Back to search resultsSummaryRMgm-5374
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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) : 38554111 |
MR4 number | |
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Parent parasite used to introduce the genetic modification | |
Rodent Malaria Parasite | P. berghei |
Parent strain/line | P. berghei ANKA |
Name parent line/clone | RMgm-4870 |
Other information parent line | The mutant (RMgm-4870) contains a gene encoding Cas9 from Streptococcus pyogenes, introduced into the c/d-ssu-rrna gene. This Cas9 nuclease does not cleave double-stranded DNA in the absence of sgRNA and is thus suitable for generating the parasite which expresses it constitutively. It does not contain a drug-selectable marker that has been removed by negative selection |
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The mutant parasite was generated by | |
Name PI/Researcher | Nishi T, Iwanaga S, Yuda M |
Name Group/Department | Department of Medicine |
Name Institute | Mie University |
City | Tsu |
Country | Japan |
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Name of the mutant parasite | |
RMgm number | RMgm-5374 |
Principal name | gARID::GFP |
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 | No GFP expression in asexual stage parasites. GFP expression in both male and female gametocytes. No GFP expression in ookinetes. |
Fertilization and ookinete | Not different from wild type |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Additional information To further confirm the absence of male gametocytes in garid(−), garid was knocked out in the gametocyte reporter line 820cl1 (garid[−]820), which have GFP and RFP expression cassettes under the control of male- and female-specific promoters, respectively. FACS analysis detected no GFP-positive parasites in the garid(−)820 parasites compared with the parental 820cl1 wherein approximately 3% of the parasites showed a GFP signal. This confirmed that disruption of garid results in the complete loss of male production. Notably, the genome-wide peak pattern of the ChIP-seq of gARID was almost identical to that of gSNF2, previously reported. gSNF2 is a core subunit of SWI/SNF2 chromatin remodeling complex expressed in gametocytes, and gsnf2 knockout parasite (gsnf2[−]) showed impaired male gametocyte development. However, the knockout phenotypes are of not the same; gsnf2(−) produces immature male gametocytes, whereas garid(−) completely loses the ability to differentiate into male gametocytes. In addition, female gametocyte development was affected in garid(−) but not in gsnf2(−). Consistent with the differences in their phenotype, the results in the differential expression analyses for garid(−) vs. wild type and gsnf2(−) vs. wild type were also different. A transgenic line expressing GFP-tagged gARID (gARID::GFP) was generated with the CRISPR/Cas9 system using Pbcas9, and then, gsnf2 was disrupted in the gARID::GFPC (gARID::GFPC_gsnf2(−)); mutant RMgm-5375). Analysis is of this mutant provided evidence that gARID is recruited by gSNF2 as a subunit of the SWI/SNF complex on the TGTCT motifs. In a previous study, it was shown that the female-specific transcriptional activator, PFG, is responsible for recognizing the TGTAYRTACA motifs. Therefore, gARID is recruited to the TGTAYRTACA motifs by PFG independent of gSNF2. In addition to the ten-base motif, the TGCACA motif was also enriched around the peak summits identified in the ChIP-seq using gARID::GFPC_gsnf2(−) suggesting that it could be another important gARID-associated sequence motif. To investigate the function of the TGCACA motif as a cis-regulatory element and the relationship between the TGTCT and TGCACA motifs in male gametocyte development, reporter assays were performed using endogenous loci. As a target for the reporter assays, the following two genes were selected: calm (PBANKA_1421000, a putative calmodulin gene) as a gene significantly down-regulated in garid(−) but only slightly in gsnf2(−) and rsph9 (PBANKA_1431500, a gene encoding a putative radial spoke head protein 9 homolog, a component of radial spokes which control axonemal dynein activity) as a gene downregulated in both garid(−) and gsnf2(−) . First, each of these genes was tagged with gfp to assess their expression using FACS (CALM::GFP and RSPH9::GFP. Then, the TGTCT and TGCACA motifs were mutated, either or both, within the peak regions located upstream of calm and rsph9 to assess the roles of these two cis-regulatory elements in the activation of male genes. |
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_0102400 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_0603600 | ||||||||||||||||||||||||||
Gene product | male development protein MD4, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | AT-rich interactive domain-containing protein, putative, gARID | ||||||||||||||||||||||||||
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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 | 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 | ||||||||||||||||||||||||||
Promoter of the selectable marker | eef1a | ||||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||||
Additional remarks genetic modification | The transgenic parasites were generated by the CRISPR/Cas9 system using the parasites expressing Cas9 (RMgm-4870). The Cas9-expressing parasite Pbcas9 has a cas9 cassette at the p230p locus. The hsp70 promoter controls the expression of Cas9, and Pbcas9 constitutively expresses Cas9 throughout the asexual blood cycle. Donor DNA for transfection was constructed by overlap PCR, cloned into pBluescript KS (+) using the XhoI and BamHI sites by In-Fusion cloning, and then amplified by PCR from the constructed plasmid. sgRNA vectors were constructed as previously described. Target sites of sgRNA were designed using the online tool CHOPCHOP (https://chopchop.cbu.uib.no/). | ||||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||||
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