||||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | hdhfr | ||||||||||||||||||||||||||
| Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||||
| Additional remarks genetic modification | The pk4 locus was modified by placing it under control of FRT-flanked 3’ TRAP regulatory sequences. The targeting vector was constructed by cloning two PCR products into a vector that carries the two Flp recognition target sequences (FRT sites) flanking 3′ UTR of TRAP gene and the hDHFR expression cassette. Primers PK4-F8 and PK4-R8 were used to amplify a 650-bp fragment encompassing the extreme C terminus of the gene. Primers PK4-F9 and PK4-R9 were used to amplify a 500-bp fragment encompassing the 3′ UTR of PbPK4. Both fragments were sequentially cloned into plasmid p3′TRAP-hDHFRflirte′3 to generate the targeting construct pUC-PbPK4cKO. The SphI/KpnI-linearized plasmid was transfected into P. berghei merozoites. | ||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
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Transgene: Mutant parasite expressing a transgene| top of page | |||||||||||||||||||
| Type and details of transgene | |||||||||||||||||||
| Is the transgene Plasmodium derived | Transgene: not Plasmodium | ||||||||||||||||||
| Transgene name | FlpL recombinase of yeast (FlpL) | ||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||
| Inducable system used | Flp/FRT | ||||||||||||||||||
| Additional remarks inducable system | The mutant expresses the yeast FlpL recombinase under the control of the promoter of trap. The mutant does not contain a selectable marker. This marker (the hdhfr gene) has been removed from the genome by using the Flp/FRT site-specific recombination (SSR) system. Removal of the hdhfr gene has been achieved by transmission of the mutant through mosquitoes, thereby activating expression of the FlpL recombinase that resulted in the excision of the hdhfr gene that was flanked by FRT sequences. | ||||||||||||||||||
| Type of plasmid/construct | 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 | hdhfr | ||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||
| Additional remarks genetic modification | The FlpL coding sequence was flanked by 1.5 kb and 0.6 kb of 5′ and 3′ regulatory sequences of trap, respectively, and associated with a FRTed hdhfr selectable marker containing its own (eef1a) promoter and terminator sequences. The plasmid was linearized in the 5′ TRAP regulatory sequence to direct gap repair and plasmid integration by single crossover recombination at the homologous chromosomal locus, leaving the endogenous trap gene intact. The plasmid contained the hdhfr selectable marker that is flanked with the 34bp FRT sequences | ||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||
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| Other details transgene | |||||||||||||||||||
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| Promoter | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1335900 | ||||||||||||||||||
| Gene product | thrombospondin-related anonymous protein | sporozoite surface protein 2 | ||||||||||||||||||
| Gene product: Alternative name | TRAP; SSP2; SSP-2 | ||||||||||||||||||
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| 3'-UTR | |||||||||||||||||||
| Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
| Gene product | sporozoite surface protein 2 thrombospondin-related anonymous protein | ||||||||||||||||||
| Gene product: Alternative name | sporozoite surface protein 2; SSP2; SSP-2 | ||||||||||||||||||
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| Insertion/Replacement locus | |||||||||||||||||||
| Replacement / Insertion | Insertion locus | ||||||||||||||||||
| Gene Model of Parasite | PBANKA_1349800 | ||||||||||||||||||
| Gene product | sporozoite surface protein 2 thrombospondin-related anonymous protein | ||||||||||||||||||
| Gene product: Alternative name | sporozoite surface protein 2; SSP2; SSP-2 | ||||||||||||||||||
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