SummaryRMgm-5423
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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) : 38824128 |
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 | Guan J, 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 |
Country | China |
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Name of the mutant parasite | |
RMgm number | RMgm-5423 |
Principal name | PY17X_1323900::6xHA |
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 | Expressed in male gametocytes. In inactivated gametocytes, these proteins were distributed in the cytoplasm, while after activation the protein displayed axoneme localization in the flagellating male gametes. |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Protein (function) Analysis of mutants with the RNA recognition motifs RRM1 (119-190 aa) and RRM2 (203-274 aa) deleted of endogenous RBPm1 in the 17XNL (∆rrm1, RMgm-5412 and ∆rrm2, RMgm-5413) showed the same phenotype as ∆Rbpm1 indicating an essential role of both two RNA recognition motifs in RBPm1 function. Genetic crosses were performed between ∆Rbpm1 mutant and the male-deficient line Δmap2 or the female-deficient line Δnek4. As expected, the cross between Δmap2 and Δnek4 produced the ookinetes in vitro. The ookinete formation was restored in the ΔRbpm1 parasites that were crossed with Δnek4 but not Δmap2, further confirming the ∆Rbpm1 defects in male gamete formation. Evidence is presented that: |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_1323900 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1456400 | ||||||||||||||||||||||||||
Gene product | conserved Plasmodium protein, unknown function | ||||||||||||||||||||||||||
Gene product: Alternative name | |||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | 6xHA | ||||||||||||||||||||||||||
Details of tagging | C-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 | The CRISPR/Cas9 plasmid pYCm was utilized for gene editing. To construct plasmids for gene tagging, the 5’- and 3’- flanking sequences (300 to 700 bp) at the designed insertion site of target genes were amplified as homologous templates. DNA fragments encoding 6HA, GFP, or 4Myc placed between them and in-frame with the target gene. To construct plasmids for N-terminal tagging, left homologous arms comprised 300 to 700 bp sequences upstream of the start codon, while right homologous arms comprised 300 to 700 bp sequences of the upstreaming sequences of the target gene. A DNA fragment encoding HA or 4Myc was then inserted between the left and right homologous arms in-frame with the target gene. To construct the plasmids for gene knockout, left and right homologous arms consisted of 400 to 700 bp sequences upstream and downstream of the coding sequences of the target gene. To construct plasmids for RRM or intron deletion, the RRM or intron and 200 to 700 bp sequences upstream and downstream of the RRM or intron were PCR-amplified and inserted into specific restriction sites in pYCm, followed by RRM or intron deletion using PCR-based site-directed mutagenesis with mutation primers. To construct the plasmids for inserting the intron into the gep1 gene, the left and right homologous arms were composed of gep1 coding sequences ranging from 300 to 600 bp upstream and downstream of the insertion site, respectively. The left homologous arm, intron, and right homologous arm were connected using overlap PCR. Subsequently, the fused fragment was inserted into specific restriction sites in pYCm. For each of the modifications mentioned above, at least two small guide RNAs (sgRNAs) were designed. Forward and reverse single sgRNA oligonucleotides were mixed, denatured at 95°C for 2 minutes, annealed at room temperature for 5 minutes, and ligated into the pYCm. To construct plasmids for the bfp reporter assay, the intact bfp reporter (717 bp) driven by the 5'-UTR (1755 bp) of the hsp70 gene and the 3'-UTR (561 bp) of the dhfr gene were inserted into specific restriction sites between the left and right homologous arms of the p230p gene deletion plasmid. The kinesin8b intron 1 (239 bp), kinesin8b intron 2 (148 bp), PF16 intron 1 (276 bp), dlc1 intron 4 (193 bp), PY17X_1109100 intron 1 (353 bp), and PY17X_1109100 intron 2 (272 bp) were inserted into the bfp reporter by overlap PCR. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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