SummaryRMgm-4820
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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) : 32273496 |
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 | Jiang Y, Yuan J |
Name Group/Department | State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signaling Network |
Name Institute | School of Life Sciences, Xiamen University |
City | Xiamen, Fujian |
Country | China |
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Name of the mutant parasite | |
RMgm number | RMgm-4820 |
Principal name | 4Myc::gep1 |
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 | The GEP1 protein is expressed in gametocytes and ookinetes, but not in asexual blood stages and other mosquito stages of the 4Myc::gep1 parasite |
Fertilization and ookinete | The GEP1 protein is expressed in gametocytes and ookinetes, but not in asexual blood stages and other mosquito stages of the 4Myc::gep1 parasite |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Additional information To investigate protein expression and localization, we tagged the endogenous GEP1 with 6HA at N-terminus, generating 6HA::gep1 parasite that had normal development throughout the life cycle. The GEP1 protein is expressed in gametocytes and ookinetes, but not in asexual blood stages and other mosquito stages of the 6HA::gep1 parasite. We also tagged the GEP1 protein with quadruple Myc (4Myc) and observed similar expression pattern in the 4Myc::gep1 parasite. In addition, mScarlet fluorescent signals driven by the endogenous gep1 promoter were detected only in gametocytes, but not in asexual blood stages of the Δgep1mScarlet parasite. Co-staining 6HA::gep1 gametocytes with anti-α-Tubulin (male gametocyte specific) and anti-HA antibody showed that GEP1 was expressed in both male and female gametocytes. Interestingly, GEP1 is not expressed in plasma membrane, but in punctate dots in the cytoplasm of gametocytes and ookinetes. Evidence is presented that: - GEP1-depleted gametocytes are viable, but lost the ability to produce functional male and female gametes.
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_1116300 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0515500 | ||||||||||||||||||||||||||
Gene product | gametogenesis essential protein 1, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | GEP1 | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | 4xMyc | ||||||||||||||||||||||||||
Details of tagging | N-terminal | ||||||||||||||||||||||||||
Additional remarks: tagging | |||||||||||||||||||||||||||
Commercial source of tag-antibodies | |||||||||||||||||||||||||||
Type of plasmid/construct | 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 | |||||||||||||||||||||||||||
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 genetic modifications. For gene deleting, 5’-genomic and 3’-genomic segments (400 to 700 bp) of the target genes were amplified as left and right homologous arms, respectively, using gene specific primers. The PCR products were digested with appropriate restriction enzymes and the digested products were inserted into matched restriction sites of pYCm. Oligonucleotides for sgRNAs were annealed and ligated into pYCm17. For each deletion modification, two sgRNAs were designed to disrupt the coding region of a target gene using the online program ZiFit47. For gene tagging, a 400 to 800 bp segment from N-terminal or C-terminal of the coding region and 400 to 800 bp sequences from 5’UTR or 3’UTR of a target gene were amplified and fused with a DNA fragment encoding 6HA or 4Myc in frame at N-terminal or C-terminal of the gene. For each tagging modification, two sgRNAs were designed to target sites close to the C-terminal or N-terminal of the gene coding region. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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