SummaryRMgm-839
|
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) : 23461714 |
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 | P. berghei ANKA 2.34 |
Other information parent line | P. berghei ANKA 2.34 is a cloned, gametocyte producer line of the ANKA strain (PubMed: PMID: 15137943). |
top of page | |
The mutant parasite was generated by | |
Name PI/Researcher | Deligianni, E.; Siden-Kiamos, I. |
Name Group/Department | Institute of Molecular Biology and Biotechnology |
Name Institute | FORTH |
City | Heraklion |
Country | Greece |
top of page | |
Name of the mutant parasite | |
RMgm number | RMgm-839 |
Principal name | pplp2(-) |
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 | Normal production of gametocytes. The production of male gametes is affected. The male gametocyte displays abnormal exflagellation; instead of forming 8 gametes, it produced only one, shared thicker flagellum. Evidence is presented that rupture of the erythrocyte membrane is blocked and thereby preventing egress of male gametes from the erythrocyte. |
Fertilization and ookinete | Strongly reduced ookinete production as a result of aberrant production of male gametes. The male gametocyte displays abnormal exflagellation; instead of forming 8 gametes, it produced only one, shared thicker flagellum. Evidence is presented that rupture of the erythrocyte membrane is blocked and thereby preventing egress of male gametes from the erythrocyte. Evidence is presented that female gametocytes/female gametes show normal egress from the erythrocyte. |
Oocyst | Strongly reduced oocyst production as a result of strongly reduced ookinete production. |
Sporozoite | Not tested |
Liver stage | Not tested |
Additional remarks phenotype | Mutant/mutation The mutant produces low numbers of ookinetes, oocysts and sporozoites. Sporozoites are infectious to mice. These results indicate that PPLP2 has a specific role in egress of male gametocytes/gametes from the erythrocyte. Crossing of pplp2(-) mutant females with wild type male gametes resulted in normal ookinete formation in vitro. This result indicates that female gametocytes/female gametes show normal egress from the erythrocyte and that the female gametes are fertile. |
top of page | |||||||||||||||||||||||||
Details of the target gene | |||||||||||||||||||||||||
Gene Model of Rodent Parasite | PBANKA_1432400 | ||||||||||||||||||||||||
Gene Model P. falciparum ortholog | PF3D7_1216700 | ||||||||||||||||||||||||
Gene product | perforin-like protein 2 | ||||||||||||||||||||||||
Gene product: Alternative name | PPLP2 | ||||||||||||||||||||||||
top of page | |||||||||||||||||||||||||
Details of the genetic modification | |||||||||||||||||||||||||
Inducable system used | No | ||||||||||||||||||||||||
Additional remarks inducable system | |||||||||||||||||||||||||
Type of plasmid/construct used | 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 | KpnI, BamHI | ||||||||||||||||||||||||
Partial or complete disruption of the gene | Complete | ||||||||||||||||||||||||
Additional remarks partial/complete disruption | |||||||||||||||||||||||||
Selectable marker used to select the mutant parasite | tgdhfr | ||||||||||||||||||||||||
Promoter of the selectable marker | pbdhfr | ||||||||||||||||||||||||
Selection (positive) procedure | pyrimethamine | ||||||||||||||||||||||||
Selection (negative) procedure | No | ||||||||||||||||||||||||
Additional remarks genetic modification | Plasmid pDpplp2 for the gene disruption was constructed in the standard vector pL0001. 579 bp from the 5’ end of the ORF of pplp2 and a 591 bp fragment of the 3’ end were amplified from P. berghei genomic DNA. The two fragments were cloned into the KpnI and HindIII sites and the EcoRI and BamHI sites, respectively. The plasmid was digested with KpnI and BamHI before transfection. After double cross-over integration of the construct 1562 bp in the middle of the target gene were replaced by the TgDHFR/TS 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 |