SummaryRMgm-4485
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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) : 30146157 |
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 | Gao H, Yuan J |
Name Group/Department | State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signal 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-4485 |
Principal name | gcβ::6HAn |
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 | Not different from wild type |
Fertilization and ookinete | Normal ookinete development. Expression of tagged protein in gametocytes, zygotes, and ookinetes and could not be detected in asexual blood stage parasites. GCb was localized in the cytoplasm of both gametocytes and zygotes but was concentrated at a site posterior to the apical structure in mature ookinetes. Because of its unique location in ookinetes, the specific location was designated as ‘ookinete extrados site’ (OES). To further investigate GCβ localization in ookinetes relative to proteins known to be expressed within specific organelles or locations, we engineered parasite clones with additional proteins tagged with quadruple Myc epitope (4Myc) from the gcb::6HAc parasite. These proteins included MTIP (glideosome), IMC1i (IMC), ARA1 (apical collar), myosin B (apical ring), and DHHC10 (crystalloid body). GCb was localized at the extrados area behind the apical collar defined by ARA1. Only P28 (plasma membrane) and MTIP showed overlapping localization with GCβ in mature ookinete. |
Oocyst | Not different from wild type |
Sporozoite | Not different from wild type |
Liver stage | Not different from wild type |
Additional remarks phenotype | Mutant/mutation For expression analyses: see above Additional information genes tagged: genes deleted: Mutated GCβ Analyses of these mutants showed the following (from the Abstract): Other mutants |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PY17X_1138200 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1360500 | ||||||||||||||||||||||||||
Gene product | guanylyl cyclase beta | ||||||||||||||||||||||||||
Gene product: Alternative name | GCbeta | ||||||||||||||||||||||||||
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Details of the genetic modification | |||||||||||||||||||||||||||
Name of the tag | HA | ||||||||||||||||||||||||||
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 | 5-fluorocytosine (5-FC) | ||||||||||||||||||||||||||
Additional remarks genetic modification | CRISPR/Cas9 plasmid pYCm was used for parasite genomic modification. To construct the vectors for gene deleting, we amplified the 5'- and 3'- genomic sequence (400 to 700 bp) of target genes as left and right homologous arms using specific primers and inserted into the restriction sites in pYCm. Oligonucleotides for guide RNAs (sgRNAs) were annealed and ligated into pYCm. For each gene, two sgRNAs were designed to target the coding region of gene using the online program ZiFit. To construct the vectors for gene tagging and T2A insertion, we first amplified the C- or N-terminal segments (400 to 800 bp) of the coding regions as left or right arm and 400 to 800 bp from 5' UTR or 3' UTR following the translation stop codon as left and right arm, respectively. A DNA fragment (encoding mCherry, mScarlet, 6HA, 4Myc, or 3V5 tag) was 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. To construct vectors for site-directed nucleotide mutations, the substitution sites were designed with a restriction site for modification detection and placed in the middle of the homologous arms. Parasite-infected red blood cells (RBC) were electroporated with 5 mg purified circular plasmid DNA using the Lonza Nucleotector. | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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