RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-202
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*RMgm-202| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Gene disruption |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 15590623 |
| MR4 number | |
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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 | Not applicable |
| Other information parent line | |
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| The mutant parasite was generated by | |
| Name PI/Researcher | P. Bhanot; 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-202 |
| Principal name | pl ko |
| 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 | Normal numbers of salivary gland sporozoites are formed that show normal gliding motility. Mutant sporozoites show an in vitro invasion rate of HepG2 cells and development within the hepatocytes that is comparable to wild type parasites. When infected by mosquito bite, the mutant parasites were detected in blood 1 day later than wild type parasites. In contrast to a bite infection, there was no difference in the prepatent period of mutant and wild type parasites when the rats were infected by intravenous injection, indicating a 90% reduction in the infectivity of the sporozoites. By real time PCR a decrease of 90% in parasite load in the liver was detected in mice infected with the mutant parasites by mosquito bite compared to mice infected with wild type parasites. |
| Liver stage | Mutant sporozoites show an in vitro invasion rate of HepG2 cells and development within the hepatocytes that is comparable to wild type parasites. When infected by mosquito bite, the mutant parasites were detected in blood 1 day later than wild type parasites, indicating a 90% reduction in the infectivity of the sporozoites. In contrast to a bite infection, there was no difference in the prepatent period of mutant and wild type parasites when the rats were infected by intravenous injection. By real time PCR a decrease of 90% in parasite load in the liver was detected in mice infected with the mutant parasites by mosquito bite compared to mice infected with wild type parasites. |
| Additional remarks phenotype | Mutant/mutation To test the cell traversal capacity of sporozoites an assay was used in which sporozoites have to traverse an epithelial cell barrier formed by a confluent monolayer of Mardin-Darby canine kidney (MDCK) cells. MDCK cells are plated in the top chamber of a Transwell filter system, whereas HepG2 cells are plated in the bottom chamber. Sporozoites are then added to the top chamber. Since confluent MDCK cells form very tight intercellular junctions, the number of EEFs formed in the HepG2 cell layer is proportional to the numbers of sporozoites that are able to traverse through the MDCK cell layer and is a reflection of the ability of sporozoites to cross cell barriers. Mutant sporozoites formed about 90% fewer EEFs compared with wild type sporozoites, suggesting that they are impaired in their ability to traverse cell layers. |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1128100 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0629300 | ||||||||||||||||||||||||
| Gene product | phosphatidylcholine-sterol acyltransferase, putative | phospholipase, putative | ||||||||||||||||||||||||
| Gene product: Alternative name | PL, phospholipase; UIS10 | ||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||
| Type of plasmid/construct used | 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 | KpnI/SacII | ||||||||||||||||||||||||
| Partial or complete disruption of the gene | Partial | ||||||||||||||||||||||||
| Additional remarks partial/complete disruption | Integration of the targeting plasmid causes reduction in size of a 2.5-kb fragment in wild type parasites to a 0.5-kb fragment in the mutant parasites. There is complete loss of PL transcripts in the mutant parasites | ||||||||||||||||||||||||
| 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 | Wild type pl was replaced by a tgdhfr/ts-gfp selection cassette. | ||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||
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Primer information: Primers used for amplification of the target sequences
 Primer information: Primers used for amplification of the target sequences
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