SummaryRMgm-5256
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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) : 36780562 |
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, Yuda M |
Name Group/Department | Laboratory of Medical Zoology, Department of Medicine |
Name Institute | Mie University |
City | Mie, Tsu |
Country | Japan |
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Name of the mutant parasite | |
RMgm number | RMgm-5256 |
Principal name | AP2-R2::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 | AP2-R2 expression was observed in the nucleus of female gametocytes |
Fertilization and ookinete | Not tested |
Oocyst | Not tested |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation Protein (function) Here, we report that PbAP2-O3 and AP2R-2 are expressed in female gametocytes and function together as a transcriptional repressor complex in P. berghei. Accordingly, we renamed PbAP2-O3 PbAP2-FG2.
Phenotype AP2-R2 expression was observed in the nucleus of female gametocytes.
Ap2r-2 knockout parasites [ap2r-2(-)] (RMgm-5033) are not able to form banana-shaped ookinetes. Additional information Evidence is presented that: - Crossing experiments showed that Pbap2-fg2(-) parasites produce fertile males; only females are 'infertile' and produce aberrant ookinetes
- Disruption of pbap2-fg2 affected the female transcriptome, causing downregulation of female-enriched genes
- Target genes of PbAP2-FG2 were upregulated in ap2-fg2(-)
- The binding motifs of PbAP2-FG2 functioned as a cis-acting repressive element. PbAP2-FG2 functions as a transcriptional repressor.
- PbAP2-FG2 requires a co-repressor AP2R-2 (PBANKA_1418100; PF3D7_1319600) to repress its target genes.
Previously two putative transcriptional regulator genes were identified, ap2r-1 and ap2r-2, as a target gene of AP2-G and AP2-FG. Of these, ap2r-1 functions as a transcriptional activator in zygotes and is renamed ap2-z, but the functional role of ap2r-2 remains unknown. AP2R-2 has an ACDC domain at its C-terminus, but no AP2 domain. It is expressed in females, and ap2r-2 knockout parasites [ap2r-2(-)] are not able to form banana-shaped ookinetes. - PbAP2-FG2 and AP2R-2 repress the target genes of AP2-G
- The function of PyAP2-FG2 is identical to that of PbAP2-FG2.
The analyses revealed that PbAP2-FG2 and PyAP2-FG2 both repress not only male genes but also a wide-variety of genes to support female differentiation.
Other mutants
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Details of the target gene | |||||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1418100 | ||||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1319600 | ||||||||||||||||||||||||||
Gene product | ACDC domain-containing protein, putative | ||||||||||||||||||||||||||
Gene product: Alternative name | AP2R-2 (AP2 Transcription Factor-related gene 2) | ||||||||||||||||||||||||||
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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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