RMgmDB - Rodent Malaria genetically modified Parasites
SummaryRMgm-5255
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Last modified: 14 February 2023, 15:00
*RMgm-5255| 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 | Not applicable |
| Other information parent line | |
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| The mutant parasite was generated by | |
| Name PI/Researcher | Nishi T, Yuda M |
| Name Group/Department | 1Laboratory 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-5255 |
| Principal name | PbAP2-FG2::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 | PbAP2-FG2 expression was observed in the nucleus of female gametocytes but not in the other stages including male gametocytes. |
| Fertilization and ookinete | PbAP2-FG2::GFP parasites produced mature, banana-shaped ookinetes, confirming that the GFP fusion did not impair PbAP2-FG2 function (see also below). During ookinete development, no fluorescence was detected at any stage of the parasites |
| 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 PbAP2-FG2 expression was observed in the nucleus of female gametocytes but not in the other stages including male gametocytes. PbAP2-FG2::GFP parasites produced mature, banana-shaped ookinetes, confirming that the GFP fusion did not impair PbAP2-FG2 function (see also below). During ookinete development, no fluorescence was detected at any stage of the parasites. These results collectively indicated that like PyAP2-FG2, PbAP2-FG2 is only expressed in female gametocytes during sexual development and presumably functions in females instead of zygotes.
Analyses of a mutant lacking expression of PbAP2-FG2 (RMgm-5254) showed the following: The mutant formed morphologically normal female and male gametocytes, and the male gametocytes showed normal exflagellation. Pbap2-fg2(-) parasites failed to produce banana-shaped ookinetes; more than half of the fertilized population stopped developing at round zygotes, and the others, at retort-form ookinetes. No oocyst/sporozoite formation. 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 |
Tagged: Mutant parasite with a tagged gene| top of page | |||||||||||||||||||||||||||
| Details of the target gene | |||||||||||||||||||||||||||
| Gene Model of Rodent Parasite | PBANKA_1015500 | ||||||||||||||||||||||||||
| Gene Model P. falciparum ortholog | PF3D7_1429200 | ||||||||||||||||||||||||||
| Gene product | AP2 domain transcription factor AP2-O3, putative | ||||||||||||||||||||||||||
| Gene product: Alternative name | ApiAP2; AP2-O3; PbAP2-FG2 | ||||||||||||||||||||||||||
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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 | (Linear) plasmid single cross-over | ||||||||||||||||||||||||||
| 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 | |||||||||||||||||||||||||||
| Additional remarks selection procedure | |||||||||||||||||||||||||||
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