RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-4723
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Last modified: 6 February 2020, 13:04
*RMgm-4723| Successful modification | The parasite was generated by the genetic modification |
| The mutant contains the following genetic modification(s) | Gene tagging |
| Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 31673027 |
| 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 | Lindner, SE, Kappe SHI |
| Name Group/Department | Department of Global Health |
| Name Institute | University of Washington |
| City | Seattle |
| Country | US |
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| Name of the mutant parasite | |
| RMgm number | RMgm-4723 |
| Principal name | UIS12::-GFP |
| Alternative name | |
| Standardized name | |
| Is the mutant parasite cloned after genetic modification | Yes |
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| Phenotype | |
| Asexual blood stage | Not tested |
| Gametocyte/Gamete | Not tested |
| Fertilization and ookinete | Not tested |
| Oocyst | Not tested |
| Sporozoite | PyUIS12 is an abundant mRNA in salivary gland sporozoites (80th percentile) but was barely detected by mass spectrometry (a single peptide spectrum match (PSM) in salivary gland sporozoites). Using live fluorescence microscopy with PyWT-GFP or PyUIS12::GFP sporozoites, we clearly observed GFP expression in control P. yoelii WT-GFP sporozoites, but did not detect UIS12::GFP protein when transcribed from its native locus. In agreement with translational repression being relieved posttransmission, IFA micrographs clearly show UIS12::GFP expression in the cytosol of 24-h old liver stage parasites. |
| Liver stage | PyUIS12 is an abundant mRNA in salivary gland sporozoites (80th percentile) but was barely detected by mass spectrometry (a single peptide spectrum match (PSM) in salivary gland sporozoites). Using live fluorescence microscopy with PyWT-GFP or PyUIS12::GFP sporozoites, we clearly observed GFP expression in control P. yoelii WT-GFP sporozoites, but did not detect UIS12::GFP protein when transcribed from its native locus. In agreement with translational repression being relieved posttransmission, IFA micrographs clearly show UIS12::GFP expression in the cytosol of 24-h old liver stage parasites. |
| Additional remarks phenotype | Mutant/mutation |
Tagged: Mutant parasite with a tagged gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PY17X_0507300 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1022000 | ||||||||||||||||||||||||||
| Gene product | RNA-binding protein, putative | ||||||||||||||||||||||||||
| Gene product: Alternative name | UIS12 | ||||||||||||||||||||||||||
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| Details of the genetic modification | |||||||||||||||||||||||||||
| Name of the tag | GFP | ||||||||||||||||||||||||||
| Details of tagging | C-terminal | ||||||||||||||||||||||||||
| Additional remarks: tagging | |||||||||||||||||||||||||||
| Commercial source of tag-antibodies | |||||||||||||||||||||||||||
| Type of plasmid/construct | (Linear) 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 | Reverse genetic modification of P. yoelii parasites. Plasmodium yoelii (17XNL strain) was genetically modified using conventional, double homologous recombination approaches with the pDEF plasmid vector. The 3′ end of py17X_1354300 or pyuis12 (PY17X_0507300) was modified by the addition of the GFPmut2 coding sequence prior to the stop codon. Transgenic parasites were identified by genotyping PCR, with independent transgenic clones being isolated by limiting dilution cloning. Clonal parasites were transmitted to A. stephensi mosquitoes to produce salivary gland sporozoites. | ||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
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