RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-5644
|
||||||||||
Last modified: 21 September 2025, 11:13
*RMgm-5644| Successful modification | The gene/parasite could not be changed/generated by the genetic modification. |
| The following genetic modifications were attempted | Gene disruption |
| Number of attempts to introduce the genetic modification | 2 |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 30700551 |
| top of page | |
| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. berghei |
| Parent strain/line | P. berghei ANKA |
| Name parent line/clone | Not applicable |
| Other information parent line | |
| top of page | |
| Attempts to generate the mutant parasite were performed by | |
| Name PI/Researcher | Nagappa LK, Balaram H |
| Name Group/Department | Jawaharlal Nehru Centre for Advanced Scientific Research |
| Name Institute | Jawaharlal Nehru Centre for Advanced Scientific Research |
| City | - |
| Country | India |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1421300 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0715000 | ||||||||||||||||||||||||
| Gene product | 4-nitrophenylphosphatase | ||||||||||||||||||||||||
| Gene product: Alternative name | phosphoglycolate phosphatase | ||||||||||||||||||||||||
| top of page | |||||||||||||||||||||||||
| Details of the genetic modification | |||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||
| Type of plasmid/construct used | (Linear) plasmid double cross-over | ||||||||||||||||||||||||
| PlasmoGEM (Sanger) construct/vector used | Yes | ||||||||||||||||||||||||
| Name of PlasmoGEM construct/vector | - | ||||||||||||||||||||||||
| Modified PlasmoGEM construct/vector used | No | ||||||||||||||||||||||||
| Plasmid/construct map | |||||||||||||||||||||||||
| Plasmid/construct sequence | |||||||||||||||||||||||||
| Restriction sites to linearize plasmid | |||||||||||||||||||||||||
| Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
| Additional remarks partial/complete disruption | |||||||||||||||||||||||||
| Selectable marker used to select the mutant parasite | hdhfr/yfcu | ||||||||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | The unsuccessful attempts to disrupt this gene indicate an essential function during asexual blood stage growth/multiplication The library clone for P. berghei PGP (PbG02_B-53b06) was obtained from PlasmoGem. Because it was not possible to obtain knockout parasites, a conditional knockdown (at the protein level) strategy was employed by tagging the gene for PbPGP with a regulatable fluorescent affinity (RFA) tag, where the stability of the fusion protein is conditional to the binding of the small molecule trimethoprim. The conditional knockdown vector was also generated by following the recombineering strategy and validated by PCR. Transgenic parasites were obtained in the first transfection attempt, and genotyping by PCR showed the presence of a single homogenous population with correct insertion of the RFA tag (Fig. 4F). Nevertheless, it was observed that the reduction in the levels of RFA-tagged protein upon removal of TMP varied between 30–60% across experiments, and complete knockdown could not be achieved. As a consequence, there was no significant difference in growth rate between parasites grown in mice fed with or without trimethoprim. The transgenic RFA-tagged P. berghei parasites were employed to determine localization of PbPGP, and upon microscopic observation, a cytosolic GFP signal was observed in all intraerythrocytic stages The pJAZZ linear knockout vector for PbPGP was generated by following the strategy described by Pfander et al. (18). Drug-resistant parasites were not obtained in the first transfection attempt. In the second attempt, although drug-resistant parasites were obtained, genotyping by PCR revealed nonspecific integration of the marker cassette. These parasites were positive by PCR for both the PbPGP gene and the human dihydrofolate reductase (hDHFR) marker but were negative for specific 5′ and 3′ integration PCRs | ||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||
|
Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
| |||||||||||||||||||||||||
| top of page | |||||||||||||||||||||||||