SummaryRMgm-5254
|
Successful modification | The parasite was generated by the genetic modification |
The mutant contains the following genetic modification(s) | Gene disruption |
Reference (PubMed-PMID number) |
Reference 1 (PMID number) : 36780562 |
MR4 number | |
top of page | |
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 | |
top of page | |
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 |
top of page | |
Name of the mutant parasite | |
RMgm number | RMgm-5254 |
Principal name | pbap2-fg2(-) |
Alternative name | |
Standardized name | |
Is the mutant parasite cloned after genetic modification | Yes |
top of page | |
Phenotype | |
Asexual blood stage | Not different from wild type |
Gametocyte/Gamete | Not different from wild type |
Fertilization and ookinete | 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. |
Oocyst | Formation of aberrant ookinetes. No oocyst/sporozoite formation |
Sporozoite | Formation of aberrant ookinetes. No oocyst/sporozoite formation |
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 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 |
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 | ||||||||||||||||||||||||
top of page | |||||||||||||||||||||||||
Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | (Linear) plasmid double 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 | |||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
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 DNA constructs for tagging PbAP2-FG2 with GFP and knocking out pbap2-fg2 were prepared as previously reported. Briefly, for gfp-tagging, two homologous regions were cloned into the gfp-fusion vector to fuse pbap2-fg2 in-frame with gfp. This vector possesses a hdhfr expression cassette next to the gfp so that mutants can be selected by treatment with pyrimethamine. The plasmid was linearized by XhoI and NotI digestion before use in transfection experiments. To knock out pbap2-fg2, the targeting construct was prepared using overlap PCR. The construct had two homologous regions around the pbap2-fg2 locus flanking a hdhfr expression cassette. | ||||||||||||||||||||||||
Additional remarks selection procedure | |||||||||||||||||||||||||
Primer information: Primers used for amplification of the target sequences
![]() Primer information: Primers used for amplification of the target sequences
![]()
| |||||||||||||||||||||||||
top of page |