RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-4318
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Last modified: 7 September 2017, 18:00
*RMgm-4318| 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 | ≥ 5 |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 28790316 |
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| Parent parasite used to introduce the genetic modification | |
| Rodent Malaria Parasite | P. yoelii |
| Parent strain/line | P. y. yoelii 17X |
| Name parent line/clone | Not applicable |
| Other information parent line | The gene was targeted both in P. y. yoelii YM and P. yoelii XL which were considered in this study the same parasite |
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| Attempts to generate the mutant parasite were performed by | |
| Name PI/Researcher | Nair SC; Su XZ |
| Name Group/Department | Malaria Functional Genomics Section, Laboratory of Malaria and Vector Research |
| Name Institute | National Institute of Allergy and Infectious Disease, National Institutes of Health |
| City | Bethesda |
| Country | USA |
Disrupted: Mutant parasite with a disrupted gene| top of page | |||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PY17X_0704800 | ||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_0826100 | ||||||||||||||||||||||||
| Gene product | HECT-like E3 ubiquitin ligase, putative | ||||||||||||||||||||||||
| Gene product: Alternative name | HEUL | ||||||||||||||||||||||||
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| 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 | 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 | ||||||||||||||||||||||||
| Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||
| Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
| Selection (negative) procedure | No | ||||||||||||||||||||||||
| Additional remarks genetic modification | Negative attempts to disrupt this gene indicates an essential role during blood stage development. This gene (PY17X_0704800; PYYM_0704700) was identified in a cross between virulent and less virulent P. yoelii lines (P. y. yoelii YM and P. y. nigeriensis N67 (using quantitative trait loci (QTL) analysis of the progeny). By exchange of (fragments of) the HEUL genes between the two different parasite lines (and by changing the expression level of HEUL) the authors provide evidence for the involvement of HEUL in virulence and growth rate of blood stages. The HEUL gene has a predicted open reading frame of 22,500 bp (182,666–205165 bp) with ~400 amino acids (AA) at the C terminus having homology to a HEUL-conserved domain. E3 ubiquitin ligases add polyubiquitin chains to proteins targeting the proteins for destruction by proteasomes or activating their targets for signal transduction and other biological processes. Protein database searches did not reveal any other known protein domains reported for the PyHEULs, suggesting a unique HEUL with unknown binding specificity. There were two polymorphic microsatellites and 225 SNPs, including 97 nonsynonymous SNPs, between YM and N67; however, no AA substitution was observed in the C-terminal HECT domain. We attempted to knockout (KO) the Pyheul gene from the parasites using a linear construct to replace the gene with a drug selection cassette and a double crossover strategy. Although we could detect integration of the cassette at the locus transiently using PCR, we were not able to obtain parasites with gene disruption after further drug selection. To further confirm the results that the Pyheul is essential for parasite viability, we also used a CRISPR/Cas9 gene-editing method described previously to disrupt the Pyheul gene. These experiments were performed in a separate laboratory using N67 and 17XL because YM was not available in the laboratory (Xiamen University, China). 17XL and YM were derived from 17X, and 17XL has the same growth and disease phenotypes as YM. the authors state: 'We consider YM and 17XL the same parasite because high day-4 parasitemia and sequencing of the Pyheul gene from the parasite supported the conclusion. We made two constructs, one to delete the whole-protein-coding region and the other to delete a segment in the N-terminal region of the PYHEUL. Both efforts failed to produce viable parasites with disrupted Pyheul gene. | ||||||||||||||||||||||||
| 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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