RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-5050
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Last modified: 29 July 2021, 14:01
*RMgm-5050| 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) : 34301597 |
| MR4 number | |
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| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. yoelii |
| Parent strain/line | P. y. yoelii 17XNL |
| Name parent line/clone | Not applicable |
| Other information parent line | |
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| The mutant parasite was generated by | |
| Name PI/Researcher | Yang Z, Yuan J |
| Name Group/Department | State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal Network, School o |
| Name Institute | Xiamen University |
| City | Xiamen, Fujian |
| Country | China |
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| Name of the mutant parasite | |
| RMgm number | RMgm-5050 |
| Principal name | Δpplp5 |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | No |
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| Phenotype | |
| Asexual blood stage | Not tested |
| Gametocyte/Gamete | Not tested |
| Fertilization and ookinete | Ookinetes are formed but no traversal through mosquito midgut cells |
| Oocyst | Ookinetes are formed but no traversal through mosquito midgut cells |
| Sporozoite | Not tested |
| Liver stage | Not tested |
| Additional remarks phenotype | Mutant/mutation
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Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PY17X_0711800 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0319000 | ||||||||||||||||||||||||
| Gene product | perforin-like protein 5 | ||||||||||||||||||||||||
| Gene product: Alternative name | PLP5, PPLP5, Plasmodium perforin-like protein 5; Membrane Attack Complex | ||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||
| Inducable system used | No | ||||||||||||||||||||||||
| Additional remarks inducable system | |||||||||||||||||||||||||
| Type of plasmid/construct used | CRISPR/Cas9 construct: integration through double strand break repair | ||||||||||||||||||||||||
| PlasmoGEM (Sanger) construct/vector used | No | ||||||||||||||||||||||||
| 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 | CRISPR-Cas9 plasmid pYCm was used for all the parasite genetic modification. To construct vectors for gene editing, we amplified 5′ and 3′ genomic sequence (400 to 500 bp) of target genes as homologous arms using specific primers and inserted the sequences into specific restriction sites in pYCm. Oligonucleotides for single guide RNAs (sgRNAs) were mixed in pairs, denatured at 95°C for 3 min, annealed at room temperature for 5 min, and ligated into pYCm. The sgRNAs were designed to target the coding region of a gene using the online program EuPaGDT. DNA fragments encoding 6HA, 4Myc, 3V5, and GFP or mCherry were inserted between the left and right arms in frame with the gene of interest. For each gene, two sgRNAs were designed to target sites close to the C- or N-terminal part of the coding region. Infected red blood cells (iRBCs) were electroporated with 5 to 10 μg of plasmid DNA using the Lonza Nucleofector . Transfected parasites were immediately intravenously injected into a naïve mouse and were exposed to pyrimethamine (6 mg/ml) 24 hours after transfection. | ||||||||||||||||||||||||
| 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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